To assess the incidence of benign and malignant peri‐implant fluid collections and/or masses on magnetic resonance imaging (MRI) in women with silicone implants who are being screened for silent implant rupture.P‐value < 0.05.The institutional review board approved this HIPAA‐compliant retrospective study and waived informed consent. Women who underwent silicone implant oncoplastic and/or cosmetic surgery and postoperative implant‐protocol MRI from 2000 to 2014 were included. Peri‐implant fluid collections and/or masses were measured volumetrically. A benign peri‐implant fluid collection and/or mass was pathologically proven or defined as showing 2 years of imaging and/or clinical stability. A malignant peri‐implant fluid collection was pathologically proven. Incidence of peri‐implant fluid collections and/or masses and positive predictive value (PPV) were calculated on a per‐patient level using proportions and exact 95% confidence intervals (CIs). Fisher's exact test was used in the analysis to test statistical significance pre‐defined as A total of 1070 women with silicone implants were included . Median time between reconstructive surgery and first MRI was 88.9 months . Eighteen women (1.7%) had a peri‐implant fluid collection and/or mass: 15/18 (83.3%) had adequate follow‐up; and only 1/15 was malignant implant associated anaplastic large cell lymphoma, with a PPV of 6.7% (95% CI: 0.003‐0.0005). The median peri‐implant fluid collection size was 89 mL .Peri‐implant fluid collections and/or masses identified at silicone implant protocol breast MR imaging are rarely seen 24 months after reconstructive surgery. Image‐guided fine‐needle aspiration with flow cytometry may be warranted to evaluate for implant‐associated lymphoma. Peri‐implant fluid collections and/or masses on implant protocol breast MRI are rarely seen 24 months after surgery. Image‐guided fine needle aspiration of delayed peri‐implant fluid collection may be considered, as there is a significant association with breast implant associated anaplastic large cell lymphoma. All women with a personal history of operable breast cancer had no clinical evidence of disease at the time of MRI. Women with locally recurrent or metastatic disease were excluded from the study. MRI studies were ordered for either (a) FDA recommended screening for silent implant rupture2.2All images were acquired with either a 1.5 or 3.0 Tesla MRI . A dedicated four‐ or eight‐channel surface breast coil was used. Sagittal T2‐weighted images were acquired using the following parameters: repetition time (msec)/echo time (msec), 3500/102; flip angle, 90°; bandwidth, 25 kHz; field of view, 20‐24 cm; matrix, 256 × 288; number of signals acquired, 2; section thickness, 4 mm; and gap, 1 mm. Axial inversion‐recovery silicone bright water saturation images were acquired using the following parameters: 5000/34; flip angle, 90°; bandwidth, 25 kHz; field of view, 20‐24 cm; matrix, 160 × 256; number of signals acquired, 2; section thickness, 5 mm; and gap, 1 mm. Axial T2‐weighted silicone‐suppressed fat‐saturated images were acquired using the following parameters: 5000/120; flip angle, 90°; bandwidth, 32 kHz; field of view, 20‐24 cm; matrix, 320 × 224; number of signals acquired, 2; section thickness, 5 mm; and gap, 1 mm. No intravenous contrast agent was administered.2.3Three radiologists  with 2, 4, and 19 years of experience, respectively, interpreted the images in consensus. The images were interpreted in random order. Trace peri‐implant fluid was considered physiologic , currently clinically used in radiation treatment planning and various radiology applications. Peri‐implant fluid collections and/or masses were segmented on each image slice of the T2‐weighted MRI where visible. All volumetric segmentations were verified by 1 of the 3 radiologists (E.J.S.) and adjusted accordingly. The volume of peri‐implant fluid collection and/or mass was calculated by adding the volume of each voxel inside the segmented structure. The total volume (in mL) represents the volume of the peri‐implant fluid and/or mass.The MRIs were imported into an FDA‐approved image processing software, 2.4Benign peri‐implant fluid collections and/or masses were pathologically proven or defined as showing at least 2 years of clinical and/or imaging stability. Malignant peri‐implant fluid collections were pathologically proven. Methods used for diagnosis were fine needle aspiration, core biopsy of a mass, or evaluation at the time of implant removal and capsulectomy. Pathologic evaluation included cytology and flow cytometry. Diagnosis of BIA ALCL required immunohistochemistry confirmation of expression of 1 or more T‐cell receptors, as well as staining positive for CD30 and negative for anaplastic lymphoma kinase‐1.2.5P‐value <0.05. The incidence of peri‐implant fluid collections/masses and the positive predictive value (PPV) were calculated on a per‐patient level using proportions and exact 95% confidence intervals (CIs). All statistical tests were 2‐tailed and performed using SAS 9.4 .We examined the distribution of clinical and demographic factors by peri‐implant fluid status Table . Continu3In total, 1070 women with silicone implants with a mean age of 50.7 years  were included in the study. The mean time between reconstructive surgery and the first MRI examination was 47.4 months . Of the 1070 women, 807 (75.4%) had 1 MRI and 263 (24.6%) had more than 1 MRI. Of the women who had more than 1 MRI, 194 (18.1%) had 2, 58 (5.4%) had 3, 9 (0.8%) had 4, 1 (0.09%) had 5, and 1 (0.09%) had 6  had a peri‐implant fluid collection and/or mass Table . Of thosThe median peri‐implant fluid collection size was 89 mL . Only 1 of the 15 peri‐implant fluid collections and/or masses with adequate follow‐up was malignant BIA ALCL, with a PPV of 6.7%. The patient diagnosed with BIA ALCL did not have a prior history of breast cancer but had a textured silicone implant. For the 8 benign peri‐implant fluid collections and/or masses that underwent biopsy, pathology findings were reported as follows: benign or acellular n = 3), benign and implant rupture (n = 1), benign with inflammatory cells n = 2), organizing hematoma (n = 1), or marked chronic inflammation and extensive foreign body giant cell/granulomatous reaction. None of the collections were infectious in etiology .Fisher's exact test confirmed that there was a significant association between peri‐implant fluid and/or mass and the presence of lymphoma (4Here we found that late peri‐implant fluid and/or masses post breast reconstruction or augmentation are very rare, identified in 1.7% of patients on routine MRI performed for silicone implant rupture screening. This is in concert with prior work by Mazzocchi et al, where late peri‐implant collections were identified in 1.7% of patients after reconstruction or augmentation with textured breast implants.The current literature suggests that late peri‐implant seromas arise from friction as the implant moves within the cavity and that this friction is increased with textured rather than smooth implants.Our study is limited for several reasons. First, we did not have adequate information on the type of silicone implant, whether textured or not textured. Second, 3 patients with peri‐implant collections and/or masses had inadequate follow‐up; thus they were excluded from the results. As such, our results may underestimate the prevalence of BIA ALCL. In addition, there was an inherent selection bias in that we evaluated only peri‐implant collections associated with silicone implants and 92.3% of our patients had a past medical history of breast cancer.In conclusion, late peri‐implant fluid collections and/or masses are rarely identified at silicone implant protocol breast MRI 24 months or more after reconstructive surgery. If a late peri‐implant fluid collection and/or mass is detected, ultrasound‐guided fine‐needle aspiration with CD30 immunohistochemistry and cell‐block cytology should be considered to exclude BIA ALCL.None.Elizabeth J Sutton MD: Conceptualization, data curation, formal analysis, funding acquisition, investigation, methodology, project administration, resources, software, supervision, validation, visualization, writing—original draft, and writing—review and editing. Brittany Z Dashevsky MD DPhil: Data curation, investigation, methodology, project administration, writing—original draft, and writing—review and editing. Elizabeth J. Watson MD: Data curation, investigation, methodology, project administration and writing—review and editing. Neelam Tyagi PhD: Data curation, formal analysis, software and writing—review and editing. Blanca Bernard‐Davila MPH MS: Formal analysis, software and writing—review and editing. Danny Martinez BSC MSc: Data curation, formal analysis, software and writing—review and editing. Ahmet Dogan MD PhD: Data curation, formal analysis, supervision and writing—review and editing. Steven M Horwitz MD: Conceptualization, formal analysis, supervision and writing—review and editing. Peter G Cordeiro MD: Data curation, formal analysis, supervision and writing—review and editing. Elizabeth A. Morris MD: Conceptualization, formal analysis, supervision and writing—review and editing.


The present study investigated possible therapeutic effects of radiofrequency or hypomagnetic fields on the growth rate of two types of implanted tumours. To this end, mice with implanted fibrosarcoma and pancreatic tumours were exposed continuously to a 2 µT, 10 MHz radiofrequency magnetic field (MF) perpendicular to a 45 µT static MF or to a hypomagnetic (~0.4–1 µT) field. The reasoning for a 10 MHz treatment was based on a current theoretical explanation for MF effects, which predicts a resonance phenomenon in this frequency range. Radiofrequency MFs reduced consistently the growth rate of two implanted tumour types (by ~30% in both cases). Also, hypomagnetic field hindered tumour growth in both tumour types, but the observation was not statistically significant with fibrosarcoma tumours. In conclusion, although experiments included a limited number of animals, the results indicate that MFs may offer a novel therapeutic strategy in the treatment of cancer. Exposure to 100–120 Hz MFs—alone or in combination with ionising radiation—has been reported to retard the development of implanted mammary3 and liver tumours,4 as well as chemically induced preneoplastic liver lesions.5 However, the underlying mechanism for therapeutic effects of MFs is not known.Research on the biological effects of time-varying magnetic fields (MFs) has mostly focused on possible carcinogenicity and other adverse health effects of environmental 50–60 Hz MFs.6 and this mechanism could also be involved in possible anti-carcinogenic effects of MFs. According to the RPM, chemical reactions with radical pairs as transient intermediates are sensitive to weak MFs, and therefore MFs could affect the concentration of intracellular free radicals.7 The RPM also predicts a resonance frequency range in the order of 0.1–10 MHz because hyperfine splittings occur in this range in most biomolecules.7 The effect should be larger when the static and oscillating field are perpendicular than when they are parallel to each other.The radical pair mechanism (RPM) is considered to be a plausible biophysical mechanism for explaining the adverse health effects of weak environmental MFs,8In this pilot study, we investigated potential tumour-suppressing effects of radiofrequency (RF) MFs. Mice bearing two types of implanted tumours were exposed to a 2 µT, 10 MHz MF perpendicular to a 45 µT static MF. Another treatment group was exposed to a hypomagnetic field (the ambient static geomagnetic field reduced to ~0.4–1 µT), based on earlier results suggesting that reduction of the geomagnetic field inhibits the growth of cultured cells.6 cells/100 µL of either human fibrosarcoma HT1080 or pancreatic AsPC-1 cells were injected intradermally into the flank of 6-week-old female severe-combined immunodeficient mice (Taconic Farms Inc.) after anaesthesia by isoflurane inhalation at 2–3%. The growth of tumour volume was determined by the standard callipre-based measuring procedure twice per week. The measurement was done blinded, without knowledge of the exposure of the animals. In the beginning of the exposure (when the average volume of fibrosarcoma tumours exceeded ~50 mm3 and that of pancreatic cell tumours exceeded ~25 mm3), the mice were randomised by tumour volume and assigned to three treatment groups: (a) control (ambient geomagnetic field 45 µT), (b) hypomagnetic field (~0.4–1 µT) and (c) a 2 µT, 10 MHz RF MF in the horizontal direction combined with a vertical 45 µT static MF. The exposure system has been described in Supplementary Fig. 2 inhalation.A total of 1 × 10p value <0.05 was considered to be statistically significant.A two- or three-way analysis of variance was used for statistical analysis, with MF treatment (RF or hypomagnetic) and a measurement time as fixed factors and cohort as a random factor (only in the case of fibrosarcoma tumours). Cohort was included as a random factor in the analysis because tumour growth rates differed statistically significantly between cohorts. Post-hoc tests were performed using Tukey’s honest significance test . A The RF MF perpendicular to the static field inhibited the growth of implanted fibrosarcoma and pancreatic tumours in comparison to controls Fig. . The eff9In this study, we observed that a 2 µT, 10 MHz RF MF perpendicular to a 45 µT static field inhibited the growth of implanted fibrosarcoma and pancreatic tumours in mice. An ~30% reduction in tumour growth was consistently seen in both xenograft models, although the results of pancreatic tumour experiments must be interpreted with caution due to small group size. To the best of our knowledge, this is the first study suggesting that RF MFs in this frequency range affect tumour growth. The present results are concordant with previous results showing that RF MFs inhibit proliferation of HT1080 fibrosarcoma cells in vitro.9 Positive findings in this frequency range fit both with the predictions of the RPM and experimental results in birds. Evidence for RPM-related biological effects of MFs is strongest in avian magnetoreception, and there is evidence that migratory birds can be prevented from using their magnetic compass when subjected to weak RF MFs with frequencies in the range 0.1–10 MHz.10 However, the current RPM theory is not able to account for the reported extraordinary sensitivity to RF fields.7The RF treatment resulted in consistent inhibition of tumour growth in two different xenograft models, but there is no clear understanding on the mechanism behind the observed effect. As the study was conducted using a frequency within the resonance range predicted by the RPM, the results can be considered to support the hypothesis that the underlying mechanism is MF effects on radical pairs. This hypothesis is supported also by the previous findings of increased radical level in HT1080 fibrosarcoma cells exposed to 10 MHz RF MFs.1Also, the reduced growth of tumours in the hypomagnetic field, although not statistically significant in the fibrosarcoma model, might be explainable by the RPM: if the geomagnetic field is biologically effective in mammalian cells (like in RPM-related avian magnetoreception), removal of the geomagnetic field should result in biological effects. However, the avian magnetoreception system may have been optimised by evolution, and it is currently unclear whether the RPM explains sensitivity to weak MFs in mammalian cells.Although the limited number of animals used in the experiments restricts definite conclusions, the results of this pilot study indicate consistently that a 2 µT 10 MHz RF MF inhibits tumour growth in mouse models. Future studies are warranted to evaluate the applicability of RF MFs in tumour control and to optimise MF parameters  for maximal therapeutic effects. Even the moderate effect size (~30%) observed in this study might be useful in combination with, for example, chemotherapy or radiotherapy.Supplementary figure 1 legendSupplementary figure 1


Characterizing the tumour microenvironment (TME) has become increasingly important to understand the cellular interactions that may be at play for effective therapies. In this study, we used a novel spatial profiling tool, the Nanostring GeoMX Digital Spatial Profiler (DSP) technology, to profile non-small-cell lung cancer (NSCLC) for protein markers across immune cell typing, immune activation, drug targets, and tumour modules. Comparative analysis was performed between the tumour, adjacent tissue, and microenvironment to identify markers enriched in these areas with spatial resolution. Our study reveals that this methodology can be a powerful tool for determining the expression of a large number of protein markers from a single tissue slide.n = 18) of matched patient compartments indicate that the TME was significantly enriched in CD27, CD3, CD4, CD44, CD45, CD45RO, CD68, CD163, and VISTA relative to the tumour. Unmatched analysis indicated that the NAT (n = 19) was significantly enriched in CD34, fibronectin, IDO1, LAG3, ARG1, and PTEN when compared to the TME (n = 32). Univariate Cox proportional hazards indicated that the presence of cells expressing CD3 (hazard ratio (HR): 0.5, p = 0.018), CD34 , and ICOS  in tumour compartments were significantly associated with improved overall survival (OS). We implemented both high-plex and high-throughput methodologies to the discovery of protein biomarkers and molecular phenotypes within biopsy samples, and demonstrate the power of such tools for a new generation of pathology research.Profiling the tumour microenvironment (TME) has been informative in understanding the underlying tumour–immune interactions. Multiplex immunohistochemistry (mIHC) coupled with molecular barcoding technologies have revealed greater insights into the TME. In this study, we utilised the Nanostring GeoMX Digital Spatial Profiler (DSP) platform to profile a non-small-cell lung cancer (NSCLC) tissue microarray for protein markers across immune cell profiling, immuno-oncology (IO) drug targets, immune activation status, immune cell typing, and pan-tumour protein modules. Regions of interest (ROIs) were selected that described tumour, TME, and normal adjacent tissue (NAT) compartments. Our data revealed that paired analysis ( Non-small-cell lung cancer (NSCLC) accounts for 85% of lung cancers, and is the leading cause of cancer-related deaths . PatientWhile traditional immunohistochemistry (IHC) techniques allow for the spatial profiling of cells in the tumour, this is often lost when tumours are analysed using bulk tissue genomic approaches. Moreover, the actual cellular proportions, cellular heterogeneity, and deeper spatial distribution are lacking in characterisation. Spatial and immunological composition with cellular status can aid in identifying micro-niches within the TME . The clan = 19) was significantly enriched in CD34, fibronectin, IDO1, LAG3, ARG1, and PTEN when compared to TME. Univariate Cox proportional hazard analysis indicated that the presence of cells expressing CD3 (hazard ratio (HR): 0.5, p = 0.018), CD34 , and ICOS  in tumour compartments was associated with improved overall survival (OS).In this study, we used the Nanostring GeoMX Digital Spatial Profiler (DSP) to measure compartment-specific expression of proteins across immune cell profiling, immuno-oncology (IO), drug targets, immune activation status, immune cell typing, and pan-tumour protein modules. We found that in paired analysis of matched compartments, the TME was enriched for CD27, CD3, CD4, CD44, CD45, CD45RO, CD68, CD163, and VISTA relative to the tumour regions. Unmatched analysis indicated that the normal adjacent tissue (NAT) (This study has QUT Human Research Ethics Committee (UHREC) approval (#2000000494). The NSCLC Tissue Micro Array (TMA) (HLugA180Su03), containing 92 cases with concordant histologically normal adjacent tissue, was obtained from US Biomax, Inc. , including associated clinical information. H&E images were demarcated by a pathologist for tumour and non-tumour regions in each core. The tissue microarray was purchased from US Biomax . These companies keep the informed consent of the patient samples used to create the microarrays.The slides were profiled using Technology Access Program (TAP) by Nanostring Technologies . In brief, immunofluorescent staining was performed on the TMA with tissue morphology markers  in parallel with DNA-barcoded antibodies within the immune cell profiling, IO drug targets, immune activation status, immune cell typing, and pan-tumour protein panels, as shown in R) between normalisation parameters using the “ggpubr” package [t-tests with a Benjamini–Hochberg correction, while differential expression between unpaired compartments was performed by a Mann–Whitney test with Benjamini–Hochberg correction, and results were plotted in R studio using “ggplot2”. Relative expression data was exported from the GeoMx DSP analysis suite, hierarchical clustering performed using the R package “complexHeatmap” [Patient data presented in  package . NormaliHeatmap” , and uniHeatmap”  package.Ninety-six ROIs in total were selected that were representative of 45 tumours, 32 TMEs, and 19 histologically normal adjacent tissues from the cohort of patients described in 2 and 103, with observably lower median counts for ROIs 13, 67, and 96  (R = 0.92) (R = 0.91), indicating that IgG controls, histone H3, and S6 were unrelated to underlying biology, and could act as appropriate normalisers across ROIs  were plotted to determine which pairs best correlated across ROIs A–C. HistR = 0.7) C, and we = 0.92) D, and thoss ROIs E.In addition to the normalisation by IgG and traditional housekeeping members, ROI area and nuclei count were inspected for their utility to normalise DSP data. K-means clustering to further group ROIs into classes showed most NATs grouping together  positive correlations.Global correlation matrices for target protein expression within the TME  and tumon = 8) did not exhibit significant differences (n = 18) indicated an expected enrichment of CD27, CD3, CD4, CD44, CD45, CD45RO, CD68, CD163, and VISTA within the TME, while tumour regions were enriched in Ki-67, EpCAM, and cytokeratin  in edgeR package , and “Ret-test to a limited number of patient pairs, NAT was indistinguishable from TME. A clear distinction between matched tumour and TME was evident, though, and was indicated by the increased presence of several key markers within the TME. Such markers included CD44, CD45, T cell lineage , memory T cells (CD45RO), monocyte/macrophage lineage , and costimulatory immune checkpoints . When incorporating all samples, irrespective of patient matching, immunosuppressive molecules LAG3 [t-tests available for single-slide analysis within the DSP analysis suite are not wholly appropriate.With this in mind, it was notable that when differential analysis was applied by paired les LAG3  and IDO1les LAG3  were, peNevertheless, the sheer scale of high-plex analyses appropriately applied to large numbers of cases through TMAs is an incredibly powerful tool for spatial biology. The DSP protein modules include key markers that describe multiple immune cell types, immune checkpoints, and experimental targets that enable a more comprehensive understanding of the immunological parameters that influence patient outcome. While overall survival was the only clinical endpoint investigated in this study, the emergence of patient cohorts treated with immunotherapies means that such assays may be used to track patient progression and outcomes, and indicate potential biomarkers for patients most likely to respond to these therapies. Despite some limitations in the absolute definition of tumour and TME compartments in our study, we were able to identify that the presence of CD3, CD34, and ICOS expressing cells in tumour compartments were associated with better patient OS in an unadjusted univariate Cox proportional hazards model. However, these findings require validation.It is interesting to note that the enrichment of CD3 in tumour regions was associated with improved OS in this study, independently of CD4 T helper cells and cytotoxic CD8 T lymphocytes. Several markers significantly correlated with CD3 expression in the tumour compartment, including CD40, CD44, CD14, B2M, Tim-3, CD8, CD45RO, and ICOS, potentially implicating other cell lineages in immune-associated anti-tumour activity. Of note is the correlation between CD3 and ICOS, both of which were independently prognostic within tumour compartments, highlighting the potential power of such multiplex discovery.Furthermore, limitations include the retrospective nature of the study, the need for orthogonal validation, and an increase in comparative groups.In summary, the application of such novel platforms to provide comprehensive snapshots of clinical material enables an unprecedented insight into molecular phenotypes that may be indicative of response to emerging therapies, and ultimately, patient outcome. We propose the development of appropriate normalization methods to overcome systematic variation and low signal-to-noise, and indicate the requirement for larger sample numbers to overcome the limitations of multiple testing in discovery approaches. By combining such high-plex approaches with TMAs and orthogonal validation through multispectral IHC, a new field of biomarker discovery is developing that offers to change the way clinical pathology is performed.


Synchrotron Microbeam Radiation Therapy (MRT) provides excellent tumour control in preclinical cancer models. Most of the results obtained in the field employ average peak doses between 300 and 600 Gy. However, despite its proven efficacy, one of the biggest challenges in translating MRT to the clinic is that these high doses can be delivered only at synchrotron facilities, and there are only a few in the world. To solve this problem, we delivered three sessions of lower peak doses of 133 Gy, which approach clinical capabilities. The results showed complete tumour remission for 18 months. In fact, it ablated 50% of the primary melanomas with no metastasis. We believe that this study is of value because (i) it increased the therapeutic index of MRT, and (ii) it demonstrates that MRT can be less dependent on synchrotron sources, which opens the path for future clinical application.Background: Synchrotron Microbeam Radiotherapy (MRT) significantly improves local tumour control with minimal normal tissue toxicity. MRT delivers orthovoltage X-rays at an ultra-high “FLASH” dose rate in spatially fractionated beams, typically only few tens of micrometres wide. One of the biggest challenges in translating MRT to the clinic is its use of high peak doses, of around 300–600 Gy, which can currently only be delivered by synchrotron facilities. Therefore, in an effort to improve the translation of MRT to the clinic, this work studied whether the temporal fractionation of traditional MRT into several sessions with lower, more clinically feasible, peak doses could still maintain local tumour control. Methods: Two groups of twelve C57Bl/6J female mice harbouring B16-F10 melanomas in their ears were treated with microbeams of 50 µm in width spaced by 200 µm from their centres. The treatment modality was either (i) a single MRT session of 401.23 Gy peak dose , or (ii) three MRT sessions of 133.41 Gy peak dose  delivered over 3 days in different anatomical planes, which intersected at 45 degrees. The mean dose rate was 12,750 Gy/s, with exposure times between 34.2 and 11.4 ms, respectively. Results: Temporally fractionated MRT ablated 50% of B16-F10 mouse melanomas, preventing organ metastases and local tumour recurrence for 18 months. In the rest of the animals, the median survival increased by 2.5-fold in comparison to the single MRT session and by 4.1-fold with respect to untreated mice. Conclusions: Temporally fractionating MRT with lower peak doses not only maintained tumour control, but also increased the efficacy of this technique. These results demonstrate that the solution to making MRT more clinically feasible is to irradiate with several fractions of intersecting arrays with lower peak doses. This provides alternatives to synchrotron sources where future microbeam radiotherapy could be delivered with less intense radiation sources. Synchrotron Microbeam Radiation Therapy (MRT) is a preclinical approach of spatial fractionation that has been found to obtain superior tumour control in different animal models relative to a homogenous radiation field ,2,3. SynRegarding MRT’s anti-tumour effects, the published literature shows that MRT targets the tumour microenvironment with exceptional efficacy and unique complexity: MRT increases tumour cell death when compared to a homogenous beam of synchrotron radiation , selectiExploring the benefits of temporally fractionating MRT was one of the aims of this manuscript. Temporal fractionation in conventional radiotherapy is known to provide better tumour control while producing lower normal tissue toxicity than a single large dose; it increases tumour damage by allowing both the redistribution of tumour cells into more vulnerable phases of the cell cycle   and the This manuscript demonstrates that three sessions of temporally fractionated MRT intersected at 45 degrees over 3 days were able to ablate 50% of the B16-F10 mouse melanomas. Moreover, the cured mice did not develop metastasis and had no behavioural abnormalities for 18 months. The histopathological assessment of the cured mice revealed that in the place where the tumour was located, a high concentration of melanin-laden macrophages was found, indicating that the immune system played a role in tumour eradication.Two groups of 12 mice harbouring B16-F10 melanomas in their ears were exposed to either a single MRT fraction A or to tIn contrast, p < 0.001). Further analysis revealed a 2.5-fold increase in the median survival after three fractions of MRT  and during this time, the mice did not have behavioural abnormalities nor signs of cancer. s of MRT B. FigureDuring the daily tumour follow-up, we observed that when the melanomas shrank, they lost all of their volume and became completely flat. Moreover, they did not regrow, and the ears displayed a distinct black pigmentation of melanin on the skin, which resembled a nevus A. At theThe absence of melanoma cells in the ears was confirmed with immunohistochemistry. The melanin-laden cells were negative for two melanoma markers: the cytoplasmatic staining of Melan-A E and theAnalysis of the 18-month-old nevus with electron microscopy revealed that melanophages were heavily loaded with melanin I, while The data in this manuscript demonstrate that three sessions of temporally fractionated MRT arrays intersecting at 45 degrees and delivered over 3 days had the unique capability of eradicating 50% of the primary melanomas and preventing organ metastases for 18 months. In the rest of the animals, this treatment scheme increased the median survival time 2.5-fold (relative to the single MRT fraction) and 4.1-fold against untreated tumours. The fractionated MRT scheme was also associated with a delay in tumour growth of 27% (from 9.8 to 12.8 days) and a reduction of 20.8% in the number of tumours that did not respond to the treatment (from 33.3 to 12.5%).The benefits of temporally fractionating the dose are well-known in conventional radiotherapy but have been scarcely explored in MRT. In 2009, Serduc and colleagues exposed 9 L gliosarcoma-bearing rats to three MRT arrays composed of microbeams 50 μm wide and 211 μm from beam centre to beam centre, with a dose rate of approximately 16,000 Gy/s at the European Synchrotron . TumoursThe geometry of the MRT array may also significantly influence tumour control. Specifically, Uyama and colleagues  spaced tTemporal fractionation of the dose also influences the tumour response. We have shown that a single peak dose of 400 Gy of MRT markedly reduced the functional vascularity and blood perfusion of the B16-F10 melanomas . The thrThe delivery of the MRT array in different anatomical planes is also a factor that needs consideration. In fact, in the present study, the proportion of tissue exposed to peak and valley doses changed as the number of intersecting MRT arrays increased. The histopathological assessment of the cured mice revealed a high concentration of melanin-laden macrophages where the melanoma was located. This suggests that the immune system may have played a role in the elimination of the tumour as published data show that fractionated radiotherapy (but not a single fraction) can sometimes trigger systemic anti-tumour immune responses, which is a phenomenon also mentioned in the literature as abscopal effects . Preclin3 or the melanoma presented skin laceration due to its rapid growth. All the procedures presented here were approved by the Swiss Animal Welfare Office from the Canton of Bern under the permit number BE61/15 and by the French Ministry of Higher Education and Research under the permit number APAFIS#9605-2017041013465762 v2.Eight to ten-week-old young-adult female C57BL/6J mice  weighing 18–20 g on arrival were housed and cared for in the animal facility of the European Synchrotron at 50% humidity following a 12 h day/night cycle. Two weeks after their arrival, mice were subcutaneously implanted with 120,000 B16-F10 melanoma cells in both ears following our published protocol . The in-®, Grand Island, NY, USA). On the day of implantation, flasks containing B16-F10 cells on the logarithmic-growth phase were selected; they had been given a medium change 24 h before the experiment. Then, cells were washed with Dulbecco’s phosphate-buffered saline(DPBS), detached with 5 mL of Trypsin-Ethylenediamine Tetraacetic Acid (EDTA) (0.25%), and the trypsin was neutralised with 10 mL of fresh growth medium. The cell suspension was immediately centrifuged for 5 min at 130 rcf  and the pellet was re-suspended in 1 mL of Dulbecco’s Modified Eagle Medium (DMEM) FBS-free. Cells were counted with a disposable Hemocytometer C-Chip , centrifuged once more, and re-suspended with the correct amount of DMEM FBS-free medium that allows having a cell concentration of 120,000 cells in 4 μL. The final cell suspension was placed on ice and proceeded immediately with the tumour implantation.The B16-F10 melanoma cells originated from the C57BL/6J mouse strain and were bought from the American Type Culture Collection . They are adherent cells with a mixed morphology of epithelial-like and spindle-shaped cells. In preparation for the tumour implantation, the B16-F10 cells were thawed 2 weeks prior to the experiments, passaged every 3–4 days or when reaching 90% confluency, and maintained in growth medium composed of 0 UK Origin; supplemented with 10% certified Fetal Bovine Serum (FBS) . After the microsurgery, each mouse was immediately labelled on its tail using coloured markers, and the anaesthesia was reversed via an IP injection of the following antagonist cocktail: 0.5 mg/kg of BW of flumazenil, 2.5 mg/kg of BW of atipamezol, and 0.1 mg/kg of BW of buprenorphine; all diluted in sodium chloride 0.9%. The measurement of the tumour size was performed daily starting on day 7 after tumour implantation. A digital calliper was used to measure the size of each tumour following the three axes . Details of the protocol can be found in our previously published paper [In preparation for the microsurgery, each mouse was anesthetised via an intraperitoneal (IP) injection with an agonist cocktail composed of 0.05 mg/kg of body weight (BW) of fentanyl, 5 mg/kg of BW of midazolam, and 0.5 mg/kg of BW of medetomidin; all were diluted in sodium chloride 0.9%. The implantation of the melanoma is made on the ventral side of the mouse ears. Three fine cuts are made on the ear’s skin along the superior, inferior, and lateral side of a square. Then, the skin is detached from the cartilage  through a movement that resembles opening a door. Then, cuts are made on the exposed cartilage following the four sides of a square. Then, the cut square-cartilage is removed, leaving a window to the dorsal side on the skin of the ear. A clot of 120,000 B16F10 cells is placed inside this window. The clot is formed with 3 µL of fibrinogen (5 mg/mL), 3 µL of thrombin (3 mg/mL), and 120,000 cells suspended in 4 µL of cell culture medium. The skin from the ventral side is immediately repositioned back (the door is closed), and the three-ventral skin cuts are sealed using Topical Skin Adhesive (Histoacryled paper .3 (SD = 5.4). Twenty-four mice were distributed among two experimental groups of 12 animals. The melanomas were exposed to either a single MRT array or three unidirectional MRT arrays that were intersected in 45 degrees over 3 days.The irradiations started 9 days after implantation, and the mean volume of the melanomas was 11.3 mm2 radiation field used was composed by an array of 38 vertical microbeams, 50 µm wide and spaced 200 µm from their centres. Considering a volume of 1 mm3 in the centre of the tumour, a peak-to-valley dose ratio (PVDR) of 54.24 was found  in Grenoble, France. As shown in After tissue harvesting, the ears bearing melanomas were collected and fixed according to the following steps. Fixation with 4% paraformaldehyde in PBS over 24 h (the volume of fixative was at least 10× the volume the ear), three washes in PBS, removal of fixative with 15% glucose over 6 h, three washes in PBS, and 70% ethanol until paraffin embedding. Serial sections of 4 μm were prepared and stained with haematoxylin and eosin. Upon observation of the melanin-laden cells, we proceeded to perform immunohistochemistry for the following sets of primary antibodies: (i) Rabbit monoclonal anti-MelanA  diluted with 3% milk at 1:1000, (ii) Rabbit monoclonal anti-CD68  diluted with 3% milk at 1:500, and (iii) Rabbit monoclonal anti-SOX10 IgG  diluted with 3% milk at 1:200. The second antibody was the ImmPRESS goat anti-rabbit Immunoglobulin G polymer  diluted with ImmPRESS reagent at 1:27.2O2 in methanol for 30 min at room temperature, washed twice in PBS for 3 min, incubated with secondary antibody for 30 min at room temperature, washed three times in PBS for 5 min, incubated in a solution of 3,3′-Diaminobenzidine (DAB) for 8 min at room temperature, and rinsed in distilled water for 5 min. Then, we proceeded to bleach the melanin by incubating the sections in 1% trichloroisocyanuric acid (TCCA) (freshly made) for 60 min at room temperature. Slides were counter-stained with Myer’s hemalum (1:1) for 2–3 min, dehydrated, and covered. The slides from the control samples were not bleached, since the amount of melanin did not interfere with the visualisation of the antibodies. In addition, the slides were stained with the NovaRED substrate kit for peroxidase Reagent  instead of DAB.Briefly, our general protocol was the following: deparaffinisation of the slides, heat-induced antigen retrieval for 20 min , cooling at room temperature for 20 min, wash in PBS, 0.5% Tween 20 in PBS for 30 min, and two washes in PBS. Blocking steps included: blocking with 2.5% normal goat blocking solution for 20 min at room temperature, blocking with 3% milk for 20 min at room temperature, and protein block  for 20 min at room temperature. Immediately after the slides were incubated with the primary antibody for 1 h at 37 °C, they were rinsed with PBS for 5 min, incubated in 0.6% HIn conclusion, the present study demonstrates that our fractionated MRT scheme intersected at 45 degrees angle over 3 days ablated 50% of the primary melanomas, whereas delivery of the total cumulative dose in a single MRT fraction did not. The fractionated scheme also increased by 2.5-fold the survival of mice in comparison to the single MRT session, and by 4.1-fold against the untreated mice (reused data from our previously published study [


C9orf72 repeat expansion in ALS patients. Specifically, we found that that the majority of the significant probes inadvertently cross-hybridized to the C9orf72 locus. Importantly, these probes were not flagged as cross-reactive in previous studies, leading to novel insights into the extent to which cross-reactivity can impact EWAS. Our findings are particularly relevant for epigenetic studies into diseases associated with repeat expansions and other types of structural variation. More generally however, considering that most spurious associations were not excluded based on pre-defined sets of cross-reactive probes, we believe that the presented data-driven flag and consider approach is relevant for any type of EWAS.Illumina DNA methylation arrays are a widely used tool for performing genome-wide DNA methylation analyses. However, measurements obtained from these arrays may be affected by technical artefacts that result in spurious associations if left unchecked. Cross-reactivity represents one of the major challenges, meaning that probes may map to multiple regions in the genome. Although several studies have reported on this issue, few studies have empirically examined the impact of cross-reactivity in an epigenome-wide association study (EWAS). In this paper, we report on cross-reactivity issues that we discovered in a large EWAS on the presence of the  DNA methylation is a key epigenetic mechanism that is involved in gene regulation by influencing transcription factor binding and recruiting histone-modifying proteins . It invoAn important driver in expanding our understanding of DNA methylation in health and disease has been the growing number of epigenome-wide association studies (EWAS) . These sAlthough several studies have shown that these arrays generally provide accurate and reproducible measures, there have been various reports on technical artefacts that can result in spurious results . Cross-rC9orf72 (C9) repeat expansion, a GGGGCC (G4C2) nucleotide repeat expansion, which is the most common mutation (±8%) in both ALS and frontotemporal dementia  and controls. Specifically, we focused on the identification of DNA methylation profiles associated with the presence of the dementia . We provWe show that these issues apply to both the most recent EPIC array as well as the older, but more widely used, 450k array. Our findings serve as a cautionary note to researchers using Illumina arrays, and we provide several recommendations to prevent spurious results caused by cross-hybridization.www.projectmine.com), described in more detail elsewhere  was bisulfite-treated using Zymo Bisulfite Conversion Kits . DNA methylation was analyzed using the Infinium Methylation450k array (NL samples) or Infinium EPIC array (UK and Ireland samples), according to the standard Infinium HD array methylation protocol Venous blood was drawn from patients and controls from which genomic DNA was isolated using standard methods. We set the DNA concentrations at 100 ng/μl as measured by a fluorometer with the PicoGreenhttp://cnsgenomics.com/software/osca)  . All othre/osca) . Figuresa et al. ,19. Figu package . Probe m package . Finallyidyverse , for a cminfi package  Samples that failed on the inbreeding and relatedness metrics in the corresponding whole-genome-sequencing (WGS) data. Quality control of the Project MinE WGS data was performed as described earlier . Finally, we removed one individual for each related pair of individuals  to obtain a set of unrelated individuals. The number of samples that failed on the various QC metrics are listed in We performed quality control and normalization separately for the 450k data (NL) and EPIC data (UK and Ireland). Identical thresholds were used for 450k and EPIC data, unless indicated. Samples that failed the following criteria were removed: (i) Samples with median methylated or unmethylated intensity <1500 (<2000 for EPIC data). (ii) Samples with median red/green ratio <0.5 or >2 as calculated in type I probes (<0.4 or >2.5 in EPIC data). (iii) Discordance between reported sex and predicted sex based on the in minfi . (iv) Sa package . (v) Sam package . (vi) SaSNP data . We perfP-value > 1 × 10−16 or measured by <3 beads to missing .The QC’ed signal intensities were normalized using the  package . For typ package   algorithm  while fitting all the other distal probes as random effects. Fitting the distal probes as random effects accounts for correlations induced by (unobserved) confounding factors. Specifically, we used the ‘LOCO’ option, which excludes all probes located on the same chromosome as the target site from the random effects terms so that the target site is not fitted twice (once as a fixed effect and once as a random effect). We included sex, experimental batch, predicted age and predicted smoking score as fixed covariates in the model.We performed an EWAS of software . BrieflyWe used the same algorithms as described above for the EWASs on OOB β-values and EWASs on total signal intensities. In case the OSCA algorithm did not converge, we used a multivariate linear model adjusting for experimental batch, age, sex, smoking score, imputed cell fractions, the first five array-wide PCs and the first five control probe PCs. This was the case for the EWAS on total signal intensities and the EWAS in the EPIC replication dataset.in silico. Although the number of repeats differs per carrier, we used a fixed number of 10 repeats (60 bp), which is sufficient for the full 50-nt sequence of a probe to match. Both strands were bisulfite-converted in silico, where all non-CpG cytosines were converted in T’s and C’s in CpG-sites were either converted to Ts (unmethylated) or Cs (methylated). We then generated the complement strands of the bisulfite-converted strands, resulting in four strands in total .We generated the forward and reverse strand of the GGGGCC hexanucleotide repeat 3) possible probe sequences per CpG-site. We generated all possible combinations for the type II probes and combined them with the type I probe sequences, resulting in 1 119 157 450k probe sequences and 1 752 933 EPIC probe sequences.We downloaded the Illumina 450k and EPIC annotations as implemented in Bioconductor . The proBiostrings package in R , or that the repeat is fully unmethylated .Allowing every combination of DNA methylation states within the repeat by converting all C’s of CpGs to Y’s (Y = T or C in IUPAC code).et al. that variation nearer to the 3′ site of the probe will not result in hybridization (Allowing a mismatch or an INDEL >5 bp of the 3′end of the probe sequence. The 5 bp cutoff is based on the finding by Zhou dization .C9orf72 (C9) repeat expansion status (wild-type or expanded) within 1748 ALS patients , with four of these located in a CpG island directly upstream of the repeat. The remaining 12 CpGs were located in trans of the C9 repeat expansion, being distributed across chromosomes  and assumed that the repeat was fully methylated  match the C9 repeat, we mapped all probes on the 450k array (485 512 probes) to the bisulfite-converted C9 repeat sequence trans probes partially match (≥14 bp) the C9 repeat, while none of the probe sequences of the significant cis associations match the repeat . We found however that, some probes that partially match to the C9 repeat failed to reach significance in the EWAS. Moreover, we note that several genome-wide significant trans probes had only a limited match to the C9 repeat . We discovered that these observations could be explained by color channel switches and imperfect matches respectively, which we discuss in more detail in the following section.As Figure trans probes that do partially match the C9 repeat could be explained by color channel switches (4 out of 10 probes with a ≥14 bp C9 match were not significant). To investigate this hypothesis we first predicted for each probe whether a color-channel switch would occur if the probe would hybridize to the C9 repeat . For probes with an expected color-channel switch upon C9 hybridization (dubbed ’OOB probes’), we expect differential signal in the OOB color channels. We therefore performed an EWAS using β-values calculated from the OOB channels  may match imperfectly (i.e. with mismatches and/or gaps). We therefore reran the mapping procedure using inexact matching where we allowed one mismatch/INDEL. We excluded matches with a mismatch/INDEL near the 3′-end of the probe (≤5 bp) since it has been previously shown that these prevent hybridization . These include the majority of the sites that were significantly associated with C9 status . However, we found that for a range of cut-offs, the significant probes from the C9 EWAS were strongly enriched for probes with longer matches to the C9 repeat  and C9 status at each probe, using a mixed linear model as implemented in OSCA. We identified 11 probes for which the total signal intensity was significantly associated with C9 status , and had a consistent direction of effect . Finally, similar to the 450k array, we found that the significant probes from the C9 EWAS in the EPIC data were enriched for probes with longer matches to the C9 repeat .We performed a replication analysis in two cohorts comprising 437 ALS patients that were profiled using the more recent Illumina EPIC array . This wat Figure . The repa Figure . To evalC9orf72 (C9) repeat expansion in ALS patients. We provide strong evidence that the majority of the significant associations were spurious due to cross-hybridization to the C9 repeat. Our findings highlight the extent to which cross-reactivity can impact EWAS findings. Although previous studies have reported on cross-reactivity in Illumina methylation arrays, only 2 out of 11 of the technical artefacts would have been removed based on existing guidelines. These findings are particularly relevant for epigenetic studies into diseases associated with repeat expansions and other types of structural variation. More generally however, we believe that the data-driven flag&consider approach we employ in this study is relevant for any type of EWAS, since we show that removing pre-defined sets of probes may miss spurious associations.In this paper, we report on hitherto undiscovered cross-hybridization issues in Illumina DNA methylation arrays. We discovered these issues in a large EWAS on the presence of the We found several convergent lines of evidence that strongly suggest that the majority of significant CpG-sites were false positives due to cross-hybridization to the C9 repeat expansion. First, among the significant sites we found a strong enrichment for probes with longer sequence matches to the C9 repeat. This enrichment was present in both the 450k and EPIC array data. Second, we show that probes predicted to cause fluorescent signal in the (unintended) OOB color channel upon cross-hybridization to the C9 repeat indeed showed differential signal in these channels. These findings indicate a novel use of the OOB signals, and adds to previous studies that have shown that OOB signals can provide valuable information ,42. ThirSpurious associations due to cross-hybridization have long been recognized and various probe filtering approaches have been proposed to prevent them . The mosIn this study, we made several novel observations that explain why the issues we discovered were not identified in any of the aforementioned studies. Specifically, we show that genetic variation should be taken into account when considering potential cross-hybridization issues, since these are not covered by existing methods that map probes to the reference genome. This is especially relevant when the phenotype being studied is associated with repeat expansions or other types of structural variation, which have been implicated in a substantial number of diseases . To exemAn issue that emerges from this study is that off-target sequence matches as low as 14 bp resulted in spurious associations. Excluding probes based on the aforementioned cutoff would lead to the removal of practically all probes, and is therefore not a sensible strategy. Whether off-target matches lead to detectable shifts in β-values will ultimately depend on several factors. These include the combination of the number of off-target matches and the lengths of these matches. Whereas one or a few long off-target matches may be sufficient for detectable cross-hybridization, a larger number of short matches will generally be required to reach detectable levels of off-target signal. Moreover, correlated off-target signals will shift β-values in the same direction and are thus more likely to cause detectable shifts. In this regard, we expect that many copies of an off-target sequence are required before matches as small as those described here (≥14 bp) will result in spurious associations. In addition, other factors may influence the likelihood of cross-hybridization, such as the experimental washing protocols and GC-content of the probe ,46.a priori which probes should be excluded, and relying on a fixed threshold to exclude cross-reactive probes can miss spurious associations. This conclusion is supported by the fact that most of the cross-reactive probes we discovered were not flagged as such in previous studies. Moreover, we note that these phenomena, although unintended, may represent biological signals of interest. We therefore argue for a ‘flag and consider’ approach rather than removing specified sets of probes beforehand, as previously suggested regarding probes that overlap with genetic variation . Second, we recommend inspecting the results of an EWAS for the presence of various ’red flags’, which can point toward cross-hybridization issues. These include (i) correlations among (near-)significant probes; (ii) similar probe sequences among (near-)significant probes; (iii) absence of regional effects at the target locus; (iv) association between the phenotype of interest and total signal intensities and (v) associations in type I OOB channels.Several analytical checks used in this study can aid in identifying probes that may be affected by cross-hybridization. First, probes identified in an EWAS should be checked for off-target sequence matches. In contrast to previous studies, we recommend considering off-target matches <30 bp and allowing for imperfect matches. In addition, any known genetic variation associated with the phenotype should be taken into account. We made scripts to map significant probes to both reference and non-reference sequences available in an R package . All downstream data underlying tables and figures presented in the manuscript are available via lqaa105_Supplemental_FileClick here for additional data file.


Choroid plexus papilloma (CPP) is a rare brain tumour occurring mostly in infants and children. Most CPPs are intraventricular and present with symptoms and signs of increased intracranial pressure (ICP). This case report describes a middle-aged female who presented with spontaneous cerebrospinal fluid (CSF) rhinorrhea from a tumour located in the cerebellopontine angle (CPA). She underwent craniotomy with subtotal tumour resection and remained progression and rhinorrhea-free for several years. Upon clinical progression, the patient was treated with Cyberknife stereotactic radiosurgery. The patient clinically improved and demonstrated a favourable radiologic response to radiosurgery. Choroid plexus tumour (CPT) is a rare epithelial intracranial extra-axial brain tumour that usually grows inside ventricles and is connected to the choroid plexus or near the natural openings of the ventricles. The annual incidence is 0.3 cases per million , and theA 43-year-old woman presented in January 2007 with cerebrospinal fluid (CSF) rhinorrhea from the left nostril on bending forward, headache, and later on with ataxia. There was no prior history of skull trauma or surgery and no significant past medical history. After the confirmation of a CSF leak with a β-2 transferrin test, a CT scan of the paranasal sinuses on December 2007 showed the enlargement of the right foramina rotundum and ovale A,B.An MRI of the brain on May 2008 A,B reveaIn September 2008, the patient underwent a right posterior fossa craniotomy with facial nerve monitoring. A frozen section of the tumour revealed a CPP. The tumour was extremely vascular, soft, friable, and adherent to some cranial nerves. A subtotal excision of the tumour was performed in an attempt to preserve cranial nerve function. Final pathology revealed a WHO grade 1 CPP. Histologically, the tumour tissue had a papillary structure with a fibrovascular backbone and was covered by cuboidal to low columnar cells. Postoperatively, her headache and CSF rhinorrhea stopped, but she developed new vocal cord palsy and partial right hearing loss and her ataxia continued. MRI at eight weeks postoperatively revealed encephalomalacia and an 11 × 9 mm residual enhancing tumour in the right internal auditory canal extending into the right CPA without mass effect and resolution of the hydrocephalus C,D. She Several years later, in mid-2015, she presented with headaches, worsening ataxia, and new-onset facial palsy. Her physical exam revealed asymmetric pupils (right 5 mm and left 3 mm), reactive to light with a horizontal beat (gaze-evoked), nystagmus, and diplopia on lateral gaze. She had right-side hearing loss and an absent gag reflex. A new MRI revealed the residual tumour’s progression (14 × 14 × 17 mm) with central cystic degeneration/necrosis. The tumour extended further into the right internal auditory canal, causing a mild mass effect on the brainstem. The patient was reluctant to consider further surgical intervention. Three subsequent MRIs performed in 2015 demonstrated the continued slow growth of the residual tumour .® (CK) as a non-invasive method of tumour control while allowing function preservation. The patient underwent CT/MR simulation and received treatment in January 2016. The gross tumour volume (GTV) was 3.5 cc and was treated to 3000 cGy in 5 fractions (GTV covered by the 99.8% isodose line), with no treatment margin. Treatment was delivered in five daily fractions over one week. A planning target volume was strictly used for treatment planning purposes. The brainstem doses were as follows: Dmax was 3045 cGy, D0.03cc was 2942 cGy, and the D1cc was 2157 cGy (The case was discussed at neuro-oncology rounds. The multidisciplinary consensus was to offer the patient a course of stereotactic radiosurgery (SRS) using the frameless CyberKnife2157 cGy . The patient tolerated SRS well, with no acute or late toxicity. Over the next five years, she had gradual clinical improvement in her gait, swallowing and facial palsy. Additionally, she had complete resolution of her headaches and improvement in her voice function. Serial MRIs revealed her tumour regression with the relief of mass effect on the brainstem . There wThis is a rare case of CPA choroid plexus papilloma presenting with CSF rhinorrhea. Initial imaging revealed a non-specific right CPA tumour and features for long-standing intracranial hypertension. The patient underwent craniotomy and the subtotal excision of her tumour. Pathology revealed a WHO grade I CPP. Seven years after initial surgery, there was a radiologic progression of her tumour, and she developed headaches, worsening ataxia, and new-onset facial palsy. Following a multidisciplinary tumour board, she was treated with salvage CK-based SRS (3000 cGy in 5 fractions). Over the next five years, the patient had gradual subjective clinical improvement. Serial MRIs revealed objective radiological response to SRS. This case highlights two interesting clinical findings: CSF rhinorrhea is a rare presentation from CPP, and CK SRS resulted in the durable local control of this rare tumour.CPT usually presents with symptoms of raised intracranial pressure (ICP) due to obstructive hydrocephalus or from CSF overproduction . HosmannCSF leak is caused when there are combined bony and dural defects and a sufficient pressure gradient that allows CSF to drain externally . This caGross total or safe-maximal resection is the gold standard of care in the treatment of this benign tumour. At operation, the tumour was compressing the brainstem and cerebellum and extended into the internal porus acusticus. The tumour was adherent to several cranial nerves, including CN VII through CN X. As a result, the patient developed some cranial nerve injuries with clinical deficits, including hearing loss (CN VIII), swallowing dysfunction , impaired voice function (CNX), impaired gag reflex (CN IX and X) and facial palsy (CN VII). Her voice and swallowing function gradually improved, indicating that these deficits were not permanent.Wolff et al. reported on 566 patients with CPP . FollowiThe utility of radiation with conventional techniques and dose fractionation is not well documented in the literature ,14. SRS CSF rhinorrhea is a rare presentation of CPP in the CPA. CPP is usually managed with a definitive maximal-safe surgical resection. Our patient developed clinical and radiologic progression following the subtotal excision of the tumour. Salvage CK resulted in a significant improvement in the patient’s neurologic deficits and durable local tumour control.


Pasos Seguros programme to prevent pedestrian injuries and deaths at dangerous road intersections, which included street-level design changes, such as visible pedestrian crossings, sidewalk widening, refuge islands, lane reductions, pedestrian signals and adjustment of traffic light timing at these intersections. Few studies in low and middle-income countries (LMICs) have evaluated the effect of such interventions on pedestrian safety.Mexico City implemented the Pasos Seguros programme at reducing total, injury and fatal pedestrian-motor vehicle crashes.Assess the effectiveness of the Two-group quasi-experimental design. Monthly pedestrian crashes were obtained from the road incident database from Mexico City’s Citizen Contact Center. The programme’s effectiveness was evaluated by comparing 12 months preintervention to 12 months postintervention implementation using a negative binomial regression with random intercept with a difference-in-difference estimation. A qualitative comparative analysis was used to find the configuration of intersection characteristics and programme components associated with a decrease in pedestrian crashes.Pasos Seguros programme. This reduction was observed for pedestrian injury crashes  and for fatal crashes  although not statistically significant for the latter. A decrease in pedestrian crashes was found at the most complex intersections where more of the programme components was implemented.Total pedestrian crashes were reduced by 21%  after implementation of Pasos Seguros programme successfully decreased total and injury pedestrian crashes. Similar interventions may improve walking safety in other LMIC cities.The  The frequency of road traffic collisions is disproportionately higher in urban areas from developing countries; pedestrians are highly affected especially at intersections.Pasos Seguros programme, a multicomponent, street-level design intervention, to make intersections safer for pedestrians.Mexico City’s government implemented the The interventions to reduce road accidents in low and middle-income countries have been directed at drivers and vehicle occupants but not at the geometric design improvement of intersections to reduce pedestrian risk.Street-level geometric design improvements and adding traffic lights and pedestrian signal traffic control devices at intersections led to a reduction in total and injury pedestrian crashes.The multicomponent intervention programmes are more effective in the most complex intersections (with four or more legs and two directions of traffic in the main road).The results of this study encourage the maintenance of intervened intersections and expansion of the programme to other intersections in Mexico City and potentially other cities in low and middle-income countries.The evaluation of the effectiveness of road interventions oriented to their geometric redesign is necessary to make the case for public investments in road infrastructure.Globally, road traffic accidents are the eighth leading cause of death for all age groups and the main cause of death in children and young people between 5 and 29 years.Pasos Seguros programme, a citywide programme targeting intersections between arterials and other primary roads, located in 12 road corridors, where 76% of road incidents take place.In Mexico City, most pedestrian crashes occur at intersections that are considered crash hotspots.These interventions can help protect pedestrians by reducing the amount of time they spend in the roadway by reducing crossing distances, providing more dedicated walking space along roadways, making pedestrians more visible to drivers and reducing motor vehicle speeds.Pasos Seguros programme was, therefore, innovative because it tackled the urban infrastructure around intersections. The main aim of this study was to assess whether the Pasos Seguros programme was associated with a decrease in total pedestrian crashes and pedestrian crashes resulting in injuries or deaths. As secondary aim, we looked to identify the configurations of characteristics of the intersections (number of legs and number of directions of traffic in the main road) and components of the Pasos Seguros programme , which were associated with the reduction in pedestrian crashes.In low and middle-income countries, specifically in Latin America, most interventions to reduce road accidents have been directed at drivers and vehicle occupants, but not at the geometric design improvement of roads to reduce pedestrian risk.Pasos Seguros programme. The Pasos Seguros intersections were georeferenced from a list of addresses requested from the Citizen Security Ministry (Secretaría de Seguridad Ciudadana in Spanish) of Mexico City. These intersections were validated using Google Street View (GSV). We included 91 Pasos Seguros intersections that were more than 80 m from another Paso Seguro intersection to avoid pedestrian crashes being included in more than one intersection . We used a Propensity Score Matching with a level=0.001, with one-to-one nearest neighbour matching without replacement to select two control intersection for each intervened intersection , which were on the same road as the rsection .16 The oPedestrian crashes included any road traffic incident involving a pedestrian struck by a motor vehicle. The data on pedestrian crashes were obtained from the road incident database from the Mexico City Command, Control, Computing, Communications and Citizen Contact Centre (C5).Pasos Seguros programme implementation and compare them using a paired t test. Central tendency and dispersion measures were used in continuous variables and frequencies and percentages in categorical variables. To assess the effectiveness of the Pasos Seguros programme, we used negative binomial regression models with a random intercept for intersections and road corridors to consider repeated measures at the intersection level and the nested intersections in the road corridors. The regression models were adjusted by the population density at the AGEB in which the intersection is placed. The models estimate risk ratios and 95% CI. We used a difference-in-difference (DiD) approach to estimate the effectiveness of the programme in which an interaction term between the group variable (intervened vs non-intervened) and the period (before vs after) was included in the models. This interaction term assessed whether the changes in the monthly number of pedestrian crashes from the before to the after period was different between Pasos Seguros and control intersections. We used a marginal postestimation to estimate the absolute effectiveness of the programme. The parallel tendency assumption was fulfilled  to identify the configurations of components of the Pasos Seguros intersections. For this analysis, the outcome variable was the difference in pedestrian crashes comparing the mean of pedestrian crashes before the implementation of the Pasos Seguros programme with the same mean after programme implementation. This difference was calibrated using the standardisation method to convert their values into an interval between 0 and 1.The QCA was done only with the Pasos Seguros programme made these intersections safer for all road users. All collisions included vehicle-to-vehicle collisions resulting in fatal, injuries or without injuries; traffic accidents involving cyclists, motorcyclists and scooters; pedestrian crashes and other traffic accidents that included car overturned and collisions with objects.As a sensitivity analysis, we analysed all collisions to evaluate whether the Stata V.14.0  and R i386 V.3.5.1 were used. An α<0.05 was statistically significant.Pasos Seguros programme implemented. 68.1% of Pasos Seguros intersections had four or more legs and 70.3% had two traffic directions on the main road. In all Pasos Seguros intersections, visible pedestrian crossings were implemented. A total of 73.6% of intersections had sidewalk widening and refuge islands. The least frequent component was traffic light installation (38.5%). Pasos Seguros programme implemented and the characteristics of intersection.Pasos Seguros intersections. As shown in Pasos Seguros intersections had a significantly higher rate of pedestrian crashes in the before period with a mean of 6.88 (SD=28.04) total pedestrian crashes per intersection in comparison with control intersections, in which the mean rate was 1.50 (SD=2.94). Similar results were found for pedestrian injury crashes . Of the total pedestrian crashes, 721 (54.2%) occurred at  crashes . Comparios group .Pasos Seguros programme, which meant an average reduction of 0.16 pedestrian crashes per intersection per month. In terms of pedestrian crashes resulting in injuries, the results were nearly identical . For fatal pedestrian crashes, a 39% decrease was observed, but it was not statistically significant  at intersections in the nificant .Pasos Seguros programme was LDCAIWTP (16.48%). This configuration represented intersections with four or more legs (L), two directions of traffic on the main road (D), and with all the components of the Pasos Seguros programme implemented, such as visible pedestrian crossings (C), sidewalk widening (A), refuge island (I), lane reduction (W), traffic lights (T), and pedestrian signals (P). The second most frequent configuration was LDCAIWtP (14.29%).Pasos Seguros intersections with four or more legs (L) and two directions of traffic (D) and in which visible pedestrian crossings (C), sidewalk widening (A), lane reduction (W), traffic lights (T) and pedestrian signals were implemented (P). This configuration had an acceptable consistency , but a low coverage (0.035), this means that this group of intersections were more complex and got most of the components of the Pasos Seguros programme, but their frequency was low.The configuration L*D*C*A*i*W*T*P was associated with a decrease in pedestrian crashes. In other words, the decrease in pedestrian crashes was associated with Pasos Seguros programme  .The sensitivity analysis showed consistent decrease in the total number of collisions associated with the Pasos Seguros programme, designed to increase the safety of intersections previously identified as dangerous for pedestrians in Mexico City, reduced total pedestrian crashes and pedestrian injury crashes by 21%. We found similar results evaluating the effectiveness of the programme on total collisions in our sensitivity analysis.We found that the The effectiveness found in this study was similar to the 30% reported in a study in New York City, in which 118 intervened intersections as part of the Vision Zero Policy were evaluated.We did not find a reduction in fatal pedestrian crashes. This was probably due to low monthly numbers of fatal pedestrian crashes and the time periods examined. Fatal pedestrian crashes were a rare event that represented only 2.4% (32/1331) of total pedestrian crashes at intersections. This low frequency in fatalities could be because we used a data set containing only events confirmed at the scene, which means an under-reporting of around 53% of deaths do not occur at the accident site.Pasos Seguros intersection’s characteristics and components associated with decreased pedestrian crashes. We found that pedestrian crashes decreased at the most complex intersections (with four or more legs and two directions of traffic in the main road), where most of the Pasos Seguros programme components were implemented . It has been reported that, in comparison with only one component, the combination of refuge islands with lane reduction has been more effective in reducing pedestrian crashes.This study identified the combinations of Pasos Seguros programme decreased all collisions. This result is consistent with a meta-analysis of evaluation studies, which reports a reduction of 15% of injury collisions associated with traffic calming measures.In the sensitivity analysis, we found that the Pasos Seguros and control group relative to the number of pedestrian crashes during the before period, controlling for the RTM.An important threat to validity in before–after studies, when the intervention is assigned to a group with higher values in the outcome, is the regression to mean effect (RTM). Individuals or groups with higher-than-average values will have measures closer to the mean on subsequent measurement.Pasos Seguros intersection and control intersections. This design allowed us to control for factors that globally affected pedestrian crashes in Mexico City. Second, the road incidents used in this study did not include mild pedestrian crashes , so the effectiveness reported here is centred on pedestrian injury crashes and fatal pedestrian crashes, which were the target outcomes of the Pasos Seguros programme.Pasos Seguros programme in Mexico City. A differential report of injuries/deaths between Pasos Seguros and control groups or before/after is unlikely. Fourth, we did not include the pedestrian and vehicular volume at the intersections studied because this information is not available. We acknowledge that these variables could be related to both; the treatment assignment and the change in the outcome over time, so in this study, we included proxies of the pedestrian, and vehicle volume in the propensity score matching to address this confusion.This study has some limitations. First, we only had 1 year as before and after period due to the data availability. However, we used a robust design that included a before and after evaluation, with monthly pedestrian crashes, for each Pasos Seguros programme implemented in Mexico City decreased total pedestrian crashes and pedestrian injury crashes. This decrease was observed especially at the most complex intersections and with almost all the components of the programme implemented. This kind of multicomponent programme could be implemented to protect the pedestrians in other cities in the Mexican and Latin-American context.This is the first study to assess the effectiveness of a multicomponent road safety programme in the Latin-American context. In conclusion, the


Analysis of tumours and tumour-infiltrating lymphocytes (TILs) may provide insight into the progression of HPV− HNSCC.The prognosis of HPV− tumours were classified into “Infiltrating” and “Pushing” subtypes based on their different tumour nest configuration and prognosis) were retrospectively analysed. HNSCC data from the Cancer Genome Atlas (n = 263) were analysed for CD8α, HPV and overall survival (OS). A murine HNSCC model was used to verify the antitumour role of PD-1+CD8+ TILs.The tumour and TIL phenotypic characteristics of 134 HNSCC specimens  of “Infiltrating” HPV− tumours. PD-1+CD8+ TILs recognised autologous HNSCC cells and showed stronger inhibition of tumour growth in a murine HNSCC model resistant to PD-1 blockade.The “Infiltrating” HPV− HNSCC and TILs is characterised by an “Infiltrating” phenotype and less tumour-specific PD-1+CD8+ TILs, which may provide a framework for further translational studies and patient stratification.Coevolution of HPV All primary or recurrent tumours from the tonsils or lymph nodes were excluded, considering that surrounding lymphocytes in these subsites may interfere with the phenotypic analysis. Ultimately, 101 primary tumour samples, 22 matched local recurrent HPV− samples and 11 primary HPV+ samples from the same centre were obtained. Histologic examination was performed by two pathologists. Tumour grade, stage and basic patient clinical information of HPV− primary tumour samples are summarised in Supplementary Table Tumour samples of patients with HNSCC were obtained between 2007 and 2017. For HPV detection of primary HNSCC, p16 immunohistochemistry  using Fiji software. For immunofluorescence-stained image acquisition, slides were scanned using the Vectra platform (PerkinElmer) or TissueFAXS slide scanning system (TissueGnostics). Analysis was performed using InForm software (PerkinElmer), TissueQuest or StrataQuest software (TissueGnostics). Cells were identified based on segmentation of DAPI-stained nuclei. When StrataQuest software was used, whole-tumour section was scanned, and cells in indicated areas were analysed.For IHC analysis, CD85 cells into the right flanks. α-PD-1 (200 μg) and IgG isotype (200 μg) were administered intraperitoneally twice weekly. In total, 3 × 106 PD-1+CD8+ or PD-1−CD8+ TILs from autologous tumours were administered intravenously on day 4 after the inoculation. Tumour size was calculated as length × width. Nasal anaesthesia (isoflurane) was used before the tumours were collected, and the mice were then killed by cervical dislocation. Mice with length greater than 2 cm were euthanized  for ethical consideration.Female C3H/He mice aged 6–8 weeks were purchased from the Charles River Laboratories. All mice were maintained under specific pathogen-free conditions in the animal facilities of the Ninth People’s Hospital. Prior to treatment, mice were randomised. Nasal anaesthesia (isoflurane) was used before the subcutaneous injection. SCC7 cells were implanted by subcutaneous injection of 2 × 10t test for pairwise comparison or ordinary one-way ANOVA for multiple comparisons); if not, the nonparametric test was used . All multiple comparisons were corrected by Bonferroni test. Overall survival (OS) was defined as the time from initial diagnosis until death or the most recent follow-up. Survival distributions were displayed using the Kaplan–Meier method, and their comparisons were conducted using Log-rank test, unless otherwise indicated. The results are presented as the mean ± standard deviation (SD). P < 0.05 was considered statistically significant.For statistical analysis, first, the normal distribution of the data was analysed using Kolmogorov–Smirnov test. If normal distribution was assumed, the parametric test was used  primary HNSCC samples were selected by the following criteria: (1) surgical resection was the primary treatment, and no preoperative chemoradiotherapy was performed, (2) HPV status was known and (3) adequate IM and tumour centre (TC) areas were available for analysis. The density of CD8+ TILs at IM and TC was quantified  , suggesting a more intense immune response in this region  with CD8α expression and HPV status were analysed.25 Three groups were divided: HPV−CD8low (n = 113), HPV−CD8high (n = 112) and HPV+ (n = 38)  samples .Considering that tumour cells in IM are frequently different from those in other parts, and according to reports on the classification of tumour shape in IM,ng” Fig. . In “Pus+ TILs in different HNSCC subtypes. Based on the differences of CD8+ TIL patterns, “Infiltrating” and “Pushing” HNSCC were further divided into four subtypes: inflamed, sloped, excluded and deserted  were similar. In addition, the ratios of IM_epithelia PD-1+CD8+ TILs to IM_stroma PD-1+CD8+ TILs were higher than those of PD-1+CD8− TILs  PD-1+CD8+ TILs were higher than those of PD-1+CD8− TILs , Tregs (FOXP3+), NK cells (CD56+) and macrophages (CD68+) were analysed. There were little NK cells  and HPV+ HNSCC (n = 11) were analysed  of this subset may enhance antitumour immunity  and “Infiltrating” tumours (n = 16) that had plenty PD-1+CD8+ TILs in IM were analysed for CD8, PD-1, TIM3, Granzyme-B (GZMB) and Ki67. The results showed that the percentage of exhausted CD8+ TILs (PD-1+TIM3+) in IM was similar in “Pushing” and “Infiltrating” subsets . There are several possible explanations for this contradiction: (1) small sample size, (2) HPV− HNSCC represents a group of tumours that require further classification and (3) compared with the total CD8+ TILs, the PD-1+CD8+ TILs may more accurately represent the tumour-specific subset and be of greater value to prognosis and immunotherapy.32Even though the detailed mechanisms remain unclear, it is widely accepted that tumour evolution is shaped by two major forces: genetic/epigenetic changes of tumour cells and selective pressure from the tumour microenvironment. Immune predation is a major form of selective pressure.+ T-cell infiltrates.35 However, there is still an urgent need for better biomarkers or combinations that improve the response rate of immune-checkpoint therapy in different cancers, including HPV− HNSCC. In this study, we performed additional analyses in the context of HPV− HNSCC to understand the importance of considering tumoral and immune factors as a whole, to gain insights into their primary resistance to immunotherapy as well as to help clinicians optimise personal therapy for cancer patients.Clinical trials targeting ICR have shown promising results for multiple cancers, and many biomarkers predict the response to PD-1-based immunotherapy, such as PD-L1 expression, tumour mutational burden  and CD813 and we proposed “Pushing” and “Infiltrating” only for the convenience of classification and understanding. Our data showed that “Infiltrating” subtype was associated with worse prognosis. From primary to local recurrent tumours, the coevolutionary trend supports the notion that adaptive immune selection is a main selective pressure for HNSCC cells. The phenotypic coevolution of HPV− HNSCC and CD8+ TILs may serve as a primary blueprint for patient stratification.In this study, we performed a proof-of-concept study in the context of HNSCC to understand the importance of considering tumoral and immune factors as a whole. Pathological features of tumour are important for two reasons: firstly, different genotypes may produce the same phenotype; secondly, phenotypic analysis of whole-tumour section, combined with multipoint sequencing, will lead to more accurate tumour typing. The terms “Pushing” and “Infiltrating” used in this study are similar to “Type A” and “Type B” proposed by Nakanishi et al.,36 From the perspective of tumour, the HNSCCs may have adopted different types of immune-escape strategies. From the perspective of TILs, CD8+ TILs functionally can be further divided into tumour-specific and non-specific groups of cells. This study suggests that the combined analysis of tumours and TILs may help to further increase the response rate of ICI therapy by providing the basis for patient stratification.Despite the presence of programmed death-ligand 1 (PD-L1) in >50% HNSCC patients, only a minority of patients with HNSCC (< 20%) respond to ICI immunotherapy.39 A potential drawback of this study is that the number of tumours is limited, except for HNSCC in the oral or oropharynx, although oral and oropharyngeal squamous cell carcinoma is the most common type of head and neck carcinomas. This is partly because all primary or recurrent tumours from the tonsils or lymph nodes were excluded, given that lymphocytes around these subsites may interfere with phenotypic analysis. The HPV− HNSCCs were divided into “Pushing” and “Infiltrating” subtypes in this study, but their clinical relevance and definite marker (based on blood or biopsy) need further study. However, the relatively low levels of B cells and PD-1+CD8+ cells in high-risk “Infiltrating” HPV− HNSCC suggest that ICI therapy may show a higher response if combined with treatments that reverse the lack of PD-1+CD8+ TILs in these patients. Such treatments are included but not limited to TGF-β blockade, immune modulation, macrophage inhibition or cancer vaccines.40HNSCC covers a lot of different subsites, and the risk factors for HNSCC at different subsites are similar: about 80% of HNSCC attributed to tobacco exposures, other risk factors include alcohol, betel nuts, foods high in nitroso compounds and HPV exposure.+CD8+ TILs in most HNSCC patients, 84.1% of the HPV− HNSCC microenvironments were rich in CD8+ TILs in this study, provides the basis for immunotherapy. First, PD-1+CD8+ TIL ACT is a new option with clinical potential for patients with α-PD-1 resistance. Second, since PD-1+CD8+ TILs are rich in tumour-specific cells, their TCRs can be obtained by single-cell sequencing and bioinformatics analysis, and new personalised TCR weapons may thus be obtained. Moreover, the results suggest that, by combined analysis of HNSCC and PD-1+CD8+ TILs, we may more accurately understand the immunotyping and escape strategy of the tumour. In summary, through the combined analysis of tumour and tumour-specific CD8+ TILs, we could understand the evolutionary status of a tumour more accurately and provide the basis for clinicians to develop personalised treatment plans.The abundance of PD-1Supplementary Materials