To evaluate the efficacy and safety of the PreserFlo MicroShunt glaucoma device in a multicentre cohort study.All consecutive patients who received the microshunt with mitomycin-C (MMC) 0.4 mg/mL from May 2019 to September 2020 in three UK tertiary centres. Primary outcome at 1 year was a complete success, with failure defined as intraocular pressure (IOP) >21 mmHg or <20% reduction, IOP≤5 mmHg with any decreased vision on two consecutive visits, reoperation or loss of light perception vision. Secondary outcomes were IOP, best-corrected visual acuity, medications, complications, interventions and reoperations. We also performed subgroup analyses for severe glaucoma and assessed risk factors for failure.104 eyes had 1-year follow-up. Complete and qualified success at 1 year were achieved in 51.9% (N=54) and 16.4% (N=17), respectively, and failure occurred in 31.7% (N=33). There was a significant reduction in IOP (mmHg) from preoperatively  to 12 months  (p<0.0001). Antiglaucoma medications also decreased from preoperatively  to 12 months  (p<0.0001). Multivariate analyses showed an association between higher mean deviation and failure . Complications were hypotony , choroidal detachments , hyphaema  and bleb leak . Needling and 5-fluorouracil injections were performed in 12.5% (N=13) and 33.7% (N=35), respectively, and 11.5% (N=12) required revision surgery.The PreserFlo MicroShunt with MMC 0.4 mg/mL showed an overall success rate of 68.3% at 1 year, and led to significant IOP and medication reduction with a low rate of adverse effects. The recently published randomised clinical trial using a lower dose of mitomycin-C (MMC) of 0.2 mg/mL for 2 min has shown a significantly lower success rate and higher mean intraocular pressure (IOP) and number of medications at 1 year compared with trabeculectomy in POAG patients. However, the results may not reflect the outcomes associated with the higher concentration of MMC 0.4 mg/mL, which is routinely used in standard of care.This study evaluates the real-world efficacy and safety of the PreserFlo MicroShunt from a multicentre perspective, based on three tertiary referral centres in the UK, and using the MMC concentration of 0.4 mg/mL in different types of glaucoma. Real-world multicentre experience of the PreserFlo MicroShunt with MMC 0.4 mg/mL shows an overall success rate of 68.3% at 1 year, 37% reduction in IOP, 79% reduction in antiglaucoma medications, and a good safety profile.This study contributes real-world experience of the PreserFlo minimally invasive glaucoma surgery device, which allows more generalisability to everyday practice than trial-reported data, is consistent with the previously reported real-world outcomes for trabeculectomy in the UK, and that would help in clinical decision making in glaucoma surgery.Glaucoma is the leading cause of irreversible blindness worldwide, currently affecting 76 million people and its prevalence is estimated to increase to 112 million by 2040.The PreserFlo MicroShunt  offers a minimally invasive glaucoma surgery (MIGS) alternative to trabeculectomy. It is a device measuring 8.5 mm long with a 350 µm outer diameter and 70 µm lumen, and is made from a biocompatible material: poly(styrene-block-isobutylene-block-styrene), alternatively known as ‘SIBS’.There have been a few studies that have investigated the effectiveness of the PreserFlo MicroShunt. These studies have reported reasonable success rates,This is a multicentre cohort study of all consecutive patients who received the PreserFlo Microshunt by 14 fellowship-trained glaucoma consultants in three tertiary referral centres from May 2019 to September 2020: Guy’s and St Thomas’ NHS Foundation Trust, King’s College Hospital NHS Foundation Trust, and Nottingham University Hospitals NHS Foundation Trust. Data were obtained from electronic and written medical records. Identifiable patient data were anonymised and categorised in excel spreadsheets.We included all patients aged over 18 years with a previous diagnosis of glaucoma, having an IOP above target despite maximal medical treatment, and receiving the PreserFlo MicroShunt with intraoperative MMC 0.4 mg/mL. Following the World Glaucoma Association guidelines, we have excluded all the second eye surgeries of patients who had microshunt surgery in both eyes during the study period.After placing a corneal traction suture, a conjunctival and Tenon’s capsule peritomy was made at the superior limbus. Dissection of both layers was performed posteriorly and haemostasis was achieved using diathermy. MMC 0.4 mg/mL was applied between 2 and 5 min onto the bare sclera and inside the conjunctival flap by placing three identical sponges (provided as standard in the microshunt pack) soaked in MMC, followed by copious irrigation with balanced salt solution. The sclera was then marked 3 mm posterior to the limbus and a superficial scleral pocket was made using the blade provided in the microshunt pack. Using a 25 G orange needle, a tunnel was created through the scleral pocket and into the anterior chamber. The microshunt was then inserted inside the tunnel, leaving approximately 2–3 mm of the tube inside the anterior chamber and allowing the wings of the device to rest inside the scleral pocket. Finally, flow through the device was confirmed by the presence of drop formation at the scleral end of the microshunt before Tenon’s and conjunctival closure.Demographic data, including age, gender and ethnicity, and any previous ocular surgeries and laser procedures were collected from the medical records. Detailed clinical data, including type of glaucoma, best-corrected visual acuity (BCVA), IOP, central corneal thickness (CCT), cup-to-disc ratio (C/D) and antiglaucoma medications  were recorded preoperatively and at each postoperative visit . Visual field testing was carried out using the Humphrey Field Analyser Mark II SITA Fast 24–2 . Optical coherence tomography (OCT) imaging of the disc was also performed using the OCT-Spectralis  or the DRI-OCT Triton machine .The grade of the surgeon, any intraoperative complications, and the length of time and concentration of intraoperative MMC application were recorded. Detailed early (<3 months) and late (>3 months) postoperative complications, including hypotony, choroidal detachments, hyphaema, bleb leak, microshunt exposure, as well as postoperative interventions, including bleb injections and needling, were collected. We also recorded early (<3 months) and late (>3 months) postoperative revision surgeries as well as reoperations with another type of glaucoma surgery.Primary outcome at 1 year was defined as a complete success (CS), qualified success (QS), or failure (F).Secondary outcomes at 1 year were IOP, BCVA, antiglaucoma medications, intraoperative and postoperative complications, postoperative interventions, revisions and reoperations. We defined the severity of the glaucoma according to the extent of visual field loss (Hodapp-Parrish-Anderson classification), and performed subgroup analyses for the patients with severe/advanced glaucoma.survminer’ in R V.4.02 (https://cran.r-project.org/). Statistically significant differences were expressed as *p<0.05; ** p<0.01; ***p<0.001.All graphs display mean and standard error of the mean (SEM). Statistical analysis was performed using the Student’s t-test to calculate statistically significant differences and p values. Survival analyses for CS and QS were performed using the Kaplan-Meier log rank test. The time to event analyses were conducted using the statistical package ‘survival’ and the results were visualised using the package ‘We identified 104 consecutive patients that had the PreserFlo MicroShunt with MMC 0.4 mg/mL in all three UK tertiary centres from May 2019 to September 2020. Baseline characteristics are detailed in Most surgeries were performed by consultants , with the remainder being done by post-CCT glaucoma fellows . All patients received intraoperative MMC 0.4 mg/mL between 2 and 5 min. The mean preoperative best-corrected visual acuity BCVA  was 0.30±0.02, with 22 eyes (21.2%) recording 0.4 logMAR or worse and the mean BCVA in this subgroup was 0.50±0.02. At baseline, mean IOP was 23.6±0.5 mmHg and C/D ratio was 0.85±0.01. Most patients were on multiple antiglaucoma medications preoperatively with a mean of 3.0±0.1. Visual field tests were available in 100 eyes (96.2%) and the average mean deviation (MD) was −14.8±0.6. We further classified the eyes as having mild , moderate , or severe/advanced glaucoma . OCT disc assessments were also available in 83 eyes (79.8%) and the mean RNFL thickness was 63.3±1.5 µm.Out of the 104 eyes, 69 eyes (66.3%) had undergone previous ocular surgery . TrabecuA total of 104 eyes had 1-year follow-up data. CS and QS at 1 year for IOP 6–21 mmHg were achieved in 51.9% (N=54) and 16.4% (N=17), respectively, and F occurred in 31.7% (N=33) . Kaplan-The reasons for treatment failure at 1 year are detailed in 10.1136/bjophthalmol-2021-320631.supp1Supplementary dataIn addition, reoperation for glaucoma was the cause of treatment failure at 1 year in 14 eyes (13.5%) . No patiFourteen eyes of the 104 eyes that had 1 year follow-up data also had reoperations for glaucoma, and we censored the IOP, BCVA and medication data at the time of the second glaucoma operation. There was a significant decrease in IOP (mmHg) from preoperatively  to 1 day , 1 week , 1 month , 3 months , 6 months , and 12 months   . BCVA deWe also performed a subgroup analysis of the 62 eyes (59.6%) with severe/advanced glaucoma (MD ≤12 dB). CS was similar in the severe/advanced glaucoma eyes  and the mild-moderate glaucoma eyes . However, QS was significantly higher in the severe/advanced glaucoma eyes  compared with the mild-moderate glaucoma eyes , leading to a lower failure rate in the severe/advanced glaucoma eyes  compared with the mild-moderate glaucoma eyes .We further assessed the hazard ratios (HRs) for various factors potentially associated with failure: ethnicity, MMC treatment time (minutes), baseline BCVA, side of operated eye, number of preoperative anti-glaucoma drugs, lens status, preoperative cup-disc ratio, preoperative MD, type of glaucoma, preoperative IOP (mmHg), gender and age . MultivaIntraoperative, early (<3 months) and late (>3 months) postoperative complications are detailed in Clinical hypotony was defined as IOP ≤5 mmHg with sequelae, for example, shallow anterior chamber, hypotonous maculopathy, or choroidal detachment. Numerical hypotony was defined as IOP ≤5 mmHg without any sequelae. Early postoperative hypotony was encountered in 20 eyes (19.2%), where all but 1 case (1.0%) resolved within 3 months. Eight eyes (7.7%) only experienced numerical hypotony with no adverse sequelae. The eye with persistent late hypotony was due to the ongoing bleb leak described above. In the early postoperative period, hypotony led to maculopathy in 1 eye (1.0%), choroidal detachments in 11 eyes (10.6%), and iridocorneal touch in 3 eyes (2.9%). Tube migration was observed in one eye (1.0%) with hypotony, where the wings had become visible under the conjunctiva from posterior dislodgement, and the tube was reinserted into place at day 1.Of the 104 eyes, 32 eyes (30.8%) lost ≥2 Snellen lines of visual acuity and 23 eyes (22.1%) lost ≥3 Snellen lines. Hyphaema/microhyphaema occurred early postoperatively in six eyes (5.8%) and all resolved within 3 months. Corneal complications were present in two eyes, with corneal oedema and corneal abrasion recorded in one eye (1.0%) each. All cases were managed medically with resolution by 3 months, at which time central visual acuity was not affected. Late tube exposure also occurred in one eye (1.0%) after 3 months and required surgical revision. There were no cases of endophthalmitis reported.The most common interventions performed postoperatively were subconjunctival injections of 5-FU or steroid (dexamethasone/betamethasone) in 35 eyes (33.7%) and 24 eyes (23.1%), respectively . SubconjNeedling was performed in 13 eyes (12.5%), of which 7 eyes (6.7%) required repeated needling. Most needling interventions were performed in clinic but 3 eyes required needling in theatre. Of the 13 eyes needled, 8 eyes (61.5%) failed at 12 months, 4 eyes (30.8%) reached QS and only 1 eye (7.7%) achieved CS. Overall, from the 13 eyes that required a needling intervention, 8 eyes (61.5%) went on to have surgical revision in theatre.Microshunts may require less intensive early post-operative management. It is interesting to note that of the 90 eyes reviewed at day 1 in our study, only two eyes (2.2%) needed an intervention. One eye had a 5-fluorouracil (5FU) injection and the other eye required the reinsertion of the microshunt tube into the scleral tunnel. The remaining 88 eyes (97.8%) did not require any intervention at day 1 after microshunt surgery.Further glaucoma surgery was recorded in two eyes (1.9%). One eye underwent Ahmed glaucoma drainage device insertion, and the previous microshunt was exposed and partially revised at the same time to counter the anticipated postoperative hypertensive phase. Another eye with a scarred bleb also underwent combined Phaco+iStent inject surgery. A total of two eyes (1.9%) underwent subsequent cataract surgery, all in the late postoperative period and combined with 5FU injection into the bleb.The aim of this multicentre study was to investigate the real-world experience of the PreserFlo MIGS device. This allows more generalisability to everyday practice than trial-reported data and is consistent with the previously reported real-world outcomes for trabeculectomy in the UK.Our results are similar to those of the recently published prospective multicentre RCT comparing the effectiveness and safety of stand-alone microshunt (N=395) to trabeculectomy (N=132) in mild-to-severe POAG patients .12 The a10.1136/bjophthalmol-2021-320631.supp2Supplementary dataInterestingly, our study suggests that higher concentrations of MMC (0.4 mg/mL for 2–5 min) used in all patients in our study only slightly increased the success rate of microshunt surgery at 1 year compared with the RCT that used 0.2 mg/mL for 2 min. Another study also reported that MMC concentration might not affect overall success (78.1% for 0.2 mg/mL and 74.4% for 0.4 mg/mL), but that there was a trend towards lower IOP and higher medication reduction in the 0.4 mg/mL MMC subgroup.et al also studied the efficacy of the SIBS microshunt in patients with refractory glaucoma (N=85), using the same IOP ranges as in our study (6–21 mmHg inclusive). The authors described CS in 61.0% and failure in 17.8% of eyes.et al similarly found that patients with advanced glaucoma achieved greater success with microshunt surgery than those with mild-to-moderate glaucoma . This might be due to factors that influence the outcomes of microshunt surgery in different types of glaucoma and disease severity, namely that clinicians might be more reluctant to leave patients with advanced glaucoma without drops after surgery so that less patients would fail from IOP lowering-related criteria. This is demonstrated in our study by the greater number of antiglaucoma medications prescribed on average at 12 months postoperatively in patients with advanced glaucoma (0.79) compared with patients with mild to moderate glaucoma (0.49).Durr aucoma N=5, using Of the 18 eyes that failed due to pressure-related criteria alone, 83% were on a single agent or less at month 12, in fact, 56% were not taking any antiglaucoma medication at all. This leads us to propose that if medications had been reinstated when IOP began to rise, many of these cases could potentially have fallen under the criteria of QS, particularly as the mean postoperative IOP was 19.3 mmHg in this subgroup. As this was a retrospective study with no strict protocol as to when anti-glaucoma medications should be added postoperatively, this decision would have been left up to the discretion of the treating clinician who may have regarded the IOP at 1 year as satisfactory in the absence of any glaucoma progression. This is certainly plausible given that the average number of medications decreased from 3.2 preoperatively to 0.7 postoperatively in this subgroup. This is consistent with clinical decisions taken in the real-world setting and reflects routine standard of care.et al also described a similar trend in IOP.IOP is a good surrogate marker for quantitative evaluation of disease control. We found that during the first year, following an initial decrease, there was a gentle upwards trend in IOP. There is a paucity of studies that enable direct comparison; yet those that do exist seem to corroborate our findings. A smaller observational study (N=23) that investigated the long-term efficacy and safety of the microshunt over a 3-year period described a similar trend in IOP.et al, comprised 43.5% Caucasian and 56.5% non-Caucasian.et al was conducted at one centre by one consultant and the fellows, while our study was conducted in three tertiary centres and included data from 14 consultants and their fellows.Variation in the results of the microshunt studies may in part be due to the differences in demographics. Our population comprised 56.7% white Caucasian and 43.3% non-Caucasian, while the population in the study by Durr et al also described a significant reduction in the use of medications, with 64.5% of their patients becoming medication free by 12 months.The use of antiglaucoma medications at 12 months was significantly decreased in our microshunt patients (0.7±0.1), compared with preoperatively (3.4±0.1). In the recent RCT, the microshunt group had a similar mean number of anti-glaucoma medications (0.6±1.1) at 1 year.In the TVT study, 41% of patients in the trabeculectomy group (N=48) and 29% in the baerveldt tube group (N=36) developed complications at 12 months.Antimetabolites, such as 5-FU and MMC, are commonly used both intraoperatively and postoperatively to supress fibroblast activity and hence the deposition of fibrotic tissue, which can lead to the obstruction of stents and failure of glaucoma filtration surgery. In our study, bleb encapsulation was reported in 1.9% and 4.8% in the early and late postoperative periods, respectively. Single and repeated 5-FU injections were given in 33.7% and 24.0% at 12 months, respectively. Needling was also performed in 12.5% at 12 months and was similar to the needling rates reported in other microshunt studies ,http://www.clinicaltrials.gov; ClinicalTrials.gov identifier: NCT01881425) investigating the cost-effectiveness of the InnFocus implant versus Trabeculectomy is underway in the Netherlands; however, the results will not be available until July 2022. The Preserflo microshunt costs £820 in the UK and the cost could be offset by a lower number of follow-up visits and early interventions (only 2.2% required an intervention at day 1 in our study), as well as a low rate of revision surgeries (only 11.5% required bleb revision in the first year). Cost-effectiveness studies of the Preserflo microshunt are warranted, but the reduced rates of early post-operative visits, interventions, and return to theatre might make it more cost-effective than other types of glaucoma surgeries.As well as efficacy, the initial and long-term costs of the Preserflo microshunt are also important to consider. The SIGHT study (Our study contributes real-world and generalisable data on the surgical outcomes of the PreserFlo MicroShunt that would help in clinical decision making in glaucoma surgery. The limitations are that it is a retrospective multicentre study and there were variations in the duration of MMC treatment intraoperatively. There was also a lack of standardisation for postoperative management, including when to inject 5-FU or steroids, when to perform needling procedures, and the criteria for restarting antiglaucoma medications.In summary, the PreserFlo MicroShunt with MMC 0.4 mg/mL showed a lower success rate at 1 year compared with published trabeculectomy figures, but a slightly higher success rate compared with the RCT using MMC 0.2 mg/mL. However, this is a new device with a learning curve for surgeons, which can affect comparison with the more established trabeculectomy surgery. Nevertheless, the PreserFlo MicroShunt led to significant IOP and medication reduction at 1 year in our three UK tertiary centres, and exhibited a good safety profile with a low rate of adverse effects.


Dear Editor,Multiple Myeloma (MM) is characterized by a great clinical and genetic heterogeneity . The disU test , suggesting that ECM mutations might be potential cancer driver mutations.Mutations in adhesion genes were detected in 58 out of 67 samples from 43 patients of our previously published WES cohort [est Fig.  which remut) (56.5% in the MMRF cohort). Collagens were the most frequently mutated gene family (30.2% of patients in our dataset and 30.4% in the MMRF cohort), followed by laminins (25.6% and 12.9%), ADAMTS (20.9% and 16.1%), integrins (20.9% and 13.3%) and ADAMs (9.3% and 7.5%)  Table .LAMA1, was mutated in 2.95% of patients within the MMRF cohort  compared to ECMWT samples in both our dataset  and in the MMRF cohort . In line with previous findings [p = 0.003) and progression-free survival (PFS)  compared to patients with a consistently low TMB.To determine factors distinguishing ECMers Fig. . We founmut samples in our dataset after Benjamini-Hochberg (BH) correction for multiple hypotheses , the occurrence of extramedullary disease or other classical cytogenetic parameters were not significantly enriched in ECMes Table .Subsequently, the MMRF cohort was used to correlate mutation status with PFS and OS. PFS was significantly shorter in patients with ECM gene mutations, while OS was not significantly affected Fig. . FurtherADAM18 and ADAMTS13 had a significantly shorter OS after correction for multiple hypotheses  using the MMRF RNA-sequencing dataset .ITGA3, ITGA4, ITGA6, ITGA8, ITGAE, ITGAL, ITGAV, ITGB1, ITGB2 and ITGB7 being the most highly expressed integrins (median 3.76–46.75 transcripts per kilobase million (TPM))   ) Fig. . Most laMC1 Fig. . ADAM gePM) Fig. . Within PM) Fig. . GE datan = 800 patients). The median GE of all longitudinal samples was used for patients with more than one sample available. High GE ( > mean and/or median) of 9/26 integrins, 15/44 collagens, 7/12 laminins, 8/20 ADAMs and 7/19 ADAMTS genes significantly correlated with OS after correction for multiple hypotheses. PFS was also significantly shorter in patients with a high GE of 10/26 integrins, 11/44 collagens, 4/12 laminins, 7/20 ADAMs and 6/19 ADAMTS genes. P values are summarized in Table The effect of a high or low GE of each single ECM gene  on PFS and OS was assessed for all patients where both GE and survival data was available (ITGAL) and correlated the corresponding categorical variable with PFS and OS , where an expression higher than the median or mean was associated with a significantly shorter PFS or OS Table . To valiOS Table . Notably13 Figs. .To our knowledge, this is the first study identifying ECM mutations as possible driver mutations. In line with that, high expression of a considerable amount of integrin and ADAM genes was associated with poor survival outcomes, suggesting that these genes may prove to be valuable prognostic markers as well as potential novel treatment targets that should be the focus of further functional research.This is especially relevant for ADAMs, which are important for the development and progression of many cancer types , but whoITGB1 and ITGB7 expression with poor survival outcomes has already been described in other studies [ITGAL), whose expression is correlated with tumor growth, induces apoptosis in MM cells [Supporting the reliability of the results described herein, a correlation of high  studies , 11. ITG studies  and its  studies . InhibitMM cells , 15.In conclusion, this study shows that ECM genes are recurrently mutated and that the expression levels of several ECM molecules might be relevant prognostic markers or therapeutic targets in MM.Supplementary Figures S1 and S2Table S1Table S2Table S3Table S4Table S5


Coronary artery bypass grafting for advanced coronary artery disease is a well-established procedure with excellent long-term results. The issue of saphenous vein graft (SVG) performance and its relation to clinical symptoms and thereby the potential for improvement by using superior grafts are still not fully understood. We aim to estimate the contribution of late SVG failure to the long-term outcome.A study population operated between 1997 and 2020, with an internal thoracic artery with a single distal anastomosis and 1, 2 or 3 distal SVG anastomoses, was isolated from the Swedish Web System for Enhancement and Development of Evidence-Based Care in Heart Disease Evaluated According to Recommended Therapies registry. Data regarding postoperative clinically driven coronary angiography and status of bypass grafts were collected.The study population consisted of 44 951 patients. Clinically driven angiography occurred in 10.1% (9.5–10.8), 7.9% (7.6–8.3) and 7.1% (6.7–7.5), respectively, of patients within 3 years and 23.6% (22.6–24.5), 20.0% (19.5–20.6) and 17.5% (16.9–18.2), respectively, of patients within 10 years after surgery. Excluding the first 3 postoperative years, no failed SVGs were found in >75%, 60% and 45%, respectively, of cases when an angiography was performed in the first 10 years after surgery.The results suggest that the risk of symptomatic graft failure due to vein graft disease during the first 10 years after surgery is in the range of 1–2% for every grafted coronary vessel and provide an estimate for the upper limit of the improvements in results that could be achieved by replacing SVGs with superior grafts. Saphenous vein grafts (SVGs), the most commonly used graft in coronary artery bypass grafting (CABG), are subject to vein graft disease [1], and their reduced long-term patency as compared to left internal thoracic artery (ITA) grafts is well established . Saphenous vein grafts (SVGs), the most commonly used graft in coronary artery bypass grafting (CABG), are subject to vein graft disease , and theWe conducted a nationwide observational study of patients operated with CABG in Sweden between 1997 and 2020. We aim to quantify the mortality, incidence of first postoperative angiography and evaluate the reported patency of the SVG and ITA graft to assess the contribution of late SVG failure to the long-term outcome. We believe that a comprehendible model for investigating the association between vein graft disease and long-term clinical outcome can be achieved by specifically investigating CABG with a single and non-sequential SVG as secondary graft to ITA in a large cohort. To confirm the validity of the results and their applicability in the more common setting of multiple SVG vessel grafting, we also study patients with 2 and 3 distal SVG anastomoses.The study was approved by the institutional review board, Swedish Ethical Review Authority, Gothenburg, Sweden (Dnr 2020-06252). Informed consent was not required and also not possible to obtain due to the anonymity of the participants.No informed consent was required. The Swedish personal identity number allows nationwide registries with the possibility of long-term follow-up. The Swedish Web System for Enhancement and Development of Evidence-Based Care in Heart Disease Evaluated According to Recommended Therapies (SWEDEHEART) registry  providesThe elapsed time from the operation to death and first clinically driven angiography was used as end points. These outcomes represent robust, easily definable, clinically relevant events that are likely to have a high correlation with symptoms related to coronary artery disease and have been used previously to evaluate results after CABG , 11. DatData management and statistical analyses were performed with the use of R version 3.1.3 . Patient characteristics were described by using frequencies and percentages for categorical variables and means and standard deviations for continuous variables. Patients were followed from the date of surgery until the date of death from any cause or the end of follow-up (15 September 2021). The Kaplan–Meier method was used to illustrate cumulative survival, and the cumulative incidence function was used to estimate the cumulative incidence of first postoperative angiography using the R package ‘survival’. Hazard functions were estimated for the risk of angiography using the R package ‘muhaz’. Indications for the first angiography and the presence of failed grafts were reported using frequencies and percentages.In total, 71 919 patients with a permanent residence in Sweden between 40 and 80 years of age, with no congenital malformations or previous cardiac surgery, underwent isolated CABG with a single ITA anastomosis between 1997 and 2020. Out of the patients with complete and incontrovertible data on graft type and number of distal anastomoses, 8518 also received 1 distal SVG anastomosis, 21 842 received 2 distal SVG anastomoses and 14 591 received 3 distal SVG anastomoses. Baseline characteristics are shown in Table n = 81), and mortality during follow-up occurred in 42.4% (n = 3616) of patients. Survival [95% confidence interval (CI)] at 3, 10 and 20 years was 94.7% (94.1–95.1), 77.1% (76.1–78.0) and 39.8% (38.3–41.2), respectively  and mortality during follow-up occurred in 47.3% (n = 10 339) of patients. Survival (95% CI) at 3, 10 and 20 years was 94.2% (93.9–94.5), 74.2% (73.5–74.8) and 34.6% (33.7–35.4), respectively  of patients. Survival (95% CI) at 3, 10 and 20 years was 94.2% (93.8–94.6), 73.0% (72.2–73.8) and 31.4% (30.4–32.5), respectively  and mortality during follow-up occurred in 49.4% (n = 2407) of patients. The cumulative incidence (95% CI) at 3, 10 and 20 years for the first clinically driven postoperative angiography was 10.1% (9.5–10.8), 23.6% (22.6–24.5) and 34.8% (33.6–36.0), respectively  of patients. The cumulative incidence (95% CI) at 3, 10 and 20 years for the first clinically driven postoperative angiography was 7.9% (7.6–8.3), 20.0% (19.5–20.6) and 31.4% (30.7–32.1), respectively  of patients. The cumulative incidence (95% CI) at 3, 10 and 20 years for the first clinically driven postoperative angiography was 7.1% (6.7–7.5), 17.5% (16.9–18.2) and 28.6% (27.8–29.5), respectively  of cases where the number of SVGs was found to be 2 or more, indicating a misclassification at time of surgery, was removed from following analysis. The targets of the ITA and SVGs are presented in Table n = 138) and a failed SVG in 31% (403) of the angiographies with patency data  of cases where the number of SVGs was found to be 3 or more were removed from the following analysis. A failed SVG was reported in 28% (n = 889) and 2 failed SVGs in 20% (627) of the angiographies  of cases where the number of SVGs was found to be 4 or more were removed from the following analysis. A failed SVG was reported in 31% (632), 2 in 16% (331) and 3 in 12% (249) of angiographies (Of 3370 angiographies, 2061 had data available regarding the degree of stenosis of individual grafts. 0.2% (graphies .We present outcome data from a large cohort on CABG-operated patients on mortality and clinically driven angiography, including reported individual graft patency. We believe that we have a comprehensible model for investigating the association between vein graft disease and clinical outcome and that we thereby can estimate the contribution from late SVG failures to long-term outcome.Failure rates of ITA grafts in early protocol angiography have been reported to be 5–8% , 12, 13 Most SVG failures occur without symptoms and do not seem to influence mortality and cardiovascular events after CABG surgery , 16. DurIn our study, a failed SVG was found in 31% of all catheterizations in the group with 1 SVG. Between 4 and 7 years after surgery, the failure rate was at its lowest with 21%, giving an estimate of the upper limit of the incidence of asymptomatic early failures. As expected the probability of finding a failed SVG increased with time and reached 50% for catheterizations performed >16 years postoperatively. The probability of finding a failed graft for different time intervals after surgery in our study D is consThe probability of finding multiple failed grafts was higher than the expected distribution of graft failures under the assumption that every SVG fails independently. If one failed graft is found, there was a relatively high probability to find that the second and third grafts had failed as well. This could imply that patients with multiple grafts develop symptoms more often or that patients with a single failure have a lower risk of developing symptoms when multiple vessels are grafted. It may also indicate that the SVGs do not fail independently, e.g. by the use of sequential grafts, factors related to the surgical technique or patient-related factors such as graft quality or tendency to develop vein graft disease.For a large part of about 30–35% of patients who will experience an angiography postoperatively, the first angiography will occur already within the first 3 years after surgery B, when sFor the patients with 1 SVG, a failed graft is found in 3% of the operated population in conjunction with a postoperative angiography between 3 and 10 years after surgery. If we estimate that between 1/3 and 2/3 of all the failures found are symptomatic, only 1–2% will experience a symptomatic late SVG failure that to some extent could be influenced by using grafts with a superior patency Fig. . With thAs most catheterizations will occur within the first 10 postoperative years B, a largThe results provide important insights into why it has been difficult to demonstrate a superior clinical outcome during the first 10 years after surgery for patients where an SVG was replaced with an arterial graft . Even ifThe study is observational and the design does not allow for any causal inference to be established. The results are limited by the availability, quality and completeness of the registry data. The long inclusion period could significantly impact the generalizability. The target vessel and whether the left or right ITA was used is not registered at base-line. The degree of stenosis in the bypassed vessels at the time of surgery is also unknown. Some of the early postoperative angiographies may have been part of a planned hybrid procedure of multivessel disease or due to an inability to find or to graft a target vessel during surgery. The classification of the degree of stenosis of grafts was completely operator dependent. It is not possible to know the importance of graft failure in patients who died suddenly from an ischaemic event without an angiography.ivad052_Supplementary_DataClick here for additional data file.


Lasting local control of brain metastases following stereotactic radiotherapy is becoming increasingly relevant since systemic treatment constantly improves the prognosis of patients with extracranial metastases.73 patients with 103 brain metastases received hypofractionated stereotactic radiotherapy (FSRT) in 6 fractions of 5 Gy between January 2017 and December 2021 at the University Hospital Regensburg, Germany. The study retrospectively evaluated local progression free survival (LPFS), overall survival (OS) and distant brain progression free survival (DPFS) of patients without prior radiotherapy of the brain. Response rate and brain radiation necrosis were reported. Cox proportional hazard models evaluated prognostic factors of OS and LPFS.The median patient age was 61.0 years . The most common tumor types were malignant melanoma (34.2%) and non-small cell lung adenocarcinoma (26.0%). The median gross tumor volume (GTV) was 0.9 cm³ . The median follow-up time of all patients was 36.3 months . The median OS was 17.4 months . Overall survival rates at 6-, 12-, 18-, 24-, and 30 months were 81.9%, 59.1%, 49.0%, 41.3%, and 37.2%, retrospectively. The mean LPFS was 38.1 months , while the median LPFS has not been reached. LPFS rates at 6-, 12-, 18-, 24- and 30 months were 78.9%, 68.7%, 64.3%, 61.6% and 58.7%, retrospectively. Median DPFS of all patients was 7.7 months . Six, 12-, 18-, 24- and 30 months DPFS rates were 62.1%, 36.3%, 31.1%, 24.8% and 21.7%. Five brain metastases (4.8%) developed brain radiation necrosis. In multivariate analysis, the number of brain metastases negatively affected LPFS. Non-melanoma and non-renal cell cancer was associated with a higher chance of LPFS in comparison to other cancer. A GTV > 1.5 cm³ translated into a higher risk of death compared to a GTV ≤ 1.5 cm³ and Karnofsky performance score was predictive of OS.FSRT in 6 fractions of 5 Gy seems to be an effective treatment with an acceptable local control for patients with brain metastases although melanoma and renal cell cancer seem to have a worse local control in comparison to other cancer.This study is retrospectively registered. Brain metastases are common neoplasms in cancer patients , 2. GiveWe retrospectively analyzed local control and overall survival of patients following FSRT of brain metastases at the Department of Radiation Oncology of the University Hospital Regensburg, Germany. Eligibility criteria for this retrospective analysis included patients with brain metastases of a solid cancer who received their first FSRT in 6 fractions of 5 Gy between January 2017 and December 2021. Patients who previously received radiotherapy of brain metastases  were excluded. All patients were reviewed at a multidisciplinary tumor conference. The choice of treatment of brain metastases  was dependent of size of brain metastases, neurological deficits due to brain edema, patient age, disease risk and/or presence of comorbidities. Clinical data were extracted from the medical charts of the University Hospital Regensburg, Germany. Variables included patient age at the time of FSRT, sex, diagnosis, initial UICC stage according to the TNM Classification of Malignant Tumours (8th edition), start date and end date of FSRT, gross tumor volume (GTV), planning target volume (PTV), location of brain metastases, control of primary cancer, presence of extracranial metastases, number of brain metastases treated with 6 × 5 Gy, Karnofsky performance score (KPS), recursive partitioning analysis (RPA), and diagnosis-specific graded prognostic assessment (ds-GPA). RPA and ds-GPA were calculated as defined by Gaspar et al.  and SperAll patients were immobilized with an individual stereotactic mask system of thermoplastic material  and received a planning computed tomogram (CT). CT slice thickness was 1 mm. Diagnostic contrast-enhanced T1-weighted MRIs were fused with CT scans. As an internal requirement, MRIs used were not allowed be older than 2 weeks. The gross tumor volume (GTV) was the demonstrable volume of the brain metastases as determined by MRI. The planning target volume (PTV) was created with a margin of 2–3 mm isotropic margin around the GTV. All brain metastases were treated with FSRT and a total dose of 30 Gy in 6 daily fractions of 5 Gy. The normalized dose (100% corresponding 30 Gy) was prescribed either to the mean value in the PTV (part I) or to the median dose (part II), while D0.03 cc < 33.0 Gy was accepted . This chPatients were followed-up with gadolinium-enhanced MRIs at 6–8 weeks after FSRT and every 3 months thereafter until the last follow-up appointment or until the date of death. We used modified definitions for response assessment of brain metastases similar to proposals from the Response Assessment in Neuro-Oncology Brain Metastases (RANO-BM) group . CompletThe primary endpoint was LPFS. Secondary endpoints were OS, DPFS and response on 1st follow-up MRI. All times to the endpoints were calculated from the last day of FSRT. LPFS was defined as the time between the last day of FSRT and the first follow-up MRI showing in-field progression of the irradiated brain metastases. DPFS was defined as the time between the last day of FSRT and the appearance of distant brain failure (appearance of new or progressive brain metastases outside the PTV). OS was defined as the time from the last day of FSRT to the date of death by any cause. If a patient was event-free for all of the endpoints, the patient was censored at the last date of MRI or follow-up with confirmation of being event-free. OS and DPFS were calculated for all patients, whereas LPFS referred to the irradiated brain metastases. OS, LPFS and DPFS were evaluated using Kaplan-Meier estimators. Comparisons between groups were performed with Log-rank tests. Multivariable regression analyses were performed for OS and LPFS. Covariates were GTV, extracranial metastases, control of primary cancer, systemic treatment 3 months before/after FSRT, Karnofsky performance score, patient age, RPA, dsGPA, histology, cerebral progression outside of the PTV and number of brain metastases.Characteristics are presented as median and interquartile range (IQR) for continuous variables and as absolute and relative frequencies for categorical variables. OS and LPFS were analyzed by univariable and multivariable Cox proportional hazard regression models. Hazard Ratio (HR) and 95% - confidence interval (95% - CI) were presented as effect estimate. Median follow-up time was estimated by the reverse Kaplan-Meier method. All P - values were two-sided and P - values < 0.05 were considered significant. Statistical analysis was performed using SPSS 26.0 .In total, 91 patients with 131 brain metastases received FSRT between January 2017 and December 2021. Eighteen patients previously treated with radiotherapy of brain metastases were excluded. In summary, 73 patients with 103 brain metastases were included in the analysis. Follow-up data were reported to January 2023. The median follow-up time of all patients was 36.3 months . The median time between the first day and the last day of FSRT were 9.0 days . Patients` characteristics (n = 73) at the time of FSRT are summarized in Table Table Median OS was 17.4 months . Overall survival rates at 6-, 12-, 18-, 24-, and 30 months were 81.9%, 59.1%, 49.0%, 41.3%, and 37.2%, retrospectively , while the median LPFS has not been reached. Local progression free survival rates at 6-, 12-, 18-, 24- and 30 months were 78.9%, 68.7%, 64.3%, 61.6% and 58.7%, retrospectively . Six- and 12 months OS rates were 81.9% and 59.1%, retrospectively. Similar median OS and 6- and 12 months OS rates were reported by Fahrig et al. . WiggenrWe performed the 1st follow-up MRIs after a median time of 48 days, retrospectively. Our data indicate that response of brain metastases in the 1st follow-up MRI seems to be predictive for OS and LPFS, although there are certainly brain metastases with delayed response following FSRT. A potential treatment effect is brain radiation necrosis. Brain radiation necrosis (4.8%) was relatively low in our study. Interestingly, all patients with brain radiation necrosis had ongoing immunotherapy at the time of diagnosis of brain radiation necrosis. It is challenging to distinguish between brain radiation necrosis and true progression/local recurrence. The diagnosis of brain radiation necrosis is mostly based on radiographically findings since biopsies or resections are only performed in cases with disabling neurological symptoms related to cerebral edema. In most cases, contrast enhancement and reactive edema resolve without change of treatment. The uncertainty of distinguishing between recurrence and brain radiation necrosis should be taken into account when considering studies that do not provide information about brain radiation necrosis. A recent study of Lesueur et al.  analyzedDPFS after FSRT seems to be poor necessitating further therapy. In our study, 68.9% of patients had received a second session of stereotactic radiotherapy of new brain metastases avoiding whole brain radiotherapy in most of patients. Due to the focus on a single center there is the possibility of selection bias. Moreover, this study is limited by its retrospective nature and the relatively small number of patients. We acknowledge that the heterogeneity of cancer types may result in biases. Nevertheless, the study provides a useful and important insight into the treatment of brain metastases with FSRT. The primary strength of the study is the consistent delivery of FSRT and the length of follow-up. Additionally, data completeness was 100% through active monitoring of all patients. Although lesion size and proximity to critical structures remain critical to the choice of fractionation, it is still being discussed whether fractionation or not is the best treatment modality for patients with brain metastases. The prospective multicenter FSRT-Trial compares FSRT  and SRS (according to RTOG 9005) in brain metastases (1–4 cm). Randomization is based on metastasis size and histology. The results of this study may help to improve local control of brain metastases following stereotactic radiotherapy finding the more effective treatment scheme.In summary, 30 Gy FSRT in 6 fractions appears to be an effective treatment modality for patients with brain metastases resulting in an acceptable local control although melanoma and renal cell cancer histology seem to be a risk for worse local control in comparison to other histologic types.


Objective To evaluate the outcomes of surgical treatment in patients with chemoradiotherapy (CRT)-resistant and locally advanced cervical cancer (LACC).Methods Patients with LACC who underwent surgery due to resistance to CRT between 2005 and 2015 were reviewed retrospectively. Disease-free survival (DFS) and overall survival (OS) related factors were analyzed.Results A total of 23 patients were included in the study and the median age was 51 years old. A total of 14 patients (60.8%) experienced recurrence; among these recurrences, 8 of them were local, 5 were distant, 1 was both distant and local. A total of 9 patients (39%) died. The Median DFS and OS durations were 15 and 32 months, respectively. A total of 17 patients (74%) had undergone simple hysterectomy, 4 (17%) radical hysterectomy, and 2 (9%) total pelvic exenteration. Postoperative grade 3 and 4 complications were seen in 12 patients (52%). Macroscopic tumor presence in the pathology specimen was associated with distant recurrence and positive surgical margins with local recurrence . The only factor associated with OS was surgical margin positivity (Log-Rank test p = 0.008). The type of surgery, grades 3 and 4 postoperative complications, brachytherapy, and tumor histology were not associated with recurrence.Conclusion In patients with LACC, hysterectomy is an option in the presence of a central residual tumor after CRT. However, the risk of grades 3 and 4 complications of performed surgery is high. The presence of macroscopic tumor in the pathology specimen and positive surgical margins are poor prognostic factors. The goal of the surgeon should be to achieve a negative surgical margin. It does not seem important if the surgery is simple or radical. The incidence of invasive cervical cancer is declining, especially in developed countries, due to successful screening programs and HPV vaccination. Introduction of chemoradiotherapy instead of radiotherapy alone ~ 20 years ago, which was shown to be beneficial to survival, is a major advance in locally advanced cervical cancer (LACC) therapy.The standard treatment approach in LACC (stage 1B2–4A) is external beam radiation therapy (EBRT) followed by intracavitary brachytherapy (ICBT) and concomitant cisplatin-based chemotherapy.In the present study, we reviewed the clinical and survival outcomes of the patients with LACC who had undergone surgery due to resistance to CRT. Clinicopathological factors such as tumor size in the pathology specimen , the presence of tumor in surgical margin, operation type, grade-3 complication, brachytherapy, and histology of the tumor were analyzed.Patients with LACC who were operated for CRT resistance at the Oncology Center of the Istanbul University between 2005 and 2015 were included in the study. Patients had received CRT for LACC. Due to lack of complete response to CRT, the patients had been referred to surgery. A total of 25 patients who had been operated with a diagnosis of residual tumor following CRT were identified. One patient was excluded from the study due to a postoperative pathological diagnosis of endometrial carcinoma and due to loss to follow-up. Demographic, clinicopathological, and follow-up data of 23 patients were recorded.All of the patients had a pathological diagnosis of invasive cervical cancer with cervical biopsy. After diagnosis, the patients were classified as stage 1B2 to 4A according to the International Federation of Gynecology and Obstetrics (FIGO) staging system by gynecological examination, magnetic resonance imaging (MRI), and positron emission tomography (PET). These patients were defined as having LACC. The patients diagnosed as having LACC had been started on a general treatment protocol including pelvic EBRT, 1.8–2 Gy per fraction, total 45–50 Gy with cisplatin 40 mg/m2/week and ‘3D conformal HDR brachytherapy 5 Gy once weekly × 5 weeks to high-risk clinical target volume’ in the Department of Radiation Oncology. These patients had been evaluated with gynecological examination and MRI at the end of EBRT, before brachytherapy. In the absence of expected tumor regression in the cervix according to postCRT MRI, the CRT-resistance of tumors was accepted. The patients with clinical and/or radiological presence of residual tumor in the cervix had been defined as patients with CRT-resistant tumors and therefore had undergone surgery. The patients were offered to have surgery if the brachytherapy could not be applied or completed. The evaluation of these patients had been performed in weekly organized gynecological oncology meetings of the faculty, with the participation of medical oncology, radiation oncology, gynecologic oncology, radiology, nuclear medicine and gynecopathology teams.External beam radiation therapy (EBRT) doses and fractions, doses of external boost radiotherapy given to patients unable to receive brachytherapy, as well as doses and fractions of brachytherapy were recorded separately for the study.Age, height, weight, parity, coitarche, smoking status of the patients, as well as the presence of chronic disease, were recorded. Duration between the last radiotherapy dose and surgery, type of the surgery, and postoperative grades 3 and 4 complications were also recorded.Several data were collected from pathology reports, including presence of tumor in the specimen , histological subtypes, and presence of tumor in surgical margins. Sites of recurrence, duration until recurrence, and recurrence treatment were recorded in patients with relapse. Recurrences were classified as local if they were detected in the pelvis, cervix, or vagina and as distant if they were detected in extrapelvic locations. Disease-free survival (DFS) and overall survival (OS) analyses were performed. The influence of the following criteria on recurrence was analyzed: 1–tumor size in the pathology specimen; 2–surgical margin; 3–operation type; 4–presence of grade 3 complication; 5–whether brachytherapy was administered or not; and 6–tumor histology. According to the Dindo et alp-value < 0.05 was considered statistically significant. Because the present study is a retrospective review, permission of the local ethics committee was not sought. However, all of the patients signed an informed consent form that allowed our center to use their clinical data for scientific trials.IBM SPSS for Windows, Version 21  was used to perform all analyses. When evaluating the study data, besides descriptive statistical methods , the Fisher exact test was used to compare two groups. To assess survival, the Kaplan-Meier survival analysis was performed. A n = 17) of the patients had undergone simple hysterectomy, 17.4% (n = 4) radical hysterectomy, and 8.7% (n = 2) total exenteration. Grade 3 surgical complications had been seen in 52.1% (n = 12) of the patients. These included gastrointestinal system (GIS) injuries requiring colostomy / ileostomy (2 patients), infection requiring relaparotomy (2 patients), urinary tract injury requiring nephrostomy (2 patients), rectovaginal fistula (2 patients) and vesicovaginal fistula (4 patients). A total of 10 out of 12 cases of grade 3 complications occurred after hysterectomy, and 2 of them were after radical surgery. The complications rate was 10/17 for simple hysterectomy, and 2/6 for radical surgery.Fewer complications were seen in patients who were operated within the first 2 months (33.3% versus 64.3%). A total of 9 patients in the first 2 months and 14 patients after 2 months were operated. A total of 3 (33.3%) out of 9 patients operated within the first 2 months and 9 (%64.3) out of 14 patients operated after 2 months had experienced grade 3 complications.n = 4) of the patients and macroscopically in 60.8% (n = 14). The pathological evaluation had revealed no tumor in 21.7% (n = 5). The surgical margins had been positive in 43.4% (n = 10) of the patients and negative in 56.5% (n = 13). The histological diagnosis had been squamous in 69.5% (n = 16) and nonsquamous in 30.5%   .p = 0.029) (p = 0.048) (Relapse had occurred in 14 (60.8%) patients, 9 of whom had deceased . No patient had died due to causes other than the disease. Five patients had been classified as living with disease  and 9 (39.1%) as healthy . At the = 0.029) . When th= 0.048) . The comThe median follow-up period of 23 patients was 20.0 months (6–118 months) and the median OS was 32.0 ± 8.1 months . The 2-year OS ratio was calculated as 63.3%. A total of 14 patients (60.8%) had disease recurrence. The median DFS was 15.0 ± 4.4 months (95% CI: 6.3–23.6). The 2-year DFS rate was 29.1%.p = 0.048) (Recurrence had occurred in 8 (80%) out of 10 patients with positive surgical margins and in 6 (46.2%) out of 13 patients with negative surgical margins. The median DFS was 7.0 ± 1.58 (95% CI: 3.9–10.0) months with positive surgical margins and 24.0 ± 3.8 (95% CI: 16.4–31.5) with negative surgical margins (log rank test = 0.048) .p = 0.008)  out of 10 patients with positive surgical margins and 10 (76.9%) out of 13 patients with negative surgical margins were alive. The median OS was estimated as 20.0 months with positive surgical margins and as 36.0 months with negative surgical margins (log rank test = 0.008) .p = 0.029) (Recurrence had occurred in 12 (85.7%) out of 14 patients with macroscopic tumor and in 2 (22.2%) out of 9 patients with microscopic or no tumor. The median DFS was 11.0 months with macroscopic tumor and 16.0 months with microscopic or no tumor (log rank test = 0.029) .The number of patients was not adequate to calculate the median overall survival duration by Long Rank test analysis with regard to the presence of macroscopic tumor .Cotreatment with radiotherapy and surgery as primary approach is avoided in patients with cervical cancer in order not to increase morbidity. However, studies published in recent years have suggested that surgical approach following radiotherapy was acceptable and had no negative impact on morbidity and survival.In the present study, we analyzed the findings in LACC patients who had undergone surgery due to persistence after CRT. Out of 23 cases assessed in the present study, 14 (60.8%) had relapsed. The median DFS duration was 15.0 ± 4.4 (95% CI: 6.3–23.6) months. All of the recurrences had happened within the first 2 years.p = 0.048 and p = 0.029, respectively). In the presence of gross tumor, distant recurrences could not be prevented even if negative surgical margins were reached. At this point, adjuvant systemic chemotherapy can be considered to be useful at preventing distant recurrences. When we analyzed for local recurrence, local control could be achieved even in the presence of gross tumor if surgical margin negativity could be achieved.Size of tumor in the pathology specimen, histological subtype of tumor, surgical margin, operation type, whether brachytherapy had been administered or not, and occurrence of grade 3 complication were analyzed in terms of relation to recurrence . Among tp = 0.048)  . AccordiOur purpose in surgical treatment should be to achieve negative surgical margins; however, ensuring it intraoperatively is difficult in this patient population. The surgeon may perform simple hysterectomy if surgical margin negativity can be ensured. Boers et alIn the present study, this surgical treatment following CRT was not quite pleasing in terms of postoperative complications. Several studies reported acceptable levels of postoperative complication rate, but in the present study, grade 3 complications were observed in 52% of the patients.Depending on the improvements in radiotherapy techniques, a better tissue-radiation dose relationship may result in less tissue damage, especially in disease-free tissues. This may be an explanation for studies in which surgical approach following radiotherapy was acceptable and had no negative impact on morbidity. The frequency of grade 3 complications was not low in our study; however, these complications had no effect on survival. Even though survival was not affected, performing surgery as soon as possible  when planned in these patients might be important to prevent complications. In the present study, complication rates were similar in patients with simple hysterectomy and in those with a radical surgery. Grade 3 complications had occurred in 10 out of 17 patients with simple hysterectomy and in 2 out of 4 patients with radical surgery. We believe that a statistical difference could not be demonstrated due to the limited number of our patients. A generally accepted view is that complications increase as surgery is extended. In 34 patients who underwent radical hysterectomy after primary radiotherapy (15 patients for persistent and 19 patients for recurrent disease), Maneo et alp = 0.029)  . The preIn the present study, the pathologic examination had reported no tumor in 21.7% of the patients, tumor cells at microscopic scale in 17.3%, and macroscopic tumor in 60.8%. In spite of being operated due to residual tumor, the pathologic examination had revealed no tumor in 21.7% of the patients. In a similar study, tumor could not be demonstrated in 28% of hysterectomy materials even though preoperative biopsy had revealed it.Brachytherapy, which is part of CRT, might not be administered for various reasons. Some studies pointed out that hysterectomy could be performed as a completion surgery in this situation.The main limitation of the present study included the absence of a comparative group . However, this problem may be overcome by comparing two groups with randomized prospective studies, but such a study is currently absent.To conclude, clinical experience in the population of LACC patients with CRT-resistance is not sufficient and there is no recommended standard treatment. We think that the treatment should be personalized. Simple hysterectomy and radical hysterectomy are options. However, grades 3 and 4 complication rate of performed surgery is high. Diagnostic techniques (imaging or biopsy) may be more misleading in the period following radiotherapy; therefore, the selection of patients who will be referred to surgery is not clear. The presence of macroscopic tumor in the pathology specimen and positive surgical margins are poor prognostic factors. The most important determinant of survival is to achieve negative surgical margins rather than radical surgery or simple hysterectomy.


ObjectiveThis study was conducted to determine the prevalence of urinary tract infection in children with severe acute malnutrition (SAM) aged between six months and five years and to identify the causative organisms and their antibiotic sensitivity pattern.Study designThis study was an observational cross-sectional study.Setting and participantsThe study was conducted in the Department of Paediatrics in a tertiary care hospital in India over a period of 18 months. A total of 140 children aged between six months and five years according to the World Health Organisation's criteria of severe acute malnutrition were included upon fulfillment of inclusion and exclusion criteria. Ultrasound of kidney, ureter, and bladder (USG-KUB) was also done to exclude children with any underlying anatomical anomaly.InterventionDetailed clinical examination was performed on each of the participants with emphasis on anthropometry. Relevant blood investigations were sent along with urine routine microscopy and culture sensitivity in all patients.ResultsThe prevalence of urinary tract infection (UTI) in our study was 23.57%. The most common organism isolated was Escherichia coli found in 54.54% of cases, followed by Klebsiella in 24.24%. Other organisms isolated were Enterococcus in 12.12%, Pseudomonas aeruginosa 6.06% and Citrobacter in 3.03%. E. coli showed high sensitivity to imipenem (88.87%), meropenem (83.84%), nitrofurantoin (77.76%) and amikacin (72.23%). Overall these organisms showed good sensitivity to amikacin (60.06%), imipenem (66.6%), meropenem (63.63%) and nitrofurantoin (72.72%). Resistance to common antibiotics like ciprofloxacin, cefotaxime and cefuroxime was seen.ConclusionChildren with malnutrition are at risk of UTI. Urine routine examination and urine culture should be performed in all these children before starting antibiotics. Selection of an antibiotic should be according to the local drug sensitivity data. These antibiotics should have good efficacy against gram-negative organisms. Urinary tract infection (UTI) is an infection in any part of the urinary system, urethra, bladder, ureter or kidneys. Most UTIs are ascending infections and the bacteria arises from the faecal flora that colonize the perineum. Male preponderance is seen in infancy and female preponderance thereafter. The risk of having a UTI before the age of 14 years is found to be 3-10% in girls and 1-3% in boys. UTI in children can present with nonspecific symptoms like unexplained fever and failure to thrive or as a part of sepsis in a newborn . Older cSevere acute malnutrition (SAM) is a grave public health issue. According to the National Family Health Survey (NFHS-4) in India, among children under five years, 7.5% in rural and 7.4% in urban areas are severely wasted . MalnutrThe response to infection is impaired or absent in children with malnutrition. So, the usual clinical features seen in UTI may not be present in these children. Hence a high suspicion is required in them. In malnutrition, urinary tract infection is very common which is 6-37% in literature reviews . Not mucThis was a hospital-based observational cross-sectional study conducted in the Department of Paediatrics in one of the largest central government hospitals of India  over a period of 18 months (from October 2019 to March 2021). The sample size was calculated using 22.4% prevalence from a previous study by Sharma et al.  with a 7Children aged between six months and five years admitted in department of paediatrics of Safdarjung Hospital and meeting the World Health Organisation's (WHO) criteria for SAM were enrolled after taking informed consent from parents or guardian.The WHO criteria for SAM are weight for height/length below -3 standard deviation of the median (using the WHO Growth Charts) or presence of visible severe wasting or presence of bipedal oedema of nutritional origin or mid-upper arm circumference less than 11.5 cm .Children having congenital anomalies of the kidney and urinary tract, vesicoureteric reflux, nephrolithiasis or obstructive uropathy were excluded from the study with a prior ultrasound. We also excluded patients having any underlying renal disease, as these patients are already at a high risk for urinary tract infection. Patients on steroids for more than two weeks or those who had taken any antibiotics within one week prior to presentation were also excluded.All subjects were evaluated on the basis of a preformed proforma including demographic details and required clinical history. Clinical examination was performed on each of the participants with emphasis on anthropometry . Blood investigations were sent which included complete blood count, C-reactive protein, kidney function test, serum electrolytes and blood culture sensitivity. Ultrasound of abdomen with kidney, ureters and bladder was done in all cases. Other specific investigations were done depending upon the history and clinical findings of the patient. Two urine samples were collected from each child before starting antibiotics for routine microscopy and culture sensitivity. In children more than two years of age, a clean-catch midstream specimen was taken after washing the genitalia with soap and water. In children less than two years of age or those in which midstream specimen could not be obtained such as the patients in shock, sample was collected using an appropriate size urinary catheter, after taking all aseptic precautions. These samples were sent to the microbiology laboratory within one hour of collection for routine microscopy, culture and antibiotic susceptibility tests. In case the child was admitted at night, the samples were stored in the refrigerator and sent the next following morning.As soon as the samples were received in the microbiology laboratory, routine microscopy was performed. A drop of well mixed, un-centrifuged urine specimen was placed on a clean glass slide using a plastic dropper and it was covered with 22x22 mm cover slip avoiding bubble formation. After this the wet mount was examined under the microscope for the presence of pus cells and microorganisms.Further, the urine sample was cultured on a sheep blood agar (SBA) and a MacConkey agar (MA) by the semi-quantitative method, wherein a one microlitre (μl) sterile nichrome loop  was used to deposit the said amount of urine on a SBA and MA. The plates were then incubated for 18 - 24 hours at 35 - 37°C in an incubator. After overnight incubation, the number of colonies that had grown on the plates are counted manually and then the number was multiplied by 1000. This gave the total number of viable bacteria present in one ml undiluted urine and expressed as colony-forming unit (CFU)/ml of urine. Based on the growth characteristics on both the SBA and MA, the bacteria/bacterium was/were further identified by various biochemical tests and the antibiotic susceptibility test were performed using the Kirby Bauer’s disk diffusion method on a Mueller Hinton agar plate. The results for the bacterial identification and susceptibility pattern were read after an incubation at 35 - 37°C for 18 - 24 hours.5 CFU/ml of a single species of bacteria in a midstream clean catch sample was considered as significant bacteriuria. For the samples collected by urethral catheterisation, a colony count of more than 5 × 104 CFU/ml of single organism was considered significant [The confirmatory diagnosis of urinary tract infection was made only on the basis of urine culture report. Colony count of more than 10nificant .All details and outcome were recorded in a predesigned proforma. ObjectiveThe objective of this study was to determine the prevalence of urinary tract infection in children with severe acute malnutrition aged between six months and five years; and to identify the causative organisms and their antibiotic sensitivity pattern.Statistical analysis The presentation of the Categorical variables was done in the form of number and percentage (%). On the other hand, the quantitative data were presented as the means ± standard deviation (SD) and as median with 25th and 75th percentiles (interquartile range). The association of the quantitative variables were analysed using independent t test and the association of the qualitative variables were analysed using Chi-Square test. If any cell had an expected value of less than five, then Fisher’s exact test was used. The data entry was done in an Excel spreadsheet  and the final analysis was done using Statistical Package for Social Sciences (SPSS) software, version 21.0 . For statistical significance, p value of less than 0.05 was considered statistically significant. In this observational cross-sectional study 142 children were initially enrolled after proper history and clinical examination; two of them were excluded due to abnormal renal ultrasound findings. One hundred forty children fulfilling the inclusion and exclusion criteria were further investigated.In our study most of the children were in the age group between six to 12 months (35% patients) followed by 13 to 24 months (29.29%), 25 to 36 months (15.00%), 37 to 48 months (12.14%) and 49 to 60 months (8.57%). Mean value of age (months) of study subjects was 24.47 ± 16.5 with median (25th-75th percentile) of 21 (11.04-36). The study population had 76 females (54.29%) and 64 males (45.71%) , height (cm) and mid-upper arm circumference (cm) of study subjects was 7.54 ± 2.11, 81.52 ± 12.54 and 10.27 ± 0.95 respectively. A total of 127 (90.7%) patients out of 140 patients had weight for height less than -3 standard deviation (SD). Mid-upper arm circumference less than 11.5 cm was seen in 122 (87.14%) patients. Edema was present in only four (2.85%) patients and visible severe wasting was seen in 63 (45%) patients. No significant association of UTI was seen with any of the above parameters  of patients did not have any growth on urine culture. Out of 140 patients, urine culture was positive in 22 (23.37%). UTI was more common in females, present in 22 out of 76 females (28.95%) compared to 11 out of 64 males (17.19%) . The most common organism was E. coli, isolated in 18 (54.54%) children, followed by Klebsiella pneumoniae in eight (24.24%), Enterococcus species in four (12.12%), Pseudomonas aeruginosa in two (6.06%) and Citrobacter koseri in one out of 33 children (3.03%) , meropenem 83.84%), nitrofurantoin (77.76%), amikacin (72.23%) and comparatively lower sensitivity was seen for cotrimoxazole (44.45%), amoxycillin-clavulanic acid (44.45%) and ciprofloxacin (22.23%). Other organisms and their sensitivity pattern are shown in Table .84%, nitIn urine microscopy, the majority of children had a pus cell count of less than five (88.57%), followed by five to 10 pus cells in 8.57% of cases. Pus cells were more than 10 in only four out of 140 patients (2.86%). The proportion of patients with UTI was proportionately higher in the group having more numbers of pus cells. Among children having five to 10 pus cells in urine, 66.67% had UTI. This proportion was even higher for the group having more than 10 pus cells as 75% of these children had UTI. This was higher as compared to only 17.74% of children with less than five pus cells had UTI. This association was statistically significant with a p value of less than 0.0001 (Table In the majority 59.29%) of patients, presenting complaint was fever followed by loose stool (27.14%), cough/fast breathing (13.57%), vomiting (10.71%), not gaining weight (7.14%), lethargy/decreased oral acceptance (5.71%), pallor (12.85), seizure (1.43%) and pain abdomen (1.43%). Burning micturition as a presenting complaint was seen in only one out of 140 patients (0.71%). However it was found to have 100% association with UTI in our study. Among presenting complaints only presence of loose stool was found to have a statistically significant association with UTI with a p value of less than 0.0239 . Most of the study population was in the age group of six to 12 months (35%) and most cases of UTI were seen in the age group of 13-24 months . In our study 76 (54.29%) patients were females and 64 (45.71%) patients were males. UTI was found to be more common in females as seen in 22 out of 54 females (28.95%) as compared to 11 out of 53 males (17.19%). However, this association was not statistically significant . This was similar to previous studies ,20,21 whIn our study fever was the most common presenting complaint (59.29%) followed by loose stool (27.14%), cough/fast breathing 13.57%), pallor (12.85%), vomiting (10.71%), not gaining weight (7.14%), lethargy/decreased oral acceptance (5.71%), seizure (1.43%) and pain abdomen (1.43%). The presenting complaint was burning micturition in only one out of 140 patients (0.71%). Fever was present as the most common presenting complaint in other studies by Sharma et al.  (84.7%),.57%, palWe found the proportion of patients having UTI was significantly higher in the group having more than five pus cells, i.e., five to 10 pus cells (66.67%), >10 pus cells (75%) as compared to less than five pus cells (17.74%). This association was found to be statistically significant in our study with a p value <.0001. Similar significant association was found in most other studies ,19,20. OThe prevalence of UTI in our study was 23.57% (33 out of 140 patients were urine culture positive). It was comparable to some other studies ,20.In our study, the most common organism isolated was E. coli, present in 18 samples (54.54%). Followed by Klebsiella in eight (24.24%), Enterococcus in four (12.12%) and Pseudomonas aeruginosa in two (6.06%) and Citrobacter koseri in only one out of 33 patients (3.03%). E. coli showed high sensitivity to imipenem (88.87%), meropenem (83.84%), nitrofurantoin (77.76%), and amikacin (72.23%). Comparatively lower sensitivity was seen for cotrimoxazole (44.45%), amoxycillin-clavulanic acid (44.45%) and ciprofloxacin (22.23%). Klebsiella also showed high sensitivity to imipenem (75%), meropenem (87.5%) and amikacin (75%) and comparatively lower sensitivity for nitrofurantoin (50%), cotrimoxazole (25%), amoxycillin-clavulanic acid (37.5%) and all were resistant to ciprofloxacin. None of the isolates of E. coli or Klebsiella showed 100% sensitivity to any of the antibiotics. Pseudomonas showed 50% sensitivity to imipenem, meropenem and amikacin. All the Enterococci were sensitive to vancomycin, linezolid and nitrofurantoin (100%) and none were sensitive to amikacin. Citrobacter was 100% sensitive to imipenem, meropenem, nitrofurantoin and amikacin and resistant to cotrimoxazole and amoxycillin-clavulanic acid. None of the isolates were sensitive to cefotaxime or cefuroxime. In contrast, previous studies showed good sensitivity to amikacin and ciprofloxacin. In study by Kumar et al.  organismStrength of our study was that it had a good sample size compared to most of the previously done studies. Ultrasound was performed in all patients to exclude patients with structural defects in kidneys or urinary tract. Demographic characteristics of the population as well as association of UTI with clinical presentation was also studied. The main limitation of this study was that it was a hospital-based study that included only admitted patients, so it failed to estimate the prevalence of UTI in asymptomatic SAM patients and in general population. Controls were also not enrolled.Our study showed a high prevalence of UTI in children with severe acute malnutrition. The most common organisms isolated were gram-negative organisms, E. coli being the most common. Most of them showed good sensitivity to antibiotics like amikacin and nitrofurantoin and resistance to cefotaxime, cefuroxime and ciprofloxacin. Presence of pus cells in urine routine microscopy was found to be positively associated with UTI however a positive urine culture is always required to make the diagnosis of UTI. In our study presence of diarrhoea was found to be positively associated with UTI. It can be due to the increased colonisation of periurethral flora in these children. Among presenting complaints fever was most common. Urinary complaints were seen in less than 1% of cases in our study, so a high degree of suspicion is required in these children for UTI.From this study we can conclude that children with malnutrition are at risk of UTI. Urine routine examination and urine culture should be performed in all these children before starting antibiotics. Selection of empirical antibiotic should be according to the local sensitivity data. If such data is not available, antibiotics having good efficacy against gram-negative organisms should be started till the time culture reports are collected.


Different types of HPV have been associated with cancer in humans, but the role of HPV in esophageal cancer (EC) is controversial. The purpose of this study was to evaluate the correlation between HPV infection and EC in the Chinese population and to provide the scientific basis for the future prevention, control, early diagnosis, and treatment strategies of EC in China.PCR detected HPV infection in 1112 esophageal cancer tissue samples, and 89 HPV-positive samples were detected by genotyping. Proximity ligation assays (PLAs) and immunohistochemistry were used to detect the expression of HPV E6 and E7 proteins. Real-time fluorescent quantitative PCR was used to detect the integration of HPV16 E6. The level of HPV-specific antibody IgG in serum was detected by ELISA and PLA.The positive rates of HPV L1, HPV16, HPV18, hpv16 + 18 E6 and hpv16/18 E6 in 1,112 EC tissue samples were 77.6%, 41.4%, 27.2%, 14.2% and 55.4% respectively. Multiple HPV subtypes were detected in HPV-positive EC samples. PLA showed that E6 and E7 were expressed in EC109 and formed complexes with p53 and pRb, respectively. Immunohistochemistry showed that the positive rates of hpv16 + 18 E6 and E7 in HPV-positive EC samples were 56.4% and 37.0%, respectively. HPV-DNA integration rate in HPV-positive EC tissues (88.79%) was higher than that in adjacent tissues (54.17%). HPV antibody was found in the serum of EC patients by a serological test.The study suggests that HPV, especially HPV16 and HPV18, the infection may be a risk factor for EC in the Chinese population and that the E6 protein may play a key role in HPV-associated malignancies. These results may be important for the prevention and treatment of HPV-positive EC in China.The online version contains supplementary material available at 10.1007/s00432-022-04322-5. Cancers of the esophagus account for over 500,000 cancer deaths annually, representing 5.3% of all global cancer deaths. However, the worldwide regional distribution varies substantially  and HPV (31.1%)  tissue specimens were taken from 10 high-risk area pathology departments in China from 2009 to 2012 . 1,112 histologically confirmed ESCC specimens (FFPE tissue blocks) were sectioned and DNA was extracted. The specific process refers to our previous research .To identify HPV types and any sequence variations, PCR products from all samples were sequenced and analyzed using the T7 Sequence Version 2.0 DNA PCR product sequencing Kit . The nucleotide sequence was then determined using BLAST  was used for rapid and accurate HPV genotyping. It can identify 21 HPV including 15 high-risk types  and 6 low-risk types .Cell culture and preparation of centrifuge cell smear. In the experimental group, EC109 cell lines infected with HPV18 were suspended in PBS at medium density, then coated on glass slides, dried at room temperature and fixed with poly-oxy-methylene (4%). The control group was treated with normal HEK 293 cells in the same way.In situ PLA (Proximity ligation assay). In the experiment, each protein molecule was detected by a monoclonal antibody for p53, pRb, HPV18 E6 and E7. HPV18 E6-p53 protein complex was detected by Rabbit anti HPV18 E6 antibody and mouse anti p53 antibody, and HPV18 E7-pRb protein complex was detected by Rabbit anti HPV18 E7 antibody and mouse anti p53 antibody. The PLA test kit was purchased from Olink Bioscience  and the specific operation was carried out by the manufacturer's operating procedures. Then the results were observed by laser confocal microscope.HPV16+18 E6 and HPV16+18 E7 monoclonal antibodies were used to detect cancer tissues. The antibodies used to detect HPV-18 and 16 are mouse monoclonal antibodies. We used the same procedure previously used in the laboratory  in the RotorGene fluorescent PCR system.One hundred and sixteen cancer tissues and 72 adjacent tissues were randomly selected for real-time fluorescent quantitative PCR to detect the integration of HPV DNA in esophageal cancer. The research is based on the following assumptions: the special region of the E2 open reading frame (ORF), which is most often missing during HPV integration, such as HPV exists in the form of free type, and the proportion of E2 and E6 copies is equal. If HPV is integrated into the host chromosome, E2 is deleted, and the copy number ratio of E2 to E6 is zero; HPV exists in a mixed form of free and integrated, and the copy number of E2 is less than E6. Refer to our previous article  were used to detect HPV-related antibodies in serum. HPV18-infected EC109 cells were detected by nasopharyngeal carcinoma detection method and PLA technology. Normal HEK293 cells are used as the negative control. Please refer to the equipment manual for detailed operation.HPV antibody was detected by ELISA. The human papillomavirus IgG antibody detection kit  was used to detect human papillomavirus-related IgG antibody levels in serum. Please refer to the instructions for specific operations.P value of the tests had to be < 0.05 to be statistically significant. The correlation coefficient of the lollipop chart and multi-type pie chart by the free online data analysis and visualization platform https://www.bioinformatics.com.cn.SPSS23.0 statistical software was used for statistical analysis of the results, and the correlation between HPV-related proteins and esophageal cancer was analyzed. The R=0.701, P=0.024 and R=0.848, P=0.26, respectively). HPV16/18 showed the strongest correlation with EC , followed by Linzhou 93.4%) and (Puyang 92.1%), and Jieyang had the lowest rate (40.9%). The positive rate of HPV16 was higher in Cixian, Linzhou, and Huaihe River Basin, with positive rates ranging from 16% to 86%. The positive rates of HPV18 in Huaihe River Basin, Tangshan, and Puyang were 50.0%, 47.47%, and 34.2% respectively. The positive rate of HPV16 E6 and HPV18 E6 was 39.4% in Huaihe River Basin, 28.9% in Puyang, and 27.8% in Tangshan. The positive rate of HPV16/18 was the highest in Cixian (86.0%), followed by Linzhou (68.0%), and Huaihe River Basin (62.1%). The HPV prevalence rates in ten examined high-risk areas of China and the details of patients with EC positive for different HPV types are shown in Figs. .4% and , and the positive detection rate of HPV16 and HPV18 was high. HPV51 was also widely distributed in EC tissues (positive rate: 15.7%). HPV45, 33, 52, 53, 58, 45, 31, 66, 68, and other types were also found  was performed in 275 EC cases positive for HPV DNA. HPV-specific proteins, including monoclonal antibodies against HPV16+18 E6 and HPV16+18 E7, were used to detect cancer tissues. Representative IHC images are shown in Fig. Of 116 HPV16 E6-positive EC specimens, 103 (88.8%) were positive for E2 gene integration, whereas, among 72 adjacent tissue specimens, 39 54.2%) were positive for E2 gene integration   antibodies labeled with single-stranded DNA. Only when a pair of probes are close enough (<40 nm), a pair of probes labeled with antibodies will combine to form a ring, which is reflected by an increase in fluorescence intensity. We observed that E7–pRb and E6–p53 protein complexes were highly prevalent in EC109 cells. This not only directly proves the interaction between the two proteins, but also indicates that the cells expressing HPV E6 and E7 proteins are carcinogenic. Therefore, we believe that HPV-positive cells or tissues play an important role in carcinogenesis. We also found that the expression of E6 was stronger than E7 after HPV18 infected EC109 cells Below is the link to the electronic supplementary material.


Glioblastoma is the most common malignant brain tumor in adults, entailing a very short survival. New therapeutic strategies are desperately needed. Immunotherapeutic approaches seem promising, yet their breakthrough is hindered by interactions of the tumor with its immunological tumor environment. In order to understand these complex interactions, innovative glioblastoma models are needed. We aimed to investigate whether patient-derived tumor models are able to maintain the tumor’s microenvironment signature and composition. Secondly, we added immune cells to our model in order to reflect a more realistic tumor microenvironment, which could be used for preclinical testing of novel immunotherapeutic approaches. Thus, we hope to contribute to the challenging task of advancing glioblastoma therapy.While glioblastoma (GBM) is still challenging to treat, novel immunotherapeutic approaches have shown promising effects in preclinical settings. However, their clinical breakthrough is hampered by complex interactions of GBM with the tumor microenvironment (TME). Here, we present an analysis of TME composition in a patient-derived organoid model (PDO) as well as in organotypic slice cultures (OSC). To obtain a more realistic model for immunotherapeutic testing, we introduce an enhanced PDO model. We manufactured PDOs and OSCs from fresh tissue of GBM patients and analyzed the TME. Enhanced PDOs (ePDOs) were obtained via co-culture with PBMCs  and compared to normal PDOs (nPDOs) and PT (primary tissue). At first, we showed that TME was not sustained in PDOs after a short time of culture. In contrast, TME was largely maintained in OSCs. Unfortunately, OSCs can only be cultured for up to 9 days. Thus, we enhanced the TME in PDOs by co-culturing PDOs and PBMCs from healthy donors. These cellular TME patterns could be preserved until day 21. The ePDO approach could mirror the interaction of GBM, TME and immunotherapeutic agents and may consequently represent a realistic model for individual immunotherapeutic drug testing in the future. Lately,promoter . Howeverpromoter , survivapromoter ,6 or an promoter ,8. New tpromoter ,10, chimpromoter ,12,13 orpromoter .Immunotherapeutic therapies for solid tumors such as GBM are more challenging than for hematological tumors . The solWhile the TME consists of only a small number of T-cells, TAMs represent up to 40% of the TME . MacrophTAMs produce IL4 and interleukin 10 (IL10) , leadingIn a preclinical setting, these complex interactions of GBM, TME and therapeutic agents cannot be mirrored by GBM cell lines alone and antigen surface expression patterns cannot be assumed to be similar in GBM cell lines and intracerebral tumor tissue. Rupture of cell–cell-contacts during lysis, duration of cultivation and hypoxic gradients might be reasons for changes in these patterns . AdditioTherefore, the aim of the present study was to characterize the ex vivo TME in PDOs and OSCs in comparison to primary tissue (PT). In a second step, we introduced cellular TME components in PDOs by co-culture with PBMCs  and thus generated enhanced PDOs (ePDOs). We compared the capability of normal PDOs (nPDOs) and ePDOs to reflect the TME antigen expression patterns as seen in PT. This approach might hold potential as an ex vivo test system mirroring complex TME–tumor–therapy interactions, e.g., for immunotherapeutic approaches.All patients were treated at the Department of Neurosurgery, University Hospital Würzburg, Germany, and gave written informed consent in accordance with the declaration of Helsinki, and as approved by the Institutional Review Board of the University of Würzburg (#22/20-me). The tumors were histologically assessed and graded on formalin-fixed and paraffin embedded tissue sections by an experienced neuropathologist, according to the most recent criteria of the World Health Organization .2 and 95% humidity. After standardized hematoxylin and eosin (HE) staining was performed for histology, the tumor content was assessed by an experienced neuropathologist. OSCs were fixed in 4% formalin  for 24 h at 4 °C and then transferred to phosphate-buffered saline (PBS)  for immunohistochemical staining.OSCs were prepared as previously described in Nickl et al.’s work . After s2 and 95% humidity on an orbital shaker at 120 rpm. After 2 weeks of cultivation, PDOs formed successfully and could be used for further experiments [PDOs were prepared according to Nickl et al.’s description . For thieriments . At the g, room temperature, 12 min) and diluted in 5 mL CTL medium containing 88% RPMI, 10% ml human serum, 1% Penicillin/Streptomycin, 1% Glutamax, 0.1% β-Mercaptoethanol . After a second centrifugation step  cells were brought to a concentration of 2 × 106/mL with CTL medium and incubated overnight with 150 U/mL IL2  at 37 °C, 5% CO2 and 95% humidity. After resuspending and counting the cells the next morning, they were washed in PBS  at 500 g, room temperature, 5 min and brought to a concentration of 1 × 106/mL utilizing the PDO medium adding 150 U/mL IL2. PDOs and PBMCs were incubated at a ratio of 1:20 and a half medium change was performed every other day adding IL2 to a final concentration of 150 U/mLPBMCs were isolated from healthy donors and cultured for ten days. The cells were then centrifuged  or 10  minutes with citrate buffer (pH = 6.0) or for 4 min with citrate buffer (pH = 7.0) (CD86). Alternatively, heat induced retrieval was performed in Tris-EDTA buffer (pH = 6.1) for 3  or 10 (PD1) minutes or Tris-EDTA buffer (pH = 9.0) for 5 min (LAG3). After blocking with 30% hydrogen peroxide , slides were treated with 10% normal goat serum  for CCR2. Subsequently, the primary antibody was applied over night at 4 °C  or 1 h at room temperature  , CD7 , CD8 , CD14 , CD25 , CD45 , CD68 , CD86 , CD163 , CCR2 , FOXP3 , LAG3 , PD1 , TIM3 ). Slides were then incubated with the secondary antibody for 20 min and labeled . Diaminobenzadine  was applied for 5 min (CCR2), 7 min  or 10 min  and after rinsing with water, cell nuclei were counterstained using hemalum solution acid and mounted. Tonsil tissue was used as a positive control.For the PT as well as the OSCs, five representative areas of view per slide were photographed using microscope (Olympus BX50), camera (Olympus DP27) and software  with standardized settings at 40× magnification. The whole PDO was photographed and analyzed. We detected positive staining intensity via the batch processing function of the open source program Fiji  by applyAnalyses were performed using IBM SPSS Statistics 27 . For patients’ characteristics we performed descriptive statistics, displaying either absolute and relative numbers or mean with range (minimum/maximum) wherever suitable. Data was examined for Gaussian distribution by Kolmogorov–Smirnov testing before testing for significance was conducted. We performed ANOVA for equally distributed data and the Friedmann’s test for non-equally distributed data in order to determine significant differences in the antigen-expression patterns during the course of time in PDOs and OSCs. Differences in antigen expression patterns in PT, nPDOs and ePDOs were calculated using t-test. Effect size was calculated by Pearson’s correlation coefficient r. Data was regarded as significant if α < 0.05. Whenever percentages are given, they refer to the total cell count.To establish the described ex vivo GBM models, tumor samples of 13 GBM IDH wildtype, CNS WHO grade 4 patients were utilized . PatientWe investigated the antigen expression patterns of cellular TME components in PT and found low abundance of CD7+ cells (0.7%). T lymphocytes were also found in low levels, especially cytotoxic CD8+ T-cells (0.6%). CD4+ T helper cells could be detected in higher numbers (2.2%). Regulatory T-cells were not present . In general, the most abundant cell type in PT were myeloid cells. CD14+ monocytes represented 20.6% and among CD68+ macrophages (30.5%), M1-macrophages (iNOS+), whose presence was rather low (1.2%), M2-macrophages (CD163+), which displayed the largest TAM fraction (16.7%) and CCR2+ peripheral macrophages (3.8%) were found. TIM3 (16.1%) showed the highest expression of all inhibitory receptors, followed by LAG3 (2.5%) and PD1 (2.0%). Data are shown in p = 0.04). While CD8+ represented 0.6% in PT, none were detectable in PDOs at day 14. CD14+ monocytes decreased significantly from PT (20.6%) after 14 days of culture (7.5%) (p = 0.03). In contrast, CD68+ macrophages increased from 30.5% at PT to 34.7% in PDOs at day 7, but dropped significantly from day 7 to 22.8% at day 14 (p = 0.001). iNOS+ M1 macrophages dropped from 1.2% in PT to 0.7% at day 14 in PDOs, as well as M2 macrophages from 16.7% in PT to 8.4% in PDOs at day 14. CCR2+ peripheral macrophages declined from PT (3.8%) to day 14 (0.7%). CD25+ regulatory T-cells were sparsely present in PT, but absent in PDOs at day 14. Concerning the checkpoint protein expression pattern, PD1 and LAG3 expression declined significantly until day 14 in PDOs compared to PT (both p = 0.03). TIM3 and FOXP3 expression dropped, but not significantly. Data are shown in There were significant differences in the CD4+ count when comparing PT (2.2%) to PDOs at day 14 (0.6%)  to day 9. Surprisingly, OSCs were able to retain TME expression patterns to a higher extent. We did not find any significant alterations up to day 9. CD45+ cells declined , as well as CD7+ cells and CD4+ cells. The monocytic marker CD14 decreased , while macrophage markers CD68, CD163 and CCR2 were still expressed at comparable levels . Interestingly, the M1-macrophage marker iNOS completely vanished (0.7% at day 0.0% at day 9). At the end of the culture, regulatory T cells were absent as both CD25 and FOXP3 were not expressed at day 8 or day 9. PD1, TIM3 and LAG3 expressions were initially at a very low level, but dropped further during culture. Data are shown in Since OSCs could not be cultured for longer than nine days, we aimed to enhance the TME in PDOs by co-culturing them with PBMCs. Compared to PDOs, more tumor material was required to generate OSCs. In addition, PDOs stayed stable in culture over several months. However, the organoids did not reflect TME expression patterns as cellular components deteriorated rapidly during culture. Thus, we aimed at enhancing PDOs to better reflect cellular TME patterns and depict a model for immunotherapeutic testing.In contrast to nPDOs, ePDOs were not affected by significant alterations in TME expression patterns until day 21 when comparing to PT . Interestingly, iNOS+, CD14+ and CD163+ cells were practically not detectable in ePDOs after 21 days of culture, despite being present in the corresponding PT. Data are shown in GBM is one of the most challenging solid tumors to treat, especially in relapse. Here, we presented analysis of TME expression markers in two 3D ex vivo patient-derived models (PDOs and OSCs) as well as a time and cost-effective way to incorporate TME cells in PDOs.We showed that the TME was not sustained in PDOs and deteriorated after a short time of culture. In contrast, the TME was maintained in OSCs, albeit on low cellular count level. Unfortunately, OSCs could not be cultured longer than 9 days, rendering this model impractical for immunotherapeutic testing over a longer period of time. We found an efficient solution by co-culturing PDOs with PBMCs from healthy donors. Cellular TME patterns could be persevered until day 21, which enables longer experimental setups.per se using IL13 and IL4, but used PBMCs after ten days of culture with supplementation of IL2, lymphocytes were preselected over macrophages.Monocytes and macrophages were not as strongly represented in the ePDO model. As we did not stimulate macrophages According to recent studies ,40,41, mThe interaction of GBM and TME is mutual. We did not evaluate how tumor metabolism is influenced by incorporating PBMCs in the ePDOs nor did we analyze whether the phenotype of immune cells is changed after incubation with PDOs. Thus, we cannot make a statement about possible alterations due to co-culturing with GBM cells and the impact of the tumor on the differentiation of immune cells and vice versa. This would be an interesting aspect to study in order to characterize GBM-TME interactions closer and to investigate possible therapeutic approaches that could enhance the efficacy of immunotherapy in GBM.As a next step, the ePDO model could be tested with various immunotherapeutic approaches in order to mimic the reality of the GBM-TME interactions even better. Manufacturing is technically feasible and cost-effective. In the future, ePDOs could be applied to draw a sophisticated conclusion whether therapies should be further explored and evaluated for a clinical setting.We evaluated cellular TME antigen expression patterns using two different ex vivo tumor models and found that PDOs were not able to sustain the cellular TME, while OSCs were able to maintain TME cell types at a low expression level. However, we did not manage to culture OSCs longer than nine days, which renders this model sufficient but impractical for testing novel immunological approaches. Thus, we aimed for an optimized 3D ex vivo model that could serve for testing new therapeutic strategies in preclinical settings. By co-culturing PDOs with PBMCs, we showed that cellular TME expression patterns could be preserved for 21 days. This optimized PDO model can be efficiently generated, is easy to maintain and can serve as an excellent ex vivo approach to mirror the complex interactions of GBM and TME in the future.