The choline oxidation pathway and metabolites involved have been linked to diseases including cardiovascular disease, type 2 diabetes and cancer. A healthy Nordic diet is a recently defined dietary pattern associated with decreased risk for these diseases. Our aim was to explore associations between adherence to a healthy Nordic diet and plasma concentrations of metabolites of the choline oxidation pathway.n = 969) from the Västerbotten Intervention Programme in Northern Sweden to score adherence to a healthy Nordic diet. Data included responses to a dietary questionnaire and blood sample analyses (1991–2008). Associations of diet scores with plasma concentrations of metabolites of the choline oxidation pathway and total homocysteine (tHcy), seven metabolites in total, were evaluated with linear regression, adjusting for age, BMI, education and physical activity.The Healthy Nordic Food Index (HNFI) and Baltic Sea Diet Score (BSDS) were applied to cross-sectional data (P < 0.05. The regression models predicted changes in plasma metabolite concentrations (± 1 SD changes in diet score) in the range of 1–5% for choline, betaine, serine and tHcy. No other statistically significant associations were observed.HNFI scores showed linear relationships with plasma choline (β = 0.11), betaine (β = 0.46), serine (β = 0.98) and tHcy (β =  − 0.38), and BSDS scores with betaine (β = 0.13) and tHcy (β =  − 0.13); unstandardized beta coefficients, all significant at A healthy Nordic diet was associated with plasma concentrations of several metabolites of the choline oxidation pathway. Although relationships were statistically significant, effect sizes were moderate. Further research is warranted to explore the underlying mechanisms and associations with health outcomes.The online version contains supplementary material available at 10.1186/s12937-023-00853-w. The choline oxidation pathway and its related metabolites have been linked to the development of cardiovascular disease (CVD), type 2 diabetes, cancer and all-cause mortality –4. This Food and nutrient intake can directly affect circulating concentrations of metabolites involved in the choline oxidation pathway. Foods rich in choline such as eggs have shown a positive association with concentrations of choline , 9. SimiA healthy Nordic diet is a recently defined dietary pattern based on traditional foods originating from the Nordic region with documented health benefits. Typical foods include apples, pears, berries, root vegetables, cabbages, legumes, fatty fish and whole grains . AdditioFew studies have investigated the associations between a healthy Nordic diet and circulating metabolite concentrations with choline oxidation as a main focus –20. The The Västerbotten Intervention Programme (VIP) is a primary care-based initiative launched in 1985 due to a higher-than-expected mortality rate from CVD in the Västerbotten region of Northern Sweden. To this day, residents are invited to receive a voluntary health examination when they turn 40, 50 and 60 years old. From 1990–2018, there have been over 184,000 visits in the program, averaging around 6,000 health examinations per year . Health The dataset for the present study was an extract from the main VIP database initially compiled for a planned nested case–control study exploring associations between folate intake, folate status and the incidence of colorectal cancer. Details of this dataset are described in a separate study on one-carbon metabolite ratios and colorectal cancer risk . Exclusin = 7), weight less than 35 kg (n = 7), BMI less than 15 kg/m2 (n = 12) or food intake level within the 1st or 99th percentiles (n = 101). Non-reporters with respect to the FFQ were also excluded (n = 124), defined as either leaving more than 10% of the questions unanswered or a missing answer for any of the three portion size questions. These exclusion criteria are in line with method recommendations from the Unit for Biobank Research, Umeå University, Sweden [The starting dataset for the present study consisted of 383 cases and 737 controls for a total of 1,120 observations with health examinations ranging from 1991 to 2008. Participants with any of the following attributes were subsequently excluded: height less than 130 cm or greater than 210 cm , 969 observations were included in the final data analyses.Three duplicate participant IDs were identified due to a second blood sample analysis, and observations connected to the second blood sampling were excluded. After exclusions for extremes, non-reporters and duplicates , medium  or high (= 5). Similarly, school education was categorized as base level, i.e., up to high school (responses 1–6), high school (responses 7 and 8) and university (response 9). With regards to smoking status, participants were categorized as 1 = present smoker, 2 = ex-smoker or 3 = never. In this study, smoking status was re-coded to present smoking (= 1) and non-smokers .http://www.bevital.no), Bergen, Norway in 2011. Plasma concentrations of choline, betaine and DMG were measured using liquid chromatography-tandem mass spectrometry [Blood samples were collected in the morning with 91% of subjects having fasted more than eight hours and 98% more than four hours Samples were collected in EDTA (ethylenediamine tetra-acetic acid) tubes, separated into plasma, erythrocyte fractions and buffy coat, and frozen within one hour of collection . All samtrometry . Plasma trometry , 28. Biotrometry . All plahttps://www.umu.se/en/biobank-research-unit/.The FFQ used to collect dietary intake data existed in two main versions during the observation period (1991–2008): the first questionnaire with 84 food items and a second, shorter version with 64 items where previous questions had been combined. In later versions, two additional questions were added for consumption of water and eggs. Three additional questions were related to portion sizes. Questions addressing specific food items were answered using a 9-level frequency scale: “never”, “a few times per year”, up to a maximum frequency response of “4 times per day or more”. Food items included various types of fat , dairy products, fruits and vegetables, soft drinks, sugar-containing snacks, bread, cereals, rice, pasta, potatoes, meat, fish, salty snacks, coffee, tea, juice and alcoholic beverages. Additional information regarding the FFQ can be found at The 84-item FFQ has been validated against repeated 24-h dietary recalls (24-HDR) and biomarkers for total energy intake as well as the intake of several nutrients and food items , 30, 31.https://www7.slv.se/SokNaringsinnehall/). Portion sizes were based on FFQ answers or standard portion sizes for sex and age according to a previous national survey [Daily energy and nutrient intakes were calculated for the major macronutrients, types of fat, vitamins and minerals. These were calculated based on the FFQ responses. All reported frequencies were converted to intakes per day, and then multiplied by portion sizes and energy or nutrient content from a food composition database maintained by the Swedish National Food Administration  Database for the Choline Content of Common Foods, release 2  was usedin cream . Total cBoth the HNFI and BSDS were used to measure participants’ adherence to a healthy Nordic diet. The purpose of using two different indexes was to assess if the study results could be affected by how one defined a healthy Nordic diet. However, this was not a primary aim of the study.The HNFI was adapted from Olsen et al.  and inclst quartile = 0, 2nd quartile = 1, 3rd quartile = 2 and 4th quartile = 3. Two exceptions were meat products and total fat consumption, which used the same scoring system but on an inverse order scale, i.e., 1st quartile = 3, 2nd quartile = 2, and so on. The ninth sub-category, alcohol consumption, was scored based on sex-specific cut-offs for moderate consumption as defined by the Nordic Nutrition Recommendations 2012 [The BSDS was based on Kanerva et al.  and consons 2012 , 10 and The relevant FFQ questions were mapped to twelve of the food sub-categories defined by the HNFI and BSDS. The remaining three sub-categories from the BSDS  were based on calculated nutrient values from FFQ responses. Separate mappings were required for each of the FFQ versions (long and short versions) to account for inconsistencies between the two versions. The detailed mappings can be found in the Online Resource 1. Participants were then grouped by FFQ version and sex, and each group scored separately according to median, quartiles or reference cut-offs as described above. All intakes, initially in grams per day (g/d), were energy-adjusted by the density method to g/d per 1,000 kcal before calculation of sex-specific medians and quartiles . Three eP < 0.05 was considered statistically significant.All computations were run in IBM SPSS Statistics version 27. All tests with regards to the correlation coefficients and regression models were two-tailed and Distributions of the two diet index scores and plasma metabolite concentrations were inspected for normality with the help of descriptive statistics and histogram plots. Linear correlation for each pair of variables (independent vs. dependent) was inspected visually via scatterplot diagrams. Two observations were classified as outliers and excluded from the analyses: one participant with plasma choline greater than 20 µmol/L and a second with plasma betaine greater than 200 µmol/L.Pearson correlation coefficients were used to evaluate linear correlations between dietary intake of choline and betaine  and the diet index scores; and between the diet index scores and plasma concentrations of the metabolites of interest. Scatterplot diagrams did not suggest non-linear relationships between the variables. Multivariable linear regression models were fitted separately for each diet index score variable (independent variables) against each of the metabolites of interest (dependent variables), 14 regression models in total. Each regression model was adjusted for six potential confounding variables, separately: age, sex, BMI, smoking status, physical activity and education level. Confounding variables having more than a 10% effect on the metabolite’s beta coefficient in any of the models were included in the final adjusted models for all metabolites. The final confounding variables included were age, BMI, physical activity level and education level.Baseline characteristics and nutrient intakes of the study population segmented by index score are shown in Tables Both indexes also showed similar trends in baseline nutrient intakes across score segments Tables  and 4. TFiber intake trended higher across index score segments as well as intakes of total choline and betaine. Similarly, folate intake showed a marked increase with daily intake at 198 ± 72 and 278 ± 97 µg/d for HNFI segments 1 and 3, respectively; and 181 ± 70 and 280 ± 102 µg/d for segments 1 and 5, respectively. Finally, intakes of other B-vitamins showed slightly upward trends across the HNFI and BSDS score segments. Food intakes across HNFI and BSDS index scores are shown in Supplementary Tables r) between dietary intake of choline and betaine, and the two healthy Nordic diet scores are presented in Fig. P < 0.01. The highest correlation with the HNFI was observed with betaine and free choline, r = 0.30 and 0.32, respectively. All nutrients except PtdCho showed significant correlations (P < 0.01) with BSDS scores. Again, betaine and free choline had the highest correlations with the BSDS, r = 0.33 and 0.39, respectively. However, SM demonstrated a negative linear relationship with r =  − 0.19. Correlation coefficients between dietary choline and plasma concentrations of metabolites are shown in Supplementary Fig. Pearson correlation coefficients . Correlation coefficients between the separate food components of the HNFI and the BSDS, and plasma concentrations of metabolites are shown in Supplementary Figs. Pearson correlation coefficients for diet index scores and plasma concentrations of metabolites are shown in Fig. P < 0.001) and 0.50 (P < 0.001), respectively. tHcy was inversely related to HNFI score with β =  − 0.33 (P < 0.001). The positive associations were attenuated after adjusting for confounding variables but remained significant. In contrast, this adjustment strengthened the negative association of tHcy with HNFI score from − 0.33 to − 0.38 (P < 0.001). After adjustment for confounders, serine showed a positive association with HNFI score, β = 0.98 (P = 0.019). Sensitivity analysis stratifying cases and controls did not materially change the results (data not shown).Results from multivariable linear regression analyses are listed in Tables P < 0.05 level in the adjusted models. Sensitivity analysis stratifying cases and controls did not materially change the results (data not shown).Similar results were obtained from regression analysis with BSDS score as the independent variable. Again, choline, betaine and tHcy showed significant associations vs. the index score before adjustment. Values of beta coefficients were lower compared to regressions with HNFI, but it should be noted that the range of the BSDS scale is roughly four times larger than the HNFI. Beta coefficient values were attenuated after adjustment for confounding except in the case of tHcy. Only betaine and tHcy remained statistically significant at the Predicted changes in plasma metabolite concentrations relative to sample population means were assessed by applying a one standard deviation increase in diet score  to the above regression equations. For the HNFI, regression models predicted increases in metabolite concentrations for choline , betaine  and serine , and a decrease for tHcy . For the BSDS, regression models predicted an increase for betaine  and a decrease for tHcy .In this cross-sectional study of a population in Northern Sweden, we identified four metabolites of the choline oxidation pathway that were associated with two healthy Nordic dietary indexes using a multivariable linear regression analysis. Choline, betaine and serine showed positive linear associations with the indexes while tHcy showed negative associations. In addition, we found statistically significant associations between choline and betaine intake and the dietary indexes.r =  − 0.11 to 0.15), which is in line with previously reported results of healthy eating indexes and concentrations of other metabolites [Correlations between the dietary indexes and the metabolites were in the lower range . Positive correlations may be attributed to food items rich in these nutrients that are included in the HNFI and BSDS food sub-categories. Salmon, broccoli, beans and dairy products are major sources of choline, and these foods are well represented in the dietary indexes. The same is true for whole grains, which are considered major dietary sources of betaine [Overall, dietary intakes of choline and betaine showed significant linear correlations with both Nordic dietary indexes, which supports the positive associations between the indexes and observed plasma metabolite concentrations. The magnitude of these correlations ranged from low to moderate , consumption data segmented by index scores. Supplementary Table 2. Food categories within the Baltic Sea Diet Score (BSDS), consumption data segmented by index scores. Supplementary Figure 1. Pearson correlation coefficients. Dietary choline and betaine intake  vs. plasma metabolite concentrations. Supplementary Figure 2. Pearson correlation coefficients. HNFI food components  vs. plasma metabolite concentrations. Supplementary Figure 3. Pearson correlation coefficients. BSDS food components  vs. plasma metabolite concentrations.


Chemotherapy‐induced cognitive impairment (“chemobrain”) is a frequent side‐effect in cancer survivors treated with paclitaxel (PTX). The mechanisms responsible for PTX‐induced cognitive impairment remain obscure, and there are no effective treatments or prevention strategies. Here, we test the hypothesis that PTX induces endothelial senescence, which impairs microvascular function and contributes to the genesis of cognitive decline. We treated transgenic p16‐3MR mice, which allows the detection and selective elimination of senescent cells, with PTX . PTX‐treated and control mice were tested for spatial memory performance, neurovascular coupling (NVC) responses , microvascular density, blood–brain barrier (BBB) permeability and the presence of senescent endothelial cells (by flow cytometry and single‐cell transcriptomics) at 6 months post‐treatment. PTX induced senescence in endothelial cells, which associated with microvascular rarefaction, NVC dysfunction, BBB disruption, neuroinflammation, and impaired performance on cognitive tasks. To establish a causal relationship between PTX‐induced senescence and impaired microvascular functions, senescent cells were depleted from PTX‐treated animals (at 3 months post‐treatment) by genetic (ganciclovir) or pharmacological (treatment with the senolytic drug ABT263/Navitoclax) means. In PTX treated mice, both treatments effectively eliminated senescent endothelial cells, rescued endothelium‐mediated NVC responses and BBB integrity, increased capillarization and improved cognitive performance. Our findings suggest that senolytic treatments can be a promising strategy for preventing chemotherapy‐induced cognitive impairment. Graphical representation depicting the proposed mechanism of chemotherapy‐induced cognitive impairment. PTX treatment induces endothelial senescence, which impairs microvascular function and contributes to cognitive decline. PTX‐treated mice exhibit microvascular rarefaction, neuroinflammation, and impaired cognitive performance. Senescent cells are eliminated from PTX‐treated animals using genetic or pharmacological means, which results in the restoration of endothelium‐mediated neurovascular coupling responses and blood‐brain barrier (BBB) integrity, increased capillarization, and improved cognitive performance. ABT263navitoclaxBBBblood‐brain barrierCICIchemotherapy‐induced cognitive impairmentCMVECscerebromicrovascular endothelial cellsGCVganciclovirLNAMEN(gamma)‐nitro‐L‐arginine methyl esterNOnitric oxideNVCneurovascular couplingOCToptical coherence tomographyPTXpaclitaxelVSMCsvascular smooth muscle cells1Approximately 30% to 50% of cancer survivors experience progressive chemotherapy‐induced cognitive impairment   , which enables us to FACS‐sort senescent cells from tissues, and the herpes simplex virus thymidine kinase, which allows us to selectively kill p16‐positive senescent cells by administering the prodrug ganciclovir (GCV).To identify and eliminate senescent cells, we used a novel transgenic mouse model  or vehicle  in 2 cycles (5 days/cycle) with a 7‐day interval between cycles. Mice were left to recover for 2 weeks in the original environment. Then, PTX treated mice were assigned randomly to three groups. Two groups received the senolytic drug ABT263   using our previously reported protocol  based imaging methods were used as previously described . At the end of the experiments, the animals were transcardially perfused with ice‐cold PBS and decapitated. The brains were immediately removed and samples were collected for subsequent studies. Whole brains were collected for FACS analysis and transcriptomics. Acute brain slices were collected for LTP measurements. Half brains were immersion fixed in 4% paraformaldehyde for 24 hours, transferred to sucrose gradients and embedded and cut for immunohistochemistry.NVC responses were assessed as described previously . Then, the ratio of RFPFluorescent activated cell sorting (FACS) with the low‐pressure WOLF Cell Sorter™ (NanoCellect) was used to obtain a cell suspension enriched in brain senescent cells. RFP+ senescent cells were stained with cell‐specific markers. Antibodies against CD31 were used to quantify the ratio of senescent endothelial cells.2.9Cellular senescence is characterized by senescence‐associated β‐galactosidase  activity. To assess the sensitivity of cultured primary cerebromicrovascular endothelial cells to PTX‐induced senescence, we measured SA‐β‐gal activity in PTX‐treated cultured human cerebromicrovascular endothelial cells (CMVECs). Human CMVECs were treated with vehicle (DMSO in media) or PTX  was used to assess spatial distribution of senescent cells in brains of PTX‐treated mice, as previously described  were cut. Sections were immunolabeled for IBA‐1  and the endothelial marker endomucin  to identify microglia and capillary endothelial cells in the brain, respectively. Confocal images were obtained using Leica SP8 MP confocal laser scanning microscope. The relative staining intensity for IBA‐1 positive perivascular microglia per region of interest was assessed.2.13t test, one‐way or two‐way ANOVA with Fisher LSD post hoc test using GraphPad Prism 7.0, as appropriate. Differences were considered significant at p < 0.05. Data are presented as means ± standard error of mean (SEM) or all measured values as bar graphs or box plots with interquartile distributions and median values. Analysis was made with GraphPad Prism.Depending on the experiment, statistical analyses were carried out by unpaired 33.1To provide prima facie evidence that PTX induces senescence in endothelial cells, we performed SA‐β‐gal staining of PTX treated and untreated cerebromicrovascular endothelial cell (CMVEC) cultures. PTX significantly increased the ratio of CMVECs with positive SA‐β‐gal staining Figure .3.2in vivo PTX treatment on cellular senescence and to determine the efficacy of senolytic treatments, we used flow cytometry to detect p16‐RFP+ senescent cells 6 months after PTX treatment  , restoring it to levels observed in control mice   Figure . The ext) Figure . Previou) Figure . Analyse) Figure . Spatial) Figure . PTX tre) Figure . Togethe4The key findings of this study are that (1) PTX induces senescence in endothelial cells, which associates with microvascular rarefaction, NVC dysfunction and BBB disruption and that (2) both pharmacological (ABT263/Navitoclax) and genetic depletion of senescent cerebromicrovascular endothelial cells rescues PTX‐induced cerebromicrovascular dysfunction and attenuates CICI.Our studies show that a PTX treatment induces senescence in CMVECs. The clinical relevance of our findings is supported by studies characterizing the effects of PTX on cultured peripheral endothelial cells in connection to the extensive use of PTX‐coated balloons and drug‐eluting stents for cardiovascular indications (including treatment of intermittent claudication) ) mice, which display senescence cell‐dependent phenotypes, provide additional evidence that cellular senescence is causally linked to BBB disruption (Yamazaki et al., Endothelial cells in the cerebral microvessels are an integral part of the BBB, where they exhibit extensive tight junctions to prevent leakage of blood borne factors into the brain parenchyma. Intact BBB function is required for normal structural and functional brain connectivity, synaptic activity, and information processing (Sweeney et al., CMVECs are connected via gap junctions and form a functional syncytium (Ungvari et al., In conclusion, our findings show that senolytics confer neurovascular protection in PTX treated mice. Removal of senescent cells by treatment with navitoclax/ABT263 rescued NVC responses and BBB integrity and increased capillary density in brains of PTX treated mice, which likely contribute to improved cognitive performance. Our findings highlight the potential use of senolytic drugs as therapies to prevent CICI along with other side effects of chemotherapy (Yao et al., The authors confirm contribution to the paper as follows: Study conception and design: CA, ANT, ST, AY, QT, PB, JC, ZU, and AC; data collection: CA, ANT, JD, RG, JF, ST, AKC, PB, AU, and FY; analysis and interpretation of results: CA, ANT, JD, RG, JF, ST, TK, AKC, PB, AU, RN, FY, QT, PM, TC, AY, JC, ZU, and AC; draft manuscript preparation: CA, ANT, JD, RG, JF, ST, TK, AKC, PB, AU, RN, FY, QT, PM, TC, AY, JC, ZU, and AC. All authors reviewed the results and approved the final version of the manuscript.None declared.Data S1Click here for additional data file.


PRDX3) have only recently been associated with autosomal recessive spinocerebellar ataxia characterized by early onset slowly progressive cerebellar ataxia, variably associated with hyperkinetic and hypokinetic features, accompanied by cerebellar atrophy and occasional olivary and brainstem involvement. Herein, we describe a further simplex case carrying a reported PRDX3 variant as well as two additional cases with novel variants. We report the first Brazilian patient with SCAR32, replicating the pathogenic status of a known variant. All presented cases from the Brazilian and Indian populations expand the phenotypic spectrum of the disease by displaying prominent neuroradiological findings. SCAR32, although rare, should be included in the differential diagnosis of sporadic or recessive childhood and adolescent‐onset pure and complex cerebellar ataxia.Bi‐allelic variants in peroxiredoxin 3 ( PRDX3 (OMIM 604769), the gene encoding for peroxiredoxin 3.SCAR32 is a novel autosomal recessive spinocerebellar ataxia caused by bi‐allelic variants in et al.PRDX3 that were found to impair the assembly of the PRDX3 protein complex, leading to the complete absence of PRDX3, impaired cellular response to oxidative stress and mitochondrial dysfunction.Rebelo PRDX3 variant was reported in a patient with infantile‐onset severe cerebellar ataxia and peripheral neuropathy, expanding the disease's clinical spectrum.et al.PRDX3 mutation p.Asp163Glu results in an unstable structure tending to form aggregates that trigger unfolded protein responses via mitochondria and endoplasmic reticulum. More recently, two further families carrying novel homozygous PRDX3 variants were identified, following a screen of >3,500 ataxia exomes.PRDX3‐associated disease may have a broader age of onset range than previously thought. In addition, there was a third report that associates a PRDX3 non‐sense variant p.Lys166* with cerebellar ataxia and profound hearing impairment.Subsequently, a novel homozygous PRDX3 as well as two additional cases with novel bi‐allelic variants. The variants were identified by screening of exome data from a large cohort of patients with cerebellar ataxia  of multi‐ethnic background.Herein, we present a further simplex case carrying a reported homozygous variant in This study was approved by the Ethics Committee of University College London Hospital NHS Foundation Trust (UCLH), the Hospital de Clínicas Ethics Committee  and the Indira Gandhi Institute of Child Health. Written informed consent was obtained from the patients and parents. To investigate the genetic cause of the disease, whole‐exome sequencing of the DNA from patients was performed as previously described.Patient I (p.Asp202Asn) is a 27‐year‐old male from a non‐consanguineous Brazilian family with pure cerebellar syndrome. He reported insidious onset of gait ataxia and dysarthria around the age of 18 years, with limb ataxia developing shortly thereafter. The disease course was slowly progressive, and the patient can still walk independently 10 years following symptom onset. He reported no symptoms of dysautonomia. There is no family history of ataxia Fig. . Both pafs*10; p.Gly180Ser) is a 46‐year‐old male, with no previous family history and a pure cerebellar ataxia phenotype. He began his symptoms at the age of 35 years old. At first, he reported dizziness and gait ataxia that slowly progressed to limb ataxia. Upon neurological examination, he displayed gait ataxia, mild cerebellar dysarthria, dysmetria and dysdiadochokinesia. He is still able to walk without support and no dysphagia was reported. Bidirectional horizontal nystagmus was also noted. His SARA score was 12/40. There was no pyramidal, extrapyramidal, cognitive, peripheral or cranial nerve involvement. A brain MRI scan performed at the age of 45 years old reported a diffuse cerebellar atrophy with no other abnormalities. A nerve conduction study (NCS) and video‐head‐impulse test were also performed without any remarkable findings. His parents are deceased so segregation studies could not be performed.Patient II (p.Leu8CysZ‐score −1.83), weight was 18.3 kg (Z‐score −0.01) and height was 99 cm (Z‐score −2.37). Serum immunoglobulin, AFP, eye examination and hearing assessment were all normal. A brain MRI scan performed at the age of 5 years old reported T2 hyperintensities at posterior pons, formation reticularis, medial part of bilateral middle cerebellar peduncles, dentate nuclei, anterior cerebellar vermis and cerebellar gyri. Additionally, frontal encephaloclastic porencephaly with thinning of the corpus callosum was identified which could be attributed to a congenital or acquired process. The patients' clinical details, along with data from previously reported cases with bi‐allelic PDRX3 mutations, are presented for comparative purposes in Table Patient III p.Arg170*) is a 5‐year‐old male child of Indian origin born to consanguineous parents (distant relatives). The birth was normal but soon after, at 6 months of age, he presented with global developmental delay, failure to achieve milestones and slowly progressive imbalance in gait at 3 years of age. He achieved walking at 2 years of age and could walk up and down stairs at 4 years old with handrail support. Upon neurological examination, he presented with axial and appendicular skeleton hypotonia, as well as brisk deep tendon reflexes and extensor plantar on both sides. Cerebellar examination showed gait ataxia and gaze‐evoked nystagmus. The skull and spine examination was normal. Cardiovascular, respiratory and abdomen examination was also unremarkable. Head circumference was 48 cm (70* is a PRDX3 (NM_006793.5): c.604G>A, p.(Asp202Asn) residing within a 6.45 Mb region of homozygosity : c.23delT p.(Leu8Cysfs*10) and c.538G>A, p.(Gly180Ser) were identified and confirmed by Sanger Sequencing in proband II : c.508C>T, p.(Arg170*) residing within a 2.22 Mb region of homozygosity was identified in Patient III : c.604G>A, p.(Asp202Asn). The allele frequency (AF) for the variant in the 100kGP data set is 0.0115% .Analysis on the 100k Genomes Project data set performed within a secure workspace called the ‘Research Environment’ identified an additional patient, a white British boy with cerebellar hypoplasia, with the homozygous missense variant in PRDX3 mutations, replicating the pathogenic status of a known variant in the homozygous state as well as identifying novel pathogenic variants. SCAR32 represents a rare form of SCA, until now, only described in India, Morocco and Europe, particularly in Turkey  with SCAR32 due to bi‐allelic ey Table . All prePRDX3, is noteworthy and remains to be adequately explained at a pathophysiological level. All PRDX3 variants reported to date act via loss‐of‐function, as the mutant proteins are absent in patients' fibroblasts, leading to complete loss of the enzyme. It is expected that in the case of family 2, with two compound heterozygous variants, the p.Leu8Cysfs*10 is a loss‐of‐function variant depleting the protein, however, it is possible that is compensated to some extent by the p.Gly180Ser variant, hence delaying the age of onset of disease. Previously, in patient fibroblasts, the absence of PRDX3 was accompanied by reductions in PRDX5, a paralogue of PRDX3, and glutathione peroxidase activity, suggesting a coregulatory connection between PRDX3 and PRDX5, as well as glutathione peroxidase.The homozygous missense variant identified in our case, p.Asp202Asn, has been previously described in two unrelated families of Kurdish origin. Its identification in a Brazilian family suggests that this is not a population‐specific variant and may in fact be more widespread than originally thought especially in Latino and UK populations as the allele frequency of the variant is highest in these populations, according to our inspection of different variant frequency databases Table .1 AddiInterestingly, patients with variants identified in the later part of the thioredoxin domain of the PRDX3 protein present with an early onset of the disease. Even if no clear genotype–phenotype correlations exist to date Figure , it is pPRDX3 mutations.The presence of cerebellar atrophy is a ubiquitous feature in patients with bi‐allelic In conclusion, our study reports three novel patients with rare PRDX3‐related SCAR, mainly in the Brazilian ataxia population, and hence expands the phenotypic spectrum of the disease, demonstrating that it can present with features suggestive of cerebellar cortical hyperintensities that link to early onset neurodegeneration.G.K. has received research grants from Genesis Pharma and Teva, consultation fees, advisory boards and honoraria from Genzyme, Genesis Pharma, Teva and Novartis. All other authors report no disclosures relevant to the manuscript.Figure S1. Genotype–phenotype correlations in recessive spinocerebellar ataxia type 32 (SCAR32). (A) Age of onset in an SCAR32 cohort. (B) variant type  carried in an SCAR32 cohort and (C) correlation between Scale for assessment and Rating of Ataxia (SARA) score and disease duration.Click here for additional data file.Table S1. In silico predictions and allele frequency of the identified PRDX3 variants in publicly available population databases and ACMG criteria classification.Click here for additional data file.


Hepatocellular carcinoma (HCC) is a major cause of death among patients with liver cirrhosis. The rise of immuno‐oncology has revolutionized treatment for advanced HCC. However, most pivotal randomized controlled trials have excluded patients with moderate liver dysfunction (Child–Pugh–Turcotte B), despite the high incidence of liver disease in patients with HCC at the time of diagnosis. Overall survival in patients with HCC and moderate liver dysfunction treated with sorafenib has been found to be only approximately 3–5 months, underlining the need for improved treatment algorithms for this increasingly important subgroup of patients. In this review, we summarize available data on the treatment of patients with HCC and moderate liver dysfunction. Opportunities, as well as clinical challenges, are discussed in detail, highlighting potential changes to the therapeutic landscape. The CP classification is based on clinical signs of liver function, including ascites, encephalopathy, bilirubin, albumin, and prothrombin time/international normalized ratio. Each measure is scored 1–3 according to severity, and patients are classified as CP A (5–6 points) for normal liver function, CP B (7–9 points) for moderate dysfunction, or CP C (10–15 points) for severe dysfunction.More recently, the albumin–bilirubin (ALBI) system for classification of liver function has been introduced as an alternative method for predicting treatment efficacy and survival in patients with liver damage and HCC.n = 3696).ALBI grading has been evaluated in multiple geographical locations and clinical settings,New drugs for HCC are initially tested in patients with adequate liver function, but results may not translate to patients with moderate liver dysfunction.1.21.2.1www.nccn.org/guidelines) recommends this drug for the treatment of advanced HCC in patients with moderate liver dysfunction (i.e. CP B7).Several MKIs have been approved for the treatment of advanced HCC based on improvements in overall survival (OS).n = 38 [28%]) fared worse than those with CP A, with 12.9 weeks therapy duration, 13 weeks median progression‐free survival (PFS), 14 weeks OS, and encephalopathy and worsening ascites in 11% and 18% of patients, respectively.n = 162) discontinued treatment early and mortality was higher in patients with worsening liver function than in those with normal function.n = 234).Sorafenib, an oral inhibitor of Raf‐1, B‐Raf, vascular endothelial growth factor receptors (VEGFRs) 1, 2, and 3, and platelet‐derived growth factor receptor (PDGFR), improved OS compared with placebo in the SHARP Phase 3 trial.GIDEON was a prospective, observational registry study to evaluate the real‐world safety of sorafenib in patients with HCC. Data from 669 treatment‐naive patients with CP B were included.The recommended continuous dose of sorafenib is 400 mg twice daily in patients with normal liver function.p < 0.01).In the REFLECT Phase 3 trial, lenvatinib, an inhibitor of VEGFRs 1, 2, and 3, fibroblast growth factor receptors 1, 2, 3, and 4, PDGFR‐α, and RET and KIT proto‐oncogenes, was noninferior to sorafenib in OS in patients with HCC.p = 0.001).n = 59), which was significantly worse than the CP A cohort .N = 440), median PFS and OS were 3.2 and 12.1 months, respectively.Regorafenib is an inhibitor of oncogenic kinases including RET, RAF‐1, and KIT and appears to be more potent than sorafenib.p = 0.001) and OS  when compared with CP A. The median PFS and OS were 2.2 and 3.8 months, respectively, in patients with CP B (n = 22), compared with 4.3 (p < 0.001) and 9.0 (p < 0.001) months, respectively, in patients with CP A (n = 88).Cabozantinib inhibits tyrosine kinases, including VEGFRs 1–3, MET, and AXL, and was approved based on the CELESTIAL Phase 3 trial.1.2.2For many years, sorafenib was the only systemic treatment for advanced HCC with proven efficacy.p ≥ 0.99) and there was a higher incidence of Grade 3 or 4 toxicity than in patients with CP A.In the REACH Phase 3 trial, ramucirumab, a recombinant immunoglobulin G1 monoclonal antibody that binds to VEGFR‐2, failed to demonstrate improvement against placebo in OS in patients with advanced HCC previously treated with sorafenib.n = 49) with CP B7–8; median OS for nivolumab was 7.6 months in this cohort, compared with a historical OS of 2.5–5.4 months for patients with CP A treated with sorafenib.The single‐agent PD‐1 inhibitors nivolumabp < 0.001) in the Phase 3 IMbrave150 trial in patients with advanced HCC and CP A who had not previously received any systemic treatment.ICIs, in combination or when combined with anti‐VEGF agents, have shown synergistic antitumor effects and better quality of life (QOL) endpoints compared with sorafenib in patients with advanced HCC and CP A.p < 0.001), while median OS in patients with ALBI 2 and 3 was 8.6 months and 3.2 months, respectively.p = 0.0003).When stratified by ALBI, the IMbrave150 trial found no difference in median OS between atezolizumab plus bevacizumab (11.7 months) and sorafenib (12.2 months) in patients with ALBI 2.Tremelimumab plus durvalumab also showed superiority over sorafenib as first‐line therapy in patients with advanced HCC and CP A in the Phase 3 HIMALAYA trial.Several studies combining IO agents with MKIs and other biological agents in first‐ and second‐line therapy are ongoing. A complete list of studies can be found in a recent review.Despite the advances in IO agents for treating advanced HCC, several challenges remain. Phase 3 trials of IO agents in HCC have typically been restricted to a group of patients with good liver function (CP A) and their use in patients with moderate liver dysfunction has not been fully evaluated. Owing to a lack of available therapies, patients with some residual liver function (CP B7) may receive prescriptions for IO agents, even though supporting data are restricted to CP A and there are few or no data for CP B.90Yttrium transarterial radioembolization.Even among patients with advanced HCC and good liver function, there remains a need for validated biomarkers for predicting and assessing responses to IO agents1.3Cirrhosis represents a late stage of progressive hepatic fibrosis and is generally considered irreversible in its advanced stages. Cirrhosis accounted for approximately 50,000 deaths in the US in 2010 and was the eighth leading cause of death.Risk factors for cirrhosis complications include bleeding, infection, alcohol consumption, medication use, dehydration, constipation, and obesity.Spontaneous bacterial peritonitis is common in patients with end‐stage liver disease and is associated with high mortality without early antibiotic treatment.1.4Complications of cirrhosis limit clinical development of treatments for HCC in patients with moderate liver dysfunction. Heterogeneity of the population and the shorter life expectancy and increased risk of sudden death events in those with CP B versus CP A limit the ability to assess efficacy in prospective clinical trials. Hepatic deterioration during treatment is also observed in patients with moderate liver dysfunction. This could be related to toxicity of treatment or progression of the tumor burden resulting in intrahepatic obstruction of the biliary tree and/or portal vein.Interventions involving a balanced approach to treatment of both cirrhosis and tumor burden would likely improve the long‐term survival and QOL of patients with HCC and moderate liver dysfunction Figure .1.5The optimal efficacy endpoint for clinical trials in patients with HCC and moderate liver dysfunction is a topic for consideration. OS is objective and clinically relevant and is the usual primary endpoint used in randomized Phase 3 clinical trials. OS is therefore the basis of most regulatory approval processes for drugs and other treatment interventions. However, OS is likely to be disproportionately affected by early death rates in patients with moderate liver dysfunction compared with those with normal liver function.Simulations of incremental treatment‐related improvements in 3‐month OS for patients with CP B predict meaningful improvements in long‐term OS, suggesting that prevention of early death may represent an improved efficacy outcome Figure . The maiApart from CP classification and comorbidities, other clinical characteristics and tumor‐related variables may contribute to early death rates.These uncertainties in interpreting clinical trial results mean that it is difficult to evaluate whether patients with CP B are likely to achieve similar benefits from treatment as patients with CP A.1.5.1A few prospective systemic treatment clinical trials including patients with CP B are ongoing, two of which restrict eligibility to patients with CP B only. Cabozantinib is being tested in a Phase 1/2 study as a first‐ and second‐line therapy in 32 patients (NCT04497038) and 20 patients (NCT04454762) with CP B7–8 and ECOG PS 0–1. The primary endpoint is maximum tolerated dose for a Phase 2 study and the secondary endpoints are PFS, OS, and median time to progression. Atezolizumab and bevacizumab are being tested in a Phase 2 study as first‐line therapies in 50 patients with CP B7 and ECOG PS 0–1 (NCT04829383). The primary endpoint is frequency and severity of toxicities, and the secondary endpoints are overall response, disease control rate, duration of response, OS, and PFS. The other study lists CP B7 or CP A as inclusion criteria (NCT03439891).1.6Ongoing symptoms, treatment‐related issues such as AEs and disease progression, and other emotional, physical, and social factors such as anxiety, fatigue, and stigmatization, all negatively impact QOL in patients with chronic liver diseases.1.7Effective treatment of patients with advanced HCC and CP B is an important unmet need. New treatment strategies are promising based on demonstrated activity and tolerability in patients with advanced HCC, including IO agents alone, in combination with existing therapies, or in combination with new targeted agents such as MKIs. However, for patients with moderate liver dysfunction, avoiding further impairment of liver function and maintaining QOL are important considerations for the development of treatments for HCC. Treatments targeting multiple immune mechanisms appear to be especially promising in CP B, although a better understanding of the mechanisms of action at molecular and cellular levels is needed. Validated biomarkers could guide the development of IO agents in patients with HCC and CP B. The use of medical devices, such as tumor‐treating fields,1.8This paper is not a systematic literature review and was not performed in accordance with the Preferred Reporting Items for Systematic Reviews and Meta‐Analyses (PRISMA) guidelines. The quality of evidence in the included articles was not assessed. The main limitation of this review is that it may be subject to selection and confirmation bias. In addition, the review may have been limited by the availability and discovery of relevant data, as well as publication bias.2There are insufficient data on the efficacy of treatments for advanced HCC in patients with moderate liver dysfunction . This is a neglected subgroup of patients and given the high frequency of liver dysfunction in patients with HCC there is a significant need for research into the efficacy and safety of new and existing treatments in this population. Sorafenib is the only available therapy for HCC with reasonable efficacy and safety data collected in patients with moderate liver dysfunction. Studies comparing sorafenib and newer treatment alternatives have yielded similar results, but systemic treatment in patients with moderate liver dysfunction is associated with high toxicity and early death rates. New prospective clinical trials focused on the CP B population are urgently needed, with study designs that consider the impact of both liver disease and tumor burden. Understanding the risk characteristics of this patient population and what is considered a meaningful clinical outcome is fundamental in the design of new clinical trials.Frederico P. Costa: Writing—original draft (lead). Bertram Wiedenmann: Writing—original draft . Christoph Roderburg: Writing—review and editing . Raphael Mohr: Writing—review and editing . Ghassan K. Abou‐Alfa: Writing—review and editing (lead).This work was funded by Autem Therapeutics.Frederico Costa and Bertram Wiedenmann are employees and shareholders of Autem Therapeutics. Ghassan K. Abou‐Alfa reports institutional research support from Arcus Biosciences, AstraZeneca, BioNtech, Bristol‐Myers Squibb, Celgene, Flatiron Health, Genentech/Roche, Genoscience Pharma, Incyte, Polaris Pharmaceuticals, Puma Biotechnology, QED Therapeutics, Silenseed, and Yiviva, and consulting support from Adicet Bio, Alnylam Pharmaceuticals, AstraZeneca, Autem Therapeutics, BeiGene, Berry Genomics, Boehringer Ingelheim, Celgene, Cend Therapeutics, CytomX Therapeutics, Eisai, Eli Lilly and Company, Exelixis, Flatiron Health, Genentech/Roche, Genoscience Pharma, Helio Health, Helsinn Healthcare, Incyte, Ipsen, Merck, Nerviano Medical Sciences, NewBridge Pharmaceuticals, Novartis, QED Therapeutics, Rafael Pharmaceuticals, RedHill Biopharma, Servier, Silenseed, Sobi, Vector BioPharma, and Yiviva. The remaining authors made no disclosures.


We studied the relationship of SARS‐CoV‐2 variants of concern (VOC) with COVID‐19 severity and mortality among hospitalized patients in Pakistan.COVID‐19 morbidity and mortality varied globally through the pandemicA retrospective review of clinical, laboratory, and vaccination data of 197 COVID‐19 adult patients at the Aga Khan University Hospital, Karachi between April 2021, and February 2022 was performed. SARS‐CoV‐2 VOC identified in respiratory samples were analyzed. Univariate and multivariate analysis was conducted to identify factors associated with COVID‐19 outcomes.The median age of cases was 55 years and 51.8% were males. Twenty‐four percent of females were pregnant. Of COVID‐19 cases, 48.2% had nonsevere disease, while 52.8% had severe/critical disease. Hypertension (48%) and diabetes mellitus (41%) were common comorbids. SARS‐CoV‐2 VOC identified comprised; Omicron (55.3%), Beta (14.7%), Alpha (13.7%), Delta (12.7%), and Gamma (3.6%) variants. Most (59.7%) study subjects were unvaccinated. Of vaccines, 88% had received inactivated virus COVID‐19 vaccines. Increased risk of severe disease was associated with age ≥50 years , as well as with diabetes mellitus . Full vaccination  or infection with Omicron  was associated with reduced disease severity. The risk of mortality increased with age ≥50 years  and a history of myocardial infarction  whilst, infection with Omicron was found to reduce the risk .Our study describes the relationship between the severity of COVID‐19, in‐hospital mortality in relation to SARS‐CoV‐2 variants, and the impact of COVID‐19 vaccination in Pakistan. Outcomes were more favorable in younger individuals, after vaccinations and with Omicron variant infections. Most cases received inactivated virus vaccines therefore these data highlight the protection provided against severe COVID‐19. In a study from our center during the early pandemic period in 2020, it was seen that 30% of patients had severe to critical disease at presentation with age and critical disease (identified by septic shock and multiorgan dysfunction) were independently associated with mortality.Within a few months of the start of the pandemic in December 2019, it was seen that SARS‐CoV‐2 acquired mutations particularly in its spike glycoprotein, which was associated with entry to host cells. Variants carrying mutations in the spike gene were described by the World Health Organization (WHO) to be variants of concern (VOC) and include, Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), and Omicron (B.1.1.529).G, L, and S clade SARS‐CoV‐2 strains were shown to be present in the first wave of COVID‐19 in Pakistan between March and August 2020.COVID‐19 vaccinations were first introduced at the start of 2021There is limited information regarding the effectiveness of vaccinations administered in the context of SARS‐CoV‐2 variants in Pakistan also, factors associated with severe COVID‐19. Here we have investigated the risk factors, clinical presentations, and outcomes in relation to the VOC in patients admitted to our center with COVID‐19 between April 2021 and February 2022.2This was a retrospective cross‐sectional cohort study of adults with a PCR confirmed COVID‐19 patients admitted to the AKUH , between April 2021 and February 2022. This study received approval by the Aga Khan University, Ethical Review Committee (study reference no. 2021‐6232‐19404).All COVID‐19 cases had a SARS‐CoV‐2 PCR positive respiratory sample using the SARS‐CoV‐2 Cobas 6800 Roche assay, at the AKUH Clinical Laboratories, Karachi, Pakistan. The CT (crossing threshold) value of each positive SARS‐CoV‐2 sample was noted. The cases chosen for review were those for whom SARS‐CoV‐2 VOC testing had been conducted as part of a WHO supported genomic surveillance effort.Data were collected on symptoms at presentation, treatment, duration of illness, outcomes and COVID‐19 vaccinations. Laboratory and radiological data were collected for all cases. COVID‐19 severity was categorized into nonsevere in patients who have oxygen saturation ≥94% on room air, severe in those who have an oxygen saturation of ˂94% and require supplemental oxygen support and critical in those who have respiratory failure, shock and/or multiorgan dysfunction.2.1Isolates were screened for VOC; Alpha, Beta, Gamma, Delta, and Omicron lineage associated mutations using a PCR‐based approach.2.2p value of <0.05 was considered significant. Logistic regression analysis was performed to identify risk factors for the severity of illness and in‐hospital mortality and adjusted odds ratios (aOR) and their 95% confidence intervals (CI) were estimated. Initially, Univariate Logistic regression analysis was done to identify variables with a p ≤ 0.1, which were then analyzed in the Multivariable Logistic regression model. Data were analyzed using STATA™ version‐14.Median and interquartile range was reported for continuous variables such as age and length of stay and frequency and percentages were used to describe categorical variables such as gender and mortality. Comparison between categorical variables was determined using Chi‐square test or Fischer exact test where appropriate. All the tests were two‐sided and a 33.1We studied the clinical characteristics and disease outcomes of 197 COVID‐19 patients admitted at AKUH between April 2021 and February 2022. Three COVID‐19 waves were observed in Pakistan during this study period Figure . COVID‐1n = 102) were males, Table The median age of the patients was 55 years (interquartile range: 34–70 years) and 51.8%  till the time of discharge while 38 (19.3%) patients died and 20 (10.2%) left against medical advice Table . Complicn = 43), single shot vector vaccines  or messenger RNA (mRNA)  vaccinations. Hence, the majority (88%) of study subjects had received inactivated COVID‐19 vaccines.Vaccination data was available for 154 (78.2%) and was missing for 43 (21.8%) patients. Available data showed that 92 (59.7%) study subjects were unvaccinated and 59 (38.3%) were fully vaccinated, while three individuals were partially vaccinated. Details of the administered vaccination were available in 49 cases; individuals had received inactivated vaccines , were male (p = 0.019), had a higher WHO ordinal score at admission or discharge (p < 0.001) and increased length of hospital stay (p < 0.001), Table p < 0.001 showed an association with nonsevere disease, while infection with the Omicron variant was linked to nonsevere disease , p = 0.003.We investigated the factors associated with severe COVID‐19 by stratifying the study group into those with nonsevere as compared with severe/critical disease. Age, clinical characteristics, vaccination status and infecting SARS‐CoV‐2 variants were all compared between the two sub‐groups. Most patients who developed severe disease were older than 50 years  and the presence of diabetes mellitus , whereas being fully vaccinated  were found to be protective against severe/critical disease (Table .45–13.7)3.3p = 0.001, Table p = 0.001; the presence of co‐infections or secondary infections , p = 0.032, presence of complications such as myocardial infarction (MI) , p = 0.004, acute kidney injury (AKI) , p = 0.005, having pneumothorax or pneumomediastinum , p = 0.019 and being unvaccinated , p = 0.044, as factors associated with greater risk of death. Infection with Omicron as compared with non‐Omicron variants was associated with survival , p = 0.001.Factors associated with in‐hospital mortality were determined . The median age of those who died was 64 years whilst those who survived was 48 years, p = 0.001, and the presence of MI , p = 0.001. Infection with the Omicron variant was associated with survival , p = 0.001.A multivariate analysis run after adjusting for confounding and effect modification from other variables further confirmed in‐hospital mortality were found to be associated with age greater than 50 years , 3.4p = 0.015, Kruskal–Wallis analysis. In particular, the CT values of Omicron variants were lower, reflecting significantly higher viral loads as compared with the Alpha variant, p = 0.0005.Higher SARS‐CoV‐2 viral loads have been associated with more severe disease. We determined the viral loads present in respiratory specimens and analyzed them according to the VOC. The CT of SARS‐CoV‐2 target gene amplification was used as a marker of viral loads. We found a significant difference between the CT values of the VOC, Figure 4The correlation between COVID‐19 severity and SARS‐CoV‐2 variants has been a topic of significant discussion with limited information in the context of Pakistan. Our study was conducted through the pandemic waves in Pakistan characterized by ancestral followed by G clade, Alpha, and then Delta followed by Omicron variants.Our results regarding risk factors associated with severity fit with previous reports that showed comorbidities including diabetes mellitus, structural lung disease, cardiovascular disease, and hypertension were associated with an increased risk of severe COVID‐19.During the first year of the pandemic (2020) in Karachi, we had observed that severe to critical disease at presentation, and age more than 60 years and critical disease to be independently associated with mortality.One quarter of females with COVID‐19 were those pregnant. This high number of detected cases was due to the diagnostic screening practice at the AKUH which required testing of women who presened to the labor room or for elective surgical deliveries. The COVID‐19 severity in pregnant women was mild. However, it highlights the increased risk to mother and child in this cohort as has been shown by global studies on the impact of COVID‐19 on maternal health.We found vaccinated individuals to have a reduced risk of mortality from COVID‐19. Our data correlates with previous reports which have shown effectiveness of COVID‐19 vaccines in protection against severe disease.The majority of our vaccinated study subjects were infected with the Omicron variant likely due to the higher risk of breakthrough infections. Importantly, the Omicron variant had lower CT values associated with higher viral loads as compared with the Alpha variant. This differs from previous variants where genome‐wise analysis of SARS‐CoV‐2 have demonstrated higher viral loads to be associated with increased disease severity.A study from the United Kingdom also reported an increase in deaths due to Alpha variants.A limitation of our study is that it is from a single‐center tertiary care facility. Further, our sample size was relatively small, and we did not have complete vaccination details for all study subjects. Most patients were vaccinated with inactivated vaccines due to the local availibility COVID‐19 vaccinations. However, we provide valuable clinical evidence regarding the factors associated with disease severity and mortality, specifically in the context of VOC and protective effect of vaccinations. The protective effect observed aligns with the efficacy studies of different vaccines used in Pakistan, which have shown inactivated type of vaccinations were moderately effective against symptomatic COVID‐19.Kandeel et al.The response to the emergence of Omicron variants was a strong recommendation to administer booster doses of vaccines.Importantly, we show that for admitted patients, comorbid conditions, older age group and absence of vaccinations were all associated with increased risk of inpatient mortality. There was a high incidence of COVID‐19 in pregnant women in the population. It is evident that in Pakistan like other countries, COVID‐19 vaccination were key in reducing disease severity regardless of the SARS‐CoV‐2 variant involved. Further, our research shows that virus inactivated vaccines improved COVID‐19 outcomes. This is important in the context of vaccine availability as whilst new vaccines are being developed, these may not be readily available especially in low‐resource settings. Our highlights the significance of correlating clinical and epidemiological data with pathogen genomic analysis to provide insights for public health decision‐making.Muhammad Zain Mushtaq: formal analysis; investigation; writing—original draft. Nosheen Nasir: conceptualization; formal analysis; investigation; writing—review & editing. Syed Faisal Mahmood: formal analysis; investigation; writing—review & editing. Sara Khan: data curation; investigation; methodology; writing—original draft. Akbar Kanji: data curation; formal analysis. Asghar Nasir: data curation; formal analysis. M. Asif Syed: resources. Uzma Bashir Aamir: funding acquisition. Zahra Hasan: conceptualization; funding acquisition; investigation; project administration; writing—review & editing.The authors declare no conflicts of interest.This work complies with the policies for Ethical Consent as per Helsinki Declaration.The lead author Zahra Hasan affirms that this manuscript is an honest, accurate, and transparent account of the study being reported; that no important aspects of the study have been omitted; and that any discrepancies from the study as planned  have been explained.Supporting information.Click here for additional data file.Supporting information.Click here for additional data file.Supporting information.Click here for additional data file.


The authors found a strong correlation between national‐level data for deaths from household pollution and eclampsia rates in the CRADLE 3  study clusters (correlation coefficient: 0.84). Indoor air pollution from solid fuels for cooking and heating predominantly takes place in low‐ and middle‐income countries (LMICs).https://ourworldindata.org/indoor‐air‐pollution#death‐rates‐from‐indoor‐air‐pollution).We correlated national‐level data for deaths caused by indoor air pollution with eclampsia rates in 10 LMIC sites across eight countries. The authors evaluated eclampsia rates prospectively determined from 2692 eclamptic cases in 536 223 deliveries obtained from our CRADLE 3  trial,P = 0.0019). This increased to 0.86  when eclampsia occurred at home (44.9% of cases). This supports an effect occurring at home, such as indoor air pollution.There was a significantly positive correlation between national‐level data for deaths attributable to indoor household pollution and eclampsia rates, with a correlation coefficient of 0.84 (95% CI, 0.468–0.963; Our data suggest that indoor household pollution may account for an acute lowering of seizure threshold in pre‐eclampsia, possibly related to hypoxia. Household air pollution using indoor cooking stoves in Nigeria has been linked to histological measures of chronic placental hypoxia,T.R. was involved in the design, planning, conduct, data analysis, and manuscript writing; K.K. was involved in the data analysis and manuscript writing; N.V. was involved in the design, planning, conduct, and manuscript writing; P.S. was involved in the design, planning, and data analysis; and A.S. was involved in the design, planning, conduct, data analysis, and manuscript writing.There are no relationships for any author that could compromise the objectivity of the paper and its review.


Sympathetic crashing acute pulmonary edema (SCAPE) complicating pacemaker implantation is a very uncommon and dangerous occurrence. Following pacemaker implantation, patients need stringent monitoring, and compelling evidence about SCAPE treatment is required.Sympathetic crashing acute pulmonary edema complicating a pacemaker insertion as the case in our patient is extremely rare. We report a case of 75‐year‐old man with a complete AV block, which requires urgent pacemaker implantation. Half an hour following the insertion of the pacemaker, an abrupt SCAPE emerged and the patient was incubated immediately.  Thus, treatment of these arrhythmias is mandatory, and should be initiated immediately once it is diagnosed.A variety of management have been described for cardiac arrhythmias in the medical literature and pacemaker implantation is considered one of these treatment when indicated,2A 75‐year‐old male presented to the cardiology center complaining of new‐onset dyspnea and lower limbs swelling associated with dizziness and syncopal attacks since 3 h ago. The patient has a medical history of hypertension for 11 years, gouty arthritis. He takes regularly ; he has otherwise no significant medical, surgical or allergic history. The patient is neither a smoker nor alcoholic.2 was 98% in room air, and reparatory rate was 16 rpm. Clinical examination of the limbs revealed severe pitting edema over the tibia. Further examination of other organ systems did not reveal any significant abnormality.Upon clinical examination of the cardiovascular system, the heart sounds were normal without any associated mummer, S4 sound was heard, blood pressure measured 140/80 mmHg, heart rate was 49 bpm. Regarding the respiratory system, the chest was clear upon auscultation, SpO3/μL, PLT: 285 × 103/μL, urea: 134 mg/dL, Cr: 2.9 mg/dL, K+: 3.1 mmol/L, Na+: 144 mmol/L, INR: 1.2.Subsequent electrocardiogram (ECG) revealed complete heart block (third degree AV block), and the patient was selected for an elective permanent pacemaker (VVIR) implantation. An echocardiography showed normal left ventricular systolic function (ejection fraction = 60%) with Grade 1 left ventricular diastolic dysfunction, mild dilation in most heart chambers , mild left ventricular hypertrophy, moderate mitral regurgitation, and moderate pulmonary hypertension. Laboratory test results prior to the operation were: HGB: 12.8 g/dL, WBC: 11.05 × 102 was 60% in room air, and blood pressure measured 200/120 mmHg, paced heart rate was 60 bpm. The patient was transported immediately to the cardiac care unit and was intubated and put on mechanical ventilation with the ventilation set on synchronized intermittent mandatory ventilation (SIMV) mode. The patient was put immediately on nitroglycerine infusion pump starting with a dose of 3 mg per hour and then modified according to vital signs to overcome the overwhelming pulmonary edema. He was also managed with 40 mg of furosemide and Meropenim (given for the increased WBCs count).The procedure went smoothly and the patient was transferred safely to the recovery room. Sudden severe dyspnea emerged half an hour following the procedure. Upon physical examination, fine crackles were heard all over the chest, SpO2: 34.7 mmHg, PO2: 50.4 mmHg, HCO3: 22 m Eq/L). The patient was monitored closely; SpO2 measurements were between 97% and 100%; Blood pressure measurements dropped soon afterwards and ranged between 150/60 and 180/95 the following 2 days, troponin test was negative; other labs did not show any significant change and WBCs were still increased. After 72 h, vital signs stabilized and the patient was extubated uneventfully. Two days later, an abdominal ultrasound showed signs of chronic kidney disease (CKD) which is the probable cause of elevated kidney function tests; a significant pleural effusion was also detected during the ultrasound, and a right inguinal hernia was also detected. Afterwards, the patient was discharged successfully with a blood pressure of 140/80 mmHg and SpO2 of 98% in room air. He was prescribed furosemide 40 mg QD, spironolactone 25 mg QD, valsartan 160 mg QD, amlodipine 10 mg QD, cefpodoxime 200 mg and ciprofloxacin 500 mg.A Chest X‐Ray (CXR) showed pulmonary hilar congestion with pleural effusion Figure . ArteriaAt 3 months follow‐up, we noticed acceptable improvement of the patient situation with no accompanying complications.3We believe that it is clinically difficult to differentiate SCAPE from acute heart failure symptoms (AHFS) as their symptoms may intersect.Nitroglycerin is being used for both SCAPE and AHFS. AHFS require doses of 10–20 μg/min to decrease preload. On the other hand, using nitroglycerin in SCAPE is mainly targeting the afterload with much higher doses.In our case, high doses of nitroglycerin (starting at 50 μg/min) were given through infusion pump that resulted in symptoms relief. Furosemide was also given as a common standard treatment for APE spectrum. Despite the above, no signs of hypotension were documented throughout the admission. Similarly, Stemple et al. have described case series of four cases of SCARE that were all managed with high doses of nitroglycerin infusions. All four patients achieved symptomatic improvement and required no additional acute pharmacologic therapy or invasive mechanical ventilation.The main target for our case is the put the spot light on the idea of SCAPE potentially complicating pacemaker insertion. We extensively searched the literature to find similar cases. We used PubMed, Google scholar, and EMBASE. We only found one case of the incidence of flash pulmonary edema (another name for SCAPE) after pacemaker insertion.SCAPE is clinical entity that is yet being completely understood in the literature. Our case's clinical presentation was unusual with unpredicted complication arising. Pacemaker insertion is known to have several common complications but SCAPE was only reported once before. We emphasize that clinical suspicion should be taken when similar symptoms to our case appear after pacemaker insertion. Additional caution is advised toward the elderly as we doubt that they should be more susceptible to SCAPE due to their generally poor compensating mechanisms. For now, nitroglycerin should be considered the first line management for SCAPE until more verified studies take place. Diagnosis timing is vital and there should be no delay in recognizing and managing SCAPE. This can be achieved by clinically observing pulmonary congestion signs in patients with postplacement of pacemaker.4SCAPE is an uncommon clinical condition with a poorly known etiology. SCAPE following a pacemaker implantation, as in our patient's case, is exceedingly rare; therefore, it highlights the need of patient strict monitoring following pacemaker insertion and the need for robust data about SCAPE therapy.Ibtehal Othman Hussain Mohammed: Conceptualization; writing – original draft; writing – review and editing. Sarya Swed: Writing – original draft; writing – review and editing. Weaam Ezzdean: Writing – original draft; writing – review and editing. Mohammad Badr Almoshantaf: Writing – original draft; writing – review and editing. Baraa Shebli: Writing – original draft; writing – review and editing. Bisher Sawaf: Writing – review and editing. Abutalib Mohamed Ahmed Hamoda: Writing – review and editing.This research did not receive any specific grant from funding agencies in the public, commercial, or not‐for‐profit sectors.All authors declared no conflict of interest.This case report did not require review by Ethics committee, University of Alemam Almahadi, Sudan.Written informed consent was obtained from the patient for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor‐in‐Chief of this journal.


There is limited evidence on the association between arterial stenosis and the risk of all‐cause mortality in cancer patients (ACMC). This study investigated whether the status of arterial function and structure measured by brachial–ankle pulse wave velocity (baPWV) is associated with ACMC.A total of 43,943 Chinese adults underwent a baPWV examination. Cox proportional hazards model was used to assess the association between the baPWV values and ACMC.p < 0.001) in those with baPWV ≥18 m/s. With a 1‐SD increase in baPWV, the HR (95% CI) for ACMC in the entire cohort, men, and patients ≤60 years old were 1.20 , 1.20 , and 1.44 , respectively.During a total follow‐up duration of 3.81 ± 2.50 years, there were 157 deaths among 553 cancer cases diagnosed during the follow‐up. Patients with baPWV ≥18 m/s showed an increased risk of ACMC compared to patients with ideal vascular function. In the multivariate‐adjusted model, we observed a significant association between arterial stiffness severity and ACMC with a hazard ratio (HR) 2.72  and risk of all‐cause mortality was prominent in men and those ≤60 years of age. The Kailuan study was assessed with questionnaires and clinical and laboratory tests every 2 years. After receiving written informed consent from the participants, the clinical and laboratory data were collected for analysis. The Kailuan cohort study specifics have been documented.n = 455), were diagnosed with serious cardiovascular and cerebrovascular diseases (n = 1684), had incomplete data (n = 261), and had underweight (n = 56). Then, 43,943 patients were further followed up for all‐cause mortality after cancer. During the follow‐up, there were 553 new cancer cases, and they were followed up for all causes of mortality. Figure Further, we excluded those who had a history of cancer  was applied to calculate the body mass index (BMI). After the participants sat for 30 minutes, the right arm was used to measure systolic blood pressure (SBP) and diastolic blood pressure (DBP) three times, and the average reading of the three blood pressure measurements were documented as the SBP and DBP readings. In this study, SBP ≥140 mmHg or DBP ≥90 mmHg, self‐reported history of HTN, or current use of antihypertensive medication were all considered HTN. The formula for determining mean arterial pressure (MAP) was (SBP + 2DBP)/3. Participants were advised to refrain from eating, smoking, and drinking for at least 8 h before a general medical checkup and blood and urine specimen collection. Biochemical analysis was carried out to assess fasting blood glucose (FBG) and high‐sensitivity C‐reactive protein (hs‐CRP) levels. FBG ≥7 mmol/L or self‐reported usage of glucose‐lowering medications indicated DM. A basic enzymatic approach was used to measure blood total cholesterol (TC) and high‐density lipoprotein (HDL) levels. The Chronic Kidney Disease Epidemiology Collaboration method was used to detect the estimated glomerular filtration rate (eGFR) from creatinine. Weight less than 30 kg was deemed underweight. Employing an enzyme‐linked immunosorbent assay (ELISA), the hepatitis B surface antigen (HBsAg) in venous blood was identified, and hepatic dysfunction was identified as alanine aminotransferase or aspartate aminotransferase amounts twice or more than the normal value. Meanwhile, the ankle–brachial index (ABI) was identified by the ratio of SBP recorded at the ankle to SBP obtained at the brachial artery. The absolute difference in SBP between the right and left brachial arteries is called interarm blood pressure differential (IAD). Smoking and drinking habits were self‐reported and categorized as never smokers/drinkers, past smokers/drinkers, or current smokers/drinkers. Physical activity was defined as engaging in any exercise for at least 30 minutes three or more times a week.Each health assessment included the distribution of self‐administration questionnaires to gather information on anthropometry, lifestyle, previous medical history, and patient history of cancer. Weight (kg)/height2.3–25°C for at least 30 minutes. Subjects were requested to lie supine and stay quiet during the assessment. Then we placed blood pressure cuffs on their arms and ankles. While the lower malleolus cuff's bottom edge was 1–2 cm just above the medial malleolus, the inferior margin of the cuff was 2–3 cm just above the fossa cubitus. A cardiac sound monitor was positioned on the left side of the sternum. The assessment was performed twice separately, with the second measurement considered the final outcome. As per the previous study, BaPWV <1400 cm/s was considered ideal vascular function, whereas 1400 ≤baPWV <1800 cm/s was considered as borderline arterial stiffness, and baPWV ≥1800 cm/s was indicated high‐grade arterial stiffness.Between January 2010 and December 2016, baPWV was performed to assess arterial stiffness using the web‐based arterial stiffness detection system BP‐203 RPE III .2.4All cancers were confirmed from hospital diagnostic data depending on the international classification of diseases‐10 (ICD‐10) code. These include cancers of the digestive system (C15–C26), respiratory system (C34 + C39), genitourinary system (C50–C58 in women and C60–C63 in men), and other system cancers . During follow‐up, hospitalization records were utilized to identify deaths. We also screen death certificates from provincial unit demographic offices annually to improve the quality of our data on outcome events. All patients were monitored until the ACMC or December 31, 2018, whichever happened first.2.5p < 0.05 in the univariate Cox analyses or clinically associated with cancer occurrence as candidates for the multivariate Cox regression analysis. We performed bootstrapping procedure in the framework of the Cox regression model to investigate stability and to select factors included in a final model. The proportional hazard assumption in the Cox model was tested and satisfied in all cases using the Schoenfeld residuals (PH hypothesis), which was used to investigate whether the impact of different baPWV levels on ACMC changed over time. To exclude the influence of age, sex, blood flow dynamics (represented by MAP), nutritional status (represented by BMI), metabolic diseases , chronic inflammation (represented by hs‐CRP levels), lifestyle behaviors , and hepatitis B (represented by HBsAg positive history), liver and kidney function, and family cancer history, we employed multivariate Cox proportional hazards regression model to analyze the impact of various baPWV cohorts on ACMC. We further determined the spline curve of baPWV on ACMC to explore whether a positive relationship existed between baPWV and ACMC. The Cox model was repeated after age and sex stratification to inspect the influence of baPWV on malignancy mortality in different age cohorts and sex cohorts. Participants with ABI ≤0.9 and/or IAD ≥15 mmHg at baseline were excluded from the sub‐group analysis to exclude the confounding effects of peripheral vascular disease on arterial stenosis. p < 0.05 (two‐sided test) was considered statistically significant.For the baseline description, the normally distributed variables were described as mean ± standard deviation (SD). ANOVA was employed to investigate the comparison between three or more groups. The categorical variables were represented as counts and percentages (%), and the chi‐square test was used to investigate the comparison between groups. The cumulative incidence of ACMC in different baPWV cohorts was compared by the Kaplan–Meier survival curves. To account for potential confounding effects, we selected variables with 33.1p < 0.05) than those with lower baPWV. Table From January 1, 2010 to December 31, 2018, 43,943 individuals were followed up for the endpoint of ACMC. During a total follow‐up duration of 172,775.69 person‐years, we detected 553 new cancer cases. Of them, 157 died. The baseline data show that the average age was 47.84 ± 12.74 years old, and 31,554 (71.81%) were male. Participants with higher baPWV had higher age, SBP, DBP, MAP, BMI, TC, HDL, FPG, UA, and eGFR. In addition, the participants with higher baPWV had a higher proportion of HTN, DM, current drinkers, current smokers, physical activity, and hepatic dysfunction . We observed significant variations in ACMC in the entire population, including in the digestive, respiratory, and urogenital systems. However, ACMC has no significant variations across the different baPWV groups in the “other systems.”There were 157 ACMC  in 153,889.58 person‐years follow‐up time. Figure 3.3p < 0.001) among those with baPWV ≥18 m/s . We observed HR of 1.20  per 1‐SD increase in baPWV among the entire cohort. Likewise, the HR of 1.44  and 1.20  in those less than 60 years old and men population , men , participants ˂60 years , and those with cancer of digestive system . The Schoenfeld residuals analysis confirmed no linear association between Schoenfeld residuals and time rank, as demonstrated by the corresponding p‐value of Pearson correlation  and other cohorts, including men (p = 0.0816), participants <60 years of age (p = 0.0317), and digestive cancer patients (p for trend = 0.1602) , the gold standard measurement for arterial stenosis, to test the association between arterial stenosis and all‐cause mortality. Therefore, we failed to validate the observed relationship between baPWV and all‐cause of death by replicating the analysis using c‐fPWV. Also, our study only registered the Chinese population living in Northern China, which may limit the generalization of our results. However, the homogeneous nature of our prospective study may provide absolute control of potential confounding effects that could result from racial disparities and health service inequalities. The Kailuan community cohort recruits study participants from a coal company. Therefore, more males than females were included in the study. Though the enrolment of only Chinese ethnicity in this study could bolster its internal validity, further research is needed to confirm whether this association exists among other ethnic groups.5In conclusion, the results of this study demonstrate a positive association between high baPWV and ACMC, with a potentially novel correlation. The association between high baPWV (≥18 m/s) and risk of all‐cause mortality was prominent in men and those ≤60 years of age. Future studies must clarify whether interventions that decrease arterial stiffness reduce or delay deaths among cancer patients. This study provides insight into the benefit of preventing arterial stenosis.Jiatian Li: Conceptualization ; methodology ; software . Tesfaldet Habtemariam Hidru: Data curation ; writing – original draft . Yajuan Lin: Investigation (lead); validation (lead). Xinying Wang: Investigation ; validation . Li Lin: Investigation (lead); validation (lead). Shuohua Chen: Supervision . Yunlong Xia: Software ; validation . Xiaolei Yang: Conceptualization ; methodology ; software . Shouling Wu: Project administration ; writing – review and editing .This work was supported by the Chang Jiang Scholars Program (T2017124 to YX); the National Natural Science Foundation of China (81900439 to XY), and Liao Ning Revitalization Talents (XLYC2002096 to YX); the Science Foundation of Doctors of Liaoning Province (2020‐BS‐197 to XY).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.Each individual signed a written agreement statement and consented to use the information for analysis. The Kailuan General Hospital Ethics Committee authorized the investigation, ensuring the study procedure complied with the Declaration of Helsinki II requirements.Clinical trial number ChiCTR‐TNRC‐11001489.According to World Health Organization (WHO), cancer is a leading cause of death worldwide. In parallel, the crude incidence and mortality rates of cancer have progressively risen since 2000 in China. According to the most recent epidemiological and statistical information, China is expected to have 3,210,000 cancer deaths and 4,820,000 new cases of malignancy in 2022. Therefore, there is an urgent need to discover risk factors influencing cancer mortality initiation and progression. Arterial stiffness is a well‐established indicator of the rigidity of the artery wall. It can be determined by a simple and noninvasive procedure known as the brachial–ankle pulse wave velocity (baPWV). Of note, baPWV is defined as the distance between the brachial and the tibial artery divided by the transit time between these two arteries. The relationship between arterial stiffness and deaths in people with Type 1 diabetes and hypertension has been investigated in the past. Similarly, a community‐based study showed that arterial stiffness was positively associated with all‐cause mortality in elderly Japanese individuals. Therefore, the purpose of this study was to investigate the association between arterial stiffness status measured by baPWV and all‐cause mortality in Chinese cancer patients.Figure S1.Click here for additional data file.


Assessing the incidence of Medication Related Osteonecrosis of the Jaw (MRONJ) in cancer patients with bone metastases receiving Denosumab (Dmab) and identifying potential risk factors.A retrospective observational study on consecutive cancer patients with bone metastases, who received at least one dose of Dmab and one follow‐up visit. MRONJ crude cumulative incidence (CCI) was estimated considering death without MRONJ as competing event. Multiple regression models were used to study the association between MRONJ incidence and potential risk factors: age, cancer diagnosis, previous bisphosphonates, dental treatments before starting Dmab, extraction or other dental treatment during Dmab, chemotherapy, hormone therapy, and antiangiogenic (AA) agents concurrent use.p = 0.029 and 0.0106). Dental treatments , dental extractions , and previous BP administration  were significantly associated with higher MRONJ incidence at multivariate Cox analysis. Although not statistically significant, MRONJ incidence was lower for patients receiving chemotherapy or hormone therapy and higher for those receiving AAs.On 780 patients included (median follow‐up 17 months), 54% and 18% had, respectively, breast and prostate cancer. The mean number of Dmab administration was 12. Fifty‐six patients developed MRONJ with a 24‐ and a 48‐month crude cumulative incidence of 5.7% (95% Cl: 4.2%–7.8%) and 9.8% (95% CI: 7.6%–12.7%), respectively. Higher MRONJ incidence was significantly associated with middle aged group (>56 and ≤73), both at univariate and multivariate analysis (The results confirm a clinically relevant incidence of Dmab‐induced MRONJ. Dental treatments, especially extraction, during and before Dmab, constitute a serious risk factor. The role of AA concurrent administration deserves further investigations. Evidences indicate that Dmab could perform better than BP for SRE prevention,The aim of the present study is to assess the incidence of MRONJ in oncological patients with bone metastases receiving Dmab and to evaluate the role of potential, concomitant local or systemic risk factors.22.1This is a retrospective observational study carried out on consecutive cancer patients with bone metastases attending the Palliative Care and Pain Outpatient Clinic at a tertiary oncological center from April 2013 to September 2018. Patients who had received at least one dose of Dmab and had at least one follow‐up visit were eligible.2.2All patients received 120 mg of subcutaneous Dmab every 28 days. Patients were scheduled to receive at least 24 administrations but this could be reduced or extended depending on disease characteristics and occurring side effects.examination by a dental specialist, with expertise in the clinical management of MRONJ, to exclude the need for dental treatments or the presence of risk factors for MRONJ. A panoramic dental x‐ray was also requested before the examination. When needed, a preventive dental treatment  was performed before starting Dmab. In this case, Dmab administration was delayed for at least 4–6 weeks after the intervention and depending on the type of intervention, prophylactic antibiotic therapy was prescribed,regular maintenance oral hygiene recommendations.Before starting Dmab, all patients underwent preventive dental care, including:Blood levels of calcium and vitamin D were also tested, and in case of abnormal values, supplementary therapies were administered before starting Dmab.During Dmab treatment, blood calcium and vitamin D levels were regularly checked every 4 weeks and clinical dental examination together with an panoramic dental x‐ray (when required by the dentist), were performed every six administrations.2.3MRONJ clinical diagnosis was based on the guidelines of the American Association of Oral and Maxillofacial Surgeons.2.4The list of patients who had received at least one dose of Dmab during the study period was obtained from the hospital administrative database. The following baseline data were retrieved from the electronic medical records: age, sex, primary tumor diagnosis, previous use of BP and, in this case, type and date of last BP administration, previous history of MRONJ, dental treatments before starting Dmab, presence of diabetes and previous administration of antiangiogenic agents (AA). Data related to the Dmab administration period included concurrent chemotherapy and hormone therapy, corticosteroids therapy for at least three consecutive months, anemia , dental treatment received and type, as well as number of Dmab administrations.For patients who developed MRONJ, the following data were recorded: MRONJ site, MRONJ staging, MRONJ treatment , outcome  and whether treatment with Dmab or BP was resumed.The study was approved by the institutional research Ethics Committee (identification number INT 191/18). Due to the retrospective nature of the study, informed consent was not requested.2.5Analyses of association between MRONJ occurrence and baseline variables were performed by estimating MRONJ crude cumulative incidence (CCI) curves. Time was computed from the date of Dmab start and MRONJ or death in the absence of MRONJ (competing event), whichever occurred first. In the absence of MRONJ or death, time was censored at the date of the last follow‐up. Univariate comparison between CCI curves according to categories of (time independent) baseline variables was performed using the Gray test.One important variable to be taken into account in the multivariate Cox model was the number of DS doses, that is a time‐dependent variable continuously changing its values with time, differently from the other above mentioned time‐dependent variables , that only change the value once. Since investigating its role was not the main aim of our analysis, the DS dose number was included in the multivariate Cox model as fixed (not time‐dependent) baseline variable, that can be methodologically acceptable if considering it as an adjustment factor.The intrinsic nature of time‐dependent variables hampered estimation of CCI curves which would treat them as baseline variables. In the Cox models, deaths in the absence of MRONJ were considered censored observations; this allowed to estimate the MRONJ‐specific hazard ratios (HR) and corresponding 95% confidence intervals (95% CI). Patient's age was modeled as continuous variables using three‐knot restricted cubic splines.https://cran.r‐project.org, last access 19 July, 2023, R version 4.2.2 Copyright (C) 2022 The R Foundation for Statistical Computing).The analyses were carried out using R software  were lost to follow‐up after one single dose and were not included in this analysis. Baseline demographic and clinical characteristics of the 780 patients included are described in Table Nineteen percent of the patients (148) had received prior therapy with oral or intravenous BP: 96% zoledronic acid, 4% intravenous or oral ibandronate. The median time (interquartile range ‐IQR) between BP last and Dmab first administration was 2.8 (1.1–14.6) months, with 26 patients starting <1 month after. Four patients had previously developed MRONJ, already resolved before starting Dmab, none of them developed MRONJ after Dmab. Seventy‐one patients out of the 780 analyzed (9%) had undergone preventive dental treatments such as oral hygiene, root canal treatment or extraction, before starting Dmab.The median number of Dmab administrations was 12 (IQR: 6–24). The median treatment duration was 12.4 (IQR: 5.1–23.7) months.Table 3.2Median follow‐up was 37.3 IQR: 21.4–53.9) months, and was slightly different in patients without and with prior BP (34.6 [20.0–51.4] months and 52.4 (35.7–60.4) months, respectively). Fifty‐six patients developed MRONJ out of 780 (7.15%) with a 24‐, 36‐, and a 48‐month crude cumulative incidence of 5.7% (95% CI 4.2%–7.8%), 7.9% (95% CI 6.0%–10.4%), and 9.8% (95% CI 7.6%–12.7%), respectively ; younger (≤56 years old) and older (>73) patients presented with similar MRONJ incidence, which was lower as compared with the middle age class (>56 and ≤73)  Figure . None of) Figure .p = 0.0972; Figure p = 0.0636; Figure p = 0.1659; Figure p < 0.0001 for both). Concomitant or follow‐up AA drug assumption was not significant (p = 0.1369) but, noteworthy, it was associated with doubled MRONJ risk.Despite a non‐significant test result, good separation between the incidence curves, suggesting a potential clinical association with MRONJ incidence, was shown for tumor site . In the same Cox multivariate model, the association between MRONJ incidence and the number of Dmab doses was statistically significant with lower doses associated with higher MRONJ risk .Cox multivariable analysis Table  and demo3.2.1Among 56 patients developing MRONJ, the mandible was the most common site (64%), followed by the maxilla (32%) and both in only 2 cases (4%). In three cases (6%), a stage 0 MRONJ was diagnosed, whereas 24 patients (43%) had stage 1 of MRONJ, 26 (45%) stage 2, 2 (4%) stage 3 MRONJ, and in one patient the staging of MRONJ was not available. As specified in the methods section, the AAOMS staging system was used.After MRONJ diagnosis, four patients were lost to follow‐up. The remaining 52 patients, 40 (77%) underwent ozone therapy, 27 (52%) surgery with sequestrectomy of the necrotic area and three patients were still awaiting treatment Table .At the end of the follow‐up period, 23 patients were still undergoing MRONJ treatment or lost to follow up, while 29 had completed MRONJ treatment and reported full MRONJ resolution. After MRONJ resolution, nine of these patients resumed Dmab (five patients) or BPs (four patients). The decision to resume BMAs was made based on the disease characteristics, symptoms presented, and patient's preferences. Specifically, in seven patients with progressive bone disease and two with hypercalcemia, treatment was proposed and resumed with their consent. In 20 cases, treatment was withdrawn, in seven of them because of worsening clinical conditions associated with disease progression, in other seven who had stable bone disease treatment was not proposed, while six patients with indication to resume BMAs refused.4In this study, we have investigated the incidence of MRONJ in patients receiving Dmab for bone metastases and described factors associated with a higher incidence. Out of 780 patients, 56 developed MRONJ, with a 24‐ and 48‐month crude cumulative incidence of 5.7% (95% CI: 4.2%–7.8%) and 9.8% (95% CI: 7.6%–12.7%), respectively.Literature data on MRONJ incidence are variable.p = 0.0001), especially dental extraction  which was found to be the most common event preceding MRONJ onset. In fact, 28 (50%) out of the 56 MRONJ patients had previously had at least one extraction.Similarly to previous reports,p = 0.0106) and previous BP administration (p = 0.0024) were also identified as significantly associated factors. However, results on the significance of previous BP administration and MRONJ incidence from other studies are mixed, although there is some evidence suggesting a higher prevalence of MRONJ for sequential BP‐Dmab therapy.Patients age  Figure . PreviouWe did not find any statistically significant sex differences, unlike a previous study that showed a correlation between MRONJ incidence and sex,To the best of our knowledge, this study is among the few estimating the incidence of MRONJ among cancer patients receiving Dmab in clinical practice, and despite the limitations due to its retrospective and single‐center nature, the sample size is considerable. One of the main results is the strong association between MRONJ onset and dental extraction or other dental intervention. The diagnosis of MRONJ was made by a dental specialist, experienced in the diagnosis and management of MRONJ, using the staging system of the AAOMS guidelines.5Incidence of MRONJ according to our data study is higher than that reported in the published guidelines.Given the continuous spread of the application of new target therapies, it will be important to establish if and how they can contribute to MRONJ development. Prospective and larger sample studies are required for better understanding the role of local and systemic risk factors that favor MRONJ development.Paola Bracchi: Conceptualization ; data curation (lead); investigation (lead); methodology (supporting); writing – original draft ; writing – review and editing . Ernesto Zecca: Conceptualization ; data curation (supporting); investigation (supporting); methodology (supporting); validation (lead); writing – original draft ; writing – review and editing . Cinzia Brunelli: Conceptualization ; data curation (supporting); formal analysis (supporting); methodology (supporting); project administration (lead); visualization (supporting); writing – original draft ; writing – review and editing . Rosalba Miceli: Conceptualization (supporting); data curation (supporting); formal analysis (lead); methodology ; visualization (lead); writing – review and editing . Gabriele Tinè: Formal analysis (supporting); methodology (supporting); visualization (supporting); writing – review and editing . Massimo Maniezzo: Conceptualization ; data curation ; investigation (supporting); validation ; writing – original draft (supporting). Silvia Lo Dico: Data curation (supporting); investigation (supporting); writing – review and editing . Mariangela Caputo: Data curation (supporting); investigation (supporting); writing – review and editing . Morena Shkodra: Conceptualization (supporting); formal analysis (supporting); writing – original draft ; writing – review and editing . Augusto T. Caraceni: Conceptualization ; formal analysis ; project administration (supporting); resources (lead); supervision (lead); writing – original draft (lead); writing – review and editing .This research did not receive any specific grant from funding agencies in the public, commercial, or not‐for‐profit sectors.AC has received honoraria from Angelini, Shionogi, Kyowa Kirin, Molteni, Pfizer/ Eli Lilly Italia Spa, Mundipharma. EZ has received honoraria from Amgen. All other authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.Figure S1.Table S1.Click here for additional data file.