Polysomnography (PSG) is the gold standard for the diagnosis of obstructive sleep apnoea (OSA). Home sleep apnoea testing with peripheral arterial tonometry (PAT) is a recommended diagnostic alternative for patients with an increased risk for OSA.In a large clinical cohort, we investigated concordance and predictors for discordance in diagnosing OSA using PAT and PSG, and three-year cardiovascular risk in patients with discordant OSA diagnosis.Retrospective monocentric cohort study. Patients with a PAT AHI ≥ 5/h followed by an in-hospital PSG within three months were included. All patients with a PAT AHI ≥ 5/h but a PSG AHI < 5/h were classified as discordant. Patients with PAT and PSG AHI ≥ 5/h were classified as concordant. To ascertain cardiovascular risk, major adverse cardiovascular events (MACE) were analyzed in discordant patients and sex, age, body mass index (BMI) and cardiovascular disease-matched concordant patients over a follow-up time of 3.1 ± 0.06 years.2 were included. Agreement in OSA diagnosis was observed in 80% of patients (55% in mild and 86% in moderate and severe OSA). Factors significantly associated with a discordant diagnosis were female sex, younger age and lower BMI, but not comorbidities. There was no significant difference in MACE (p = 0.920) between discordant patients (n = 155) and matched concordant patients (n = 274) with or without therapy.A total of 940 patients, 66% male with an average age of 55 ± 0.4 years and BMI of 31 ± 0.2 kg/mConcordance between PAT and PSG diagnosis of sleep apnoea is good, particularly in moderate and severe OSA. Predictors for discordant results between PAT and PSG were age, sex and BMI. MACE risk is similar in those with OSA diagnosed by PAT or PSG.The online version contains supplementary material available at 10.1186/s40001-023-01164-w. Obstructive sleep apnoea (OSA) is an important but frequently undiagnosed source of morbidity and mortality , 2. It iIn-laboratory overnight polysomnography (PSG) is generally referred to as the gold standard for the diagnosis of sleep apnoea but availability is limited due to both its complexity and cost . The AmeWe hypothesized that PAT and PSG identify the same patients as having OSA. In this study we therefore assessed the accuracy of positive PAT (AHI ≥ 5/h) compared to PSG in a large clinical cohort. We also aimed to determine predictors of discordant diagnosis of sleep apnoea between PAT and PSG. Finally, using sex-, age-, body mass index- and cardiovascular disease-matched patients, we analysed three-year cardiovascular risk in discordant patients.This is a retrospective, monocentric study performed at the Clinic of Respiratory Medicine and Pulmonary Cell Research at the University Hospital of Basel, Switzerland. The Study was approved by the Ethics Committee northwest/central Switzerland (EKNZ 2018-01789) and was carried out according to the Declaration of Helsinki and Good Clinical Practice guidelines. Only patients who provided written informed consent for data analysis were included in the study.Data from patients with a PAT AHI of ≥ 5/h between January 2016 and December 2019 and a follow-up PSG within 3 months of the PAT were included in the study  is a four-channel device worn around the wrist with a finger PAT sensor. The WatchPAT software zzzPAT version 4.4.64.4 was applied. The threshold for apnoea-hypopnoea index (AHI) and respiratory disturbance index (RDI) was a drop in oxygen saturation of ≥ 3%. The oxygen desaturation index (ODI) threshold was automatically set at an oxygen desaturation of ≥ 4%. In contrast, an oxygen desaturation of ≥ 3% is standard for setting the ODI in PSG.All patients underwent in-laboratory overnight PSG. PSG was performed and scored (by a trained sleep physician) according to the AASM 2012 criteria . Sleep a2, 18.5–24.9 kg/m2, 25–29.9 kg/m2, ≥ 30 kg/m2) and cardiovascular disease  were sourced from the concordant patients. In these matched discordant and concordant groups, all available data for the follow-up period until 31 March 2021 were collected. This included use of PAP therapy , hospitalizations, MACE and mortality. MACE were defined as fatal and non-fatal cardiovascular events leading to hospitalization, including stroke, transient ischemic attack, myocardial infarction, hypertensive emergency as well as acute decompensated heart failure.In the analysis, all patients with a positive PAT (AHI ≥ 5/h) but negative PSG (AHI < 5/h) were classified as discordant. Patients with a positive PAT (AHI ≥ 5/h) and PSG (AHI ≥ 5/h) were classified as concordant. For discordant patients 1–2 comparable controls matched for sex, age (± 3 years), BMI . A p < 0.05 was considered significant, and all tests were two tailed. Group comparisons were performed using the Chi-squared test if the variables were categorical and using the Mann–Whitney U-test if continuous. The agreement between PAT and PSG measurements was evaluated using the Lin’s concordance [Statistical analyses were performed with cordance  coefficin = 7295) in our clinic compared to polysomnography was also due to regular PAT follow-up examinations of patients under PAP therapy.Between January 2016 and December 2019, 7295 PAT were performed of which 940 patients were included for analysis . 60 measurements were performed in hospitalised patients, but not in the sleep laboratory. The patient population was predominantly male (66%). The average age and BMI were 55 ± 0.4 years and 31 ± 0.2 kg/m2, respectively (Table n = 362/754) of the patients as assessed with an ESS of ≥ 10 points. Various comorbidities were present in the patient population. The most prevalent being arterial hypertension (57%) followed by diabetes mellitus (17%) and depression (16%).The vast majority of the PAT measurements included was performed at home  was confirmed by PSG in 748 patients (80%) Table . Sleep an = 362/754) based on PAT and 38.6% (n = 291/754) based on PSG  or PSG AHI .Using an ESS ≥ 10 points as an indicator of symptomatology and an AHI ≥ 5/h, we calculated the prevalence of obstructive sleep apnoea syndrome (OSAS) in our population. It was 48.0% (n = 56/940) of the data points fall outside the limits of agreement which were set at mean + 1.96 SD and − 1.96 SD. Further, it is shown that the PAT AHI measurements are in general higher (AHI + 8.6 ± 0.6/h) than that of PSG  indicating a poor to moderate agreement between both sleep studies. The Pearson correlation between PAT and PSG AHI was 0.654 Fig. a. In a BPSG Fig. b.Fig. 2Cn = 111/202) had sleep apnoea (of any severity) in the PSG, whereas when the PAT classified sleep apnoea as moderate or severe, the concordance with the PSG diagnosis of sleep apnoea was 86% .In contrast to an overall good agreement in the diagnosis of sleep apnoea, our data showed differences in the classification of sleep apnoea severity between the two sleep studies. Based on PAT results, there was 37% (75/202) concordance for mild, 28% (93/330) for moderate, and 46% (189/408) for severe sleep apnoea Sleep variables assessed with the two sleep studies are shown in Table For 155 patients with discordant diagnosis for sleep apnoea between PAT and PSG (matched discordant), we found 274 controls (matched concordant) matched for age, sex, BMI and cardiovascular disease. There was a significant difference in baseline ESS score, coronary artery disease and liver disease between the groups  were protected against MACE and death (of any cause) compared to matched concordant cases without treatment (n = 122), p = 0.03.The mean follow-up time was 3.1 ± 0.66 years. We detected 22 MACE, which is equivalent to an average of 1.5 MACE per 100 person years. There was no significant difference in the occurrence of MACE between matched concordant and matched discordant patients with or without prescribed PAP therapy Table . Kaplan p < 0.001), sex (p < 0.001), BMI (p < 0.001), arterial hypertension (p < 0.001), renal disease (p = 0.012), asthma (p = 0.044) and depression (p = 0.046). In a multivariate analysis adjusting for these seven parameters, only female sex (p < 0.001), younger age (p < 0.001) and lower BMI (p < 0.001) were statistically significant predictors of discordance.Factors significantly associated with a discordant or concordant diagnosis of sleep apnoea between PAT and PSG were age  and congestive heart failure were significantly associated with AHI as assessed by PAT but not with AHI as assessed by PSG. Diabetes mellitus and atrial fibrillation were associated with both PAT AHI and PSG AHI Table .Table 5ATo date, this is the largest study comparing PAT with in-laboratory PSG in a representative clinical cohort of 940 patients. We show that PAT has a good concordance of 80% with PSG in diagnosing sleep apnoea. PAT AHI was on average higher than that of PSG (AHI + 8.6 ± 0.6/h). Several factors, including age, sex, and BMI, but not comorbidities, predicted diagnostic discordance between the two sleep studies. The MACE rate was similar in those with OSA diagnosed by PAT or PSG.In most studies examining the accuracy of PAT, PSG was performed concurrently. Our sleep studies were performed on two different nights within a three-month period, which is in line with clinical practice when PSG is performed to objectify sleep apnoea diagnosis. This clinical approach explains why the correlation coefficient for AHI (PAT/PSG) in our study was 0.595 and markedly lower than reported in other studies , 31. NevRelevant night-to-night variability of respiratory events must be considered if the sleep studies are not performed synchronously –40. PosiThe pronounced differences in our study could also be partly related to a selection bias. We retrospectively analyzed real-life data reflecting medical decision making with patients. In our clinic, PAT was generally used as a screening device for patients with suspected OSA. Based on symptoms, comorbidities, patient preferences and PAT results, further diagnostic testing with PSG was discussed. OSA treatment was rarely initiated without confirmatory PSG. It can be assumed that there is a selection for more symptomatic patients with comorbidities and abnormal PAT findings.Patient characteristics that were significantly associated with discordance in the diagnosis of sleep apnoea between PAT and PSG included sex , younger age and lower BMI. The influence of comorbidities on the validity of the PAT assessment is still controversial. While Kinoshita et al. demonstrated a limited AHI correlation with increased arterial stiffness , Zhang eDiscordance in sleep apnoea diagnosis was particularly high in mild sleep apnoea around the AHI cut-off value of 5/h, so it is not surprising that younger age and lower BMI were associated with diagnostic divergence. Based on several population prevalence studies women  tend to have a lower mean prevalence of OSA than men , 51.The AASM recommends HSAT as a diagnostic test for OSA in uncomplicated patients with an increased pre-test probability . IncreasIn our clinical cohort, PAT led to a diagnosis of sleep apnoea in 940 patients, 20% of whom had a negative PSG and were classified as discordant. Performing a follow-up we analysed whether these discordant patients showed a different cardiovascular risk. Our data indicate a similar MACE risk in matched-discordant  and matched-concordant  patients with or without PAP therapy. In the context of this study, we could not distinguish whether the impact of OSA (PSG AHI > 5/h) was too small to cause a significant difference in cardiovascular outcomes , 35 or wThe present study has some limitations. Since PAT was used to screen for OSA, we excluded patients with a PAT AHI < 5/h for further analysis. Therefore, we cannot make any reliable statement about the sensitivity or specificity. In 30 patients with a negative PAT a PSG was performed within 3 months due to increased clinical suspicion of sleep apnoea . In this subgroup 8 (27%) had mild and 3 (10%) had moderate OSA. No severe sleep apnoea could be objectified in this subgroup. Comparable data for negative PAT were found in the PATER study . PreviouIn our mean follow-up period of 3.1 ± 0.06 years, MACE occurred at an average of 1.5 events per 100 person years, which is lower than in other studies of sleep apnoea patients in which 2.1–4.5 events per 100 person years were observed , 53. ConHome sleep apnoea testing with PAT is increasingly used in clinical studies and practice for the diagnosis of OSA. In a representative, heterogeneous and large clinical population we found a good concordance of 80% in sleep apnoea diagnosis with a consecutive PSG, which increased to 86% with a PAT AHI of ≥ 15/h. In a retrospective follow-up of MACE, PAT positive (AHI ≥ 5/h) and PSG negative (AHI < 5/h) patients showed a similar cardiovascular risk as patients with PSG-confirmed OSA.Additional file 1:Table S1. Sleep apnoea syndrome calculated using AHI ≥5 or 15 and ESS ≥10. Table S2. Descriptive statistics for patients characteristics stratified by matched discordant and concordant cases. Table S3. Comparison of sleep parameters between matched concordant and discordant patients stratified by measurement method used.


Immune checkpoint inhibitors (ICIs) have significantly improved survival in advanced melanoma but are associated with immune‐related adverse events (irAEs). This single center, cross‐sectional survey aimed to describe the long‐term symptom burden and impact on health‐related quality of life (HRQL) of advanced melanoma patients with sustained disease control following treatment with ICIs.Advanced melanoma patients , treated with anti‐PD1‐based ICIs, who were off‐treatment and had at least 6 months follow‐up from their last infusion with an ongoing response in the metastatic setting or no evidence of disease recurrence in the adjuvant setting. A paper‐based questionnaire, consisting of the EORTC QLQ‐C30, EORTC QLQ‐FA12, and the PRO‐CTCAE was administered.n = 12, 20%). In the PRO‐CTCAE questionnaire, muscle ache  and joint ache  were commonly reported. Despite this, participants reported overall good health  and reasonable level of HRQL .Of 90 participants, 61 (68%) completed the questionnaire; 40 received single‐agent anti‐PD1, and 21 anti‐PD1/anti‐CTLA4. Thirty‐three (54%) were treated in the adjuvant setting. At the time of enrolment, 31 (51%) participants had active treatment for a previous irAE. Overall, 18/61 (30%) participants reported long‐term symptoms and trouble in physical and emotional functioning. Physical fatigue was common and interfered with daily activities (Melanoma survivors experience long‐term symptoms in physical and psychosocial HRQL domains after ICI treatment. These results underline the importance to address existing gaps in survivorship care, implement these findings in clinical practice and increase awareness for long‐term symptoms in these patients. Previous studies collecting patient‐reported outcome measures (PROMs) in people with advanced melanoma have demonstrated multiple symptoms including arthralgias, muscle ache and generalized pain or discomfort.In the present study, we aim to describe the symptom burden and impairments in HRQL in melanoma patients treated either in the adjuvant or in the metastatic setting, with sustained disease control and without disease recurrence for at least 6 months after the last infusion of anti‐PD1‐based ICIs using PROMs. We further analyze these results according to treatment type, in order to unravel the long‐term symptoms of patients that discontinued treatment due to irAEs.2This is a single‐institution, cross‐sectional study with both retrospective chart review and prospective data collection. The primary objective of the study was to describe the symptom burden and HRQL in patients alive and off‐treatment, with sustained disease control at least 6 months after the last dose of ICIs. The secondary objectives were to assess the differences in the symptom burden and HRQL according to ICI treatment type, age and sex. Ethics approval and consent to participate was obtained from the local ethics committee . Patients' enrollment followed upon collection of written informed consent.2.1Patients diagnosed with advanced melanoma  and treated either with single‐agent anti‐PD1 or anti‐PD1 in combination with anti‐CTLA4 (ipilimumab/nivolumab) were included. Of note, there was one patient with locally advanced mucosal melanoma (stage IIB) that was treated with single‐agent anti‐PD1. Patients were treated according to the proposed recommendations for the management of advanced melanoma and upon interdisciplinary tumorboard discussion; patients in the adjuvant setting were treated with single‐agent anti‐PD1, while patients in the metastatic setting received either single‐agent anti‐PD1 or anti‐PD1/anti‐CTLA4.2.2Patients' enrollment was performed between January 2022 and July 2022 (data cut‐off). Institutional database for medical records was reviewed to identify eligible participants. Participants identified as eligible were contacted by phone or approached in the clinic.2.3Baseline patient characteristics, including sex, age, ECOG performance status (PS), presence of autoimmune diseases and active immunosuppression, were retrieved from the available medical records. Disease and treatment characteristics, as well as type and grade of irAEs and reason for treatment discontinuation, were retrospectively collected. In patients that discontinued treatment due to irAEs, duration of symptoms after ICI discontinuation, and onset of new toxicities while off‐treatment were documented. In patients that discontinued treatment for reasons other than irAEs , onset of new toxicities while off‐treatment and duration of toxicities were also collected. The severity of irAEs was graded according to the Common Terminology Criteria for Adverse Events version 5 (CTCAEv5).2.4Study participants received a study‐specific, paper‐based questionnaire. The questionnaire consisted of the validated questions provided by the European Organization for Research and Treatment of Cancer (EORTC) and included the EORTC QLQ‐FA12 questionnaire, measuring the cancer‐related fatigue, and the EORTC QLQ‐C30 questionnaire, developed to assess the quality of life of cancer patients.2.5p‐value <0.05 was considered statistically significant. The questionnaire responses were evaluated using descriptive analyses.Demographic and clinical characteristics were summarized using descriptive statistics, including mean (range) and frequency (percentage). For the statistical analysis, results from the EORTC questionnaire were: not at all/very little (1–2) and quite a bit/very much (3–4). For the PRO‐CTCAE questionnaire, results were collapsed: none/mild (1–2), moderate (3) and severe/very severe (4–5). Subgroup analysis according to treatment agent, sex and age, with a cut‐off of 65 years, in accordance with melanoma phase 3 clinical trials , was performed. Differences between the treatment groups were compared using Fisher's exact test or Pearson's chi squared test for discrete variables , and Wilcoxon rank sum test for continuous variables . All analyses were conducted using statistical language R version 4.2.1 . A two‐sided 3N = 4, 14%) or distress due to confronting questions .Of the 177 patients considered for eligibility, 99 patients fulfilled the eligibility criteria and were subsequently approached for participation in the study. Of the 90 patients reached by phone, 61 patients completed the questionnaire, corresponding to a survey response rate of 68%  or irAEs . The median time of follow‐up (FU) after treatment discontinuation was 26 months (range 6–73 months).Baseline participant characteristics are summarized in Table 3.2, available online). At the time of enrolment, 31 (51%) patients had either ongoing symptoms or required active treatment for a previous irAE. Endocrinopathies were commonly associated with ongoing active treatment and included thyroiditis/hypothyroidism (20%), adrenal insufficiency (7%), hypophysitis (5%), and type 1 diabetes (2%). Notably, patients with ongoing endocrine irAEs were more likely to report fatigue in the PRO‐CTCAE (p = 0.01) and EORTC QLQ‐FA12 questionnaires . Table irAEs of any grade occurred in 56 92%) patients during treatment; 35 (88%) treated with anti‐PD1 and 21 (100%) with ipilimumab/nivolumab. Twenty‐one patients (34%) of the overall population experienced grade 3–4 irAEs; 17 (81%) were treated with ipilimumab/nivolumab and four (10%) with single‐agent anti‐PD1. In patients treated with ipilimumab/nivolumab, the most common irAEs included dermatitis (52%), hepatitis (52%) and colitis (43%), whereas anti‐PD1‐related irAEs included thyroiditis (30%), hepatitis (23%), and dermatitis (23%). In 18 (30%) patients, irAEs led to treatment discontinuation and common irAEs included pneumonitis (17%), arthritis (17%), and nephritis (11%) (Table % patient3.3p = 0.01). Symptoms and problems associated with tiredness and fatigue were common between the two treatments. In all, patients treated with ipilimumab/nivolumab rated their HRQL with a lower overall score compared to those treated with anti‐PD1 .In the questions investigating the overall health and HRQL in the EORTC QLQ‐C30 questionnaire, patients rated their overall health during the past week with 6.00  and their overall HRQL during the past week with 6.00   and having trouble getting things started . The level of fatigue interfered with daily activities in 12 (20%) patients. The level of fatigue did not differ significantly in patients who received ipilimumab/nivolumab than in those who received single‐agent anti‐PD1. Nevertheless, overall fatigue was more prominent in the former than the latter. Notably, although physical fatigue was common in both treatments, emotional fatigue was higher in patients treated with ipilimumab/nivolumab  and cognitive fatigue in patients treated with single‐agent anti‐PD1 . Other differences were noted in the proportion of patients that reported exhaustion  and interference of tiredness with daily activities . Results from the Cancer Related Fatigue Module (EORTC QLQ‐FA12) are summarized in Table Fatigue, and particularly physical fatigue, was a common symptom with frequency that was overall similar across the different questionnaires. In the QLQ‐FA12 questionnaire, 46/61 (75%) patients reported some extent of fatigue, and the corresponding frequencies were 36/61 (59%) in the QLQ‐C30 and 37/61 (61%) in the PRO‐CTCAE questionnaires. In the overall population, 14 (23%) patients reported lack of energy, 13 (21%) exhaustion and 14 (23%) sleepiness during the day. Other symptoms included feeling slowed down , joint ache , and generalized pain , whereas sadness  and depression  were also occasionally reported , joint ache , and decreased sexual interest . Although there were no significant differences with regards to their frequency and severity between the two treatment regimens, patients treated with ipilimumab/nivolumab were more likely to report severe skin itchiness (24% vs. 8%), joint ache (24% vs. 15%), fatigue (24% vs. 12%), and decreased sexual interest (24% vs. 15%). In contrast, severe muscle ache was more prominent in single‐agent anti‐PD1 (5% vs. 20%). Similarly, rates of difficulties that interfered with physical activities did not differ between ipilimumab/nivolumab and single‐agent anti‐PD1, but generalized pain (10% vs. 15%) and muscle ache (5% vs. 15%) were commonly noted in single‐agent anti‐PD1 and joint ache (19% vs. 10%) in ipilimumab/nivolumab.The most commonly reported symptoms on the PRO‐CTCAE questionnaire were muscle ache  than in men   and muscle ache  than men. Nevertheless, symptoms were not significantly different when analyzed according to age, although muscle ache significantly interfered with physical activities in patients ≥65 years (p = 0.04).The results in the overall health and HRQL in the EORTC QLQ‐C30 and EORTC QLQ‐FA12 questionnaires did not differ when analyzed according to age and sex (results not shown). On the PRO‐CTCAE questionnaire, joint ache was more frequent in women  and especially fear of melanoma recurrence or disease progression have been previously reported in approximately 30–80% of the patients.We recognize existing study limitations, which include the single‐center study design, as well as the modest sample size (response rate to survey 68%), which also precludes from applying any multivariable models to analyze how the time on treatment and other confounders affect the reported results. Furthermore, although the study population only consisted of melanoma patients that received their last ICI infusion more than 6 months ago, there was heterogeneity in the time since treatment discontinuation, which prohibits drawing any conclusions on patients' symptoms at specific points in time during follow‐up.The low number of missing data, the use of multiple validated questionnaires, and the long median time from treatment discontinuation to study enrollment are strengths of the present study. Additionally, these data provide more insight into the long‐term symptom burden and HRQL in melanoma survivors with results separated per treatment type.5The advent of anti‐PD1‐based ICIs has significantly improved treatment outcomes in advanced melanoma with many patients experiencing long‐term disease control. With these innovative treatments, the number of patients living beyond treatment cessation is gradually increasing. Taking into account the increasing incidence of melanoma diagnosis, as well as the effective treatment options available, the number of melanoma survivors will continue to increase in the next few years. Cancer survivors, and particularly melanoma survivors, are being confronted with several issues, such as anxiety of disease recurrence, fertility issues, subsequent malignancies and long‐term side effects due to the previous systemic treatments. As such, there is an emerging need to address existing gaps in survivorship care and implement these findings in clinical practice, in order to improve patients' HRQL. These results suggest the importance to increase awareness for long‐term symptoms in melanoma survivors.Esmée L. Looman: Investigation ; project administration ; writing – original draft . Phil Cheng: Formal analysis (lead). Julia Lai‐Kwon: Conceptualization ; methodology . Linda Morgan: Investigation . Marliess Wakkee: Investigation . Reinhard Dummer: Resources . Florentia Dimitriou: Conceptualization ; formal analysis ; investigation ; methodology ; supervision (lead); writing – original draft ; writing – review and editing (lead).This research did not receive any specific grant from funding agencies in the public, commercial, or not‐for‐profit sectors.FD receives/received honoraria and travel support from Merck Sharp & Dohme, Bristol Myers Squibb and Sun Pharma. RD declares intermittent, project focused consulting and/or advisory relationships with Novartis, Merck Sharp & Dhome (MSD), Bristol‐Myers Squibb (BMS), Roche, Amgen, Takeda, Pierre Fabre, Sun Pharma, Sanofi, Catalym, Second Genome, Regeneron, Alligator, T3 Pharma, MaxiVAX SA and touchIME outside the submitted work. All other authors have declared no conflicts of interest.Ethics approval and consent to participate was obtained from the local ethics committee . Patients' enrollment followed upon collection of written informed consent.Tables S1–S2.Click here for additional data file.Data S1.Click here for additional data file.


Saccharina latissimi) is of great interest to increase the circular food production in the ocean. Sugar kelp does, however, contain high levels of carbohydrates and iodine and does not have considerable levels of protein and lipids, which may make it less suitable as a feeding ingredient. A 10-week feeding trial was done to investigate the effect of graded dietary inclusion levels of fermented sugar kelp (FSK), on growth performance, digestibility, retention of nutrients, and mineral composition in postsmolt Atlantic salmon . The experimental diets were made to simulate a standard grower feed for salmon postsmolts in SW with ∼63% plant-based ingredients vs ∼34% marine ingredients and increasing concentrations of FSK between 0% (control feed) and 4% of the diet. During the feeding trial, the weight gain and specific growth rate (SGR) decreased linearly with increasing dietary FSK levels, where the SGR was slightly reduced from 1.2% for the fish given the control feed to 1.1% in the fish given feeds containing 3% and 4% FSK. This resulted in a lower weight gain of up to 9% in the fish given 4% FSK compared to the control. Feed intake and feed conversion ratio were, however, similar in all diet groups, and FSK inclusion did not influence the digestibility of macronutrients or minerals, except for lipid. The reduced growth is likely related to a lower digestible energy level in the diets, and the retention of both lipids and energy was affected by FSK inclusion. Inclusion of FSK also influenced iodine availability and retention, as well as increasing iodine status in whole body and muscle in a dose-dependent manner until reaching a plateau, which corresponds to 124 mg I kg−1 WW (135 mg I kg−1 DW), at 3% FSK inclusion.Using low trophic marine resources such as sugar kelp ( The aquaculture industry is rapidly expanding and is expected to continue growing worldwide. However, the industry faces a major challenge due to a shortage of available feed resources . TherefoNorway aims to produce 5 million metric tons (MT) of salmonids by 2050, which would require 6 million MT of feed . Sugar kOncorhynchus mykiss) including up to 2% dried sugar kelp in the diet did not reduce growth or feed utilization, whereas both weight gain and specific growth rate (SGR) were reduced when including 4%, likely due to decreased protein digestibility [Previous feeding studies have proven that overall performance of fish on macroalgae in the diet depends on the fish species (species specific) and inclusion level (dose dependent) of macroalgae –24. As stibility . Hence, tibility , 30.−1 dry weight (DW) [Brown macroalgae such as sugar kelp are known as iodine-rich sources containing up to 10,000 mg iodine kgght (DW) , 32. HowTo achieve large-scale use of macroalgae as a feed resource, it is crucial to address the challenge of a steady supply of biomass. Seasonal harvesting necessitates proper processing, preservation, and long-term storage . FermentThere are few available publications on the inclusion of sugar kelp or other brown macroalgae in feed for fish particularly salmonids as one of the most important groups of aquaculture fish species. Moreover, the generation of novel feed products by fermentation technology has yet only been developed for a few macroalgae species, particularly red algae , 39. TheThe feeding trial was conducted at Matre Research Station, Norway, according to the Norwegian regulations on animal experimentation. The experimental protocol was approved by the Norwegian Food Safety Authority (FOTS ID # 25202).Lactobacillus bacteria (LAB) delivered by European Protein  per 1,000 kg of finely chopped sugar kelp. The pH dropped to below 4.0 within a span of 3 weeks, which was sustained thereafter. The composition of both fresh sugar kelp, sampled prior to the fermentation process, as well as the FSK is presented in −1 WW (15% DW) crude protein and less than 1% g 100 g−1 WW lipid content, while FSK contained somewhat less carbohydrate than fresh sugar kelp. The cellulose level was similar in both fresh and FSK. Both groups showed the same concentration of manganese (Mn) and zinc (Zn), while copper (Cu), selenium (Se), iron (Fe), and iodine levels were different after fermentation.The FSK was provided by Ocean Forest AS, Lerøy Seafood Group. The sugar kelp was cultivated and harvested by Ocean Forest AS at Trollsøy, Austevoll  in May 2020. The fermentation process was initiated immediately after harvest on fresh material at ambient temperature (8–14°C) in closed intermediate bulk containers, by adding 10 g of a commercial blend of −1 WW (−1 WW) and lower digestible energy (DE) (19–18 MJ kg−1 WW) in FSK4% diet compared with the control group. Diets with a higher FSK contained lower NDF and hemicellulose content, while the others were comparable between the experimental diets. Some variations were seen in dietary Fe and Se levels and iodine ranged between 4 and 138 mg kg−1 WW in the experimental diets. The experimental diets were produced by Cargill . To determine apparent digestibility/availability of nutrients, yttrium oxide (0.02% ≈ 200 mg/kg) was added as an inert marker to all diets.The feeding trial was designed as a dose–response study using graded inclusion levels of FSK. The control diet was formulated as a commercially relevant reference feed for postsmolt in seawater. In the experimental diets, FSK was added to reach the target levels of 1%–4% in the finished extruded pellets . All die−1 WW . The ami−1 WW . The FSKSalmo salar L.) postsmolts with an average weight of 204 ± 37 g (mean ± SD) were randomly distributed in 15 quadrangular 1.5 m3 glass fiber tanks, in total 975 fish, and the five experimental diets were each randomly assigned to triplicate tanks. The postsmolt used in the present study originated from Aqua Gen strain, Agua Gen AS, Trondheim, Norway. In each tank, 55 fish were produced from commercially available eggs obtained in the fall of 2019 (mixed population) and 10 fish were from an isogenic salmon line  produced at Matre, also originally made from the Aqua Gen strain in 2011 [−3 (mean ± SD).At the start of the experiment, 65 Atlantic salmon  during the experimental period, under continuous (24 hr) light.The fish were given two meals per day (between 9 : 30 to 11 : 00 and 12 : 30 to 14 : 00) for 10 weeks. The fish were fed in excess with automatic feeders (Arvotec TD 2000) to ensure enough feed for all the fish, and the feeding rate was adjusted according to the increase in fish biomass. The uneaten feed pellets were collected 15min after each meal to estimate feed intake according to Helland et al. .n = 45) were sampled to determine the whole-body proximate composition and were divided into three pools each . At the end of the experiment, weight and length were recorded on all fish. From each tank, 10 fish from mixed population and 10 fish from all-male population were sampled for determination of whole-body and organ-specific nutrient compositions, where five whole fish from each were pooled for determination of whole-body composition  and five fish were dissected individually for registrations of viscera, liver, and heart to calculate somatic indices . The whole fish, as well as the whole muscle, were frozen in dry ice, homogenized, and stored at −20°C for determination of nutrient composition .All sampled fish were euthanized with an overdose of tricaine methane sulfonate . At the start of the experiment, 45 fish  were sampled to register organ weights , as well as determination of organ-specific nutrient compositions. The same number of fish  prior to dissection to monitor standard welfare indicators and operational indicators, including eye status, jaw wound and deformity, opercula status, spine deformation, gill condition, condition factor, and skin and fin damage according to a standard scoring system (SWIM) [A visual evaluation was done on the 20 individuals sampled from each tank (m (SWIM) , 45. Catm (SWIM) . Cataracm (SWIM) .n = 45 fish from the mixed population per tank, n = 3 per diet, pooled and n = 10 fish from the all-male population per tank, n = 3 per diet, pooled).Feces were collected by stripping (gently expelled using light pressure on the abdomen near the vent) from 55 fish per tank  at the end of the trial, and feces of each population  were separately pooled for a composite sample used to determine the apparent digestibility (ADC)/availability coefficient (AAC) of nutrients (2SO4 was included (NSPGlucose (12 M H2SO4)) or omitted (NSPGlucose (2 M H2SO4)). The sum of glucose, galactose, xylose, arabinose, mannose, rhamnose, fucose, and uronic acids shows T-NCP. Insoluble residue after hydrolysis with 12 M H2SO4 determined the lignin-like substances. The fractions in macroalgae that were insoluble in sulfuric acid and consequently indigestible and not fermentable were recognized as lignin. However, it could not be determined whether it is lignin or other acid-insoluble components in macroalgae the fraction will be referred to as the lignin-like substance. The sum of lignin-like substances and T-NSP corresponds to total dietary fiber (T-DF). Neutral detergent fiber (NDF), acid detergent fiber (ADF), acid detergent lignin (ADL), hemicellulose, and cellulose were measured in the feed and feces samples under a carbohydrate analyzer. The list of analyzed polysaccharides in each group is presented in DM, crude protein, crude fat, ash, gross energy, and carbohydrate content were determined in the raw materials (fresh and FSK), experimental diets, whole body, and feces samples. Briefly, DM was measured after drying to constant weight at 105°C for 24 hr . Crude pThe microminerals, yttrium oxide, and iodine concentrations in diets, and pooled samples of whole body, muscle, and feces were determined by inductively coupled plasma mass spectrometry (ICP-MS), as described by Long and Martin  and JulsThe following variables were calculated :(1)Digest is the sum of feeding days (70 days). In the current study, the mean SGR of the fish from mixed population was determined for each tank. In addition, individual SGR was also calculated on the 10 pit-tagged fish from the all-male population per tank.As described by Hopkins , where lA is the weight of air-dry feed (g), ADW is the DM content of air-dry feed (%), W is the wet weight of waste feed collected (g), WDW is the DM content of waste feed (%), and R the is recovery of DM of waste feed (%) that was calculated as follows:As described by Helland et al. , total fW dayx is the natural logarithm of biomass on a given day [Average daily feed intake per kg biomass (DFI–% biomass) was calculated from recorded daily feed intake and estimated daily biomass from SGR using the following equation:iven day .(8)CondiThe hepatosomatic indexes (HSI), cardio somatic indexes (CSI), and visceral somatic indexes (VSI) were calculated as percentages of the final weight:To understand how much of the ingested feed ingredient is absorbed by the animal and retained in their body, the apparent digestibility/availability coefficient (ADC/AAC), and retention of nutrients were measured as described by Cho :(12)ADC Nutrient retention (%) was calculated from fish biomass and nutrient content of the fish at the start and end of each growth period and nutrient intake:n = 3 for all the experimental diets and n = 2 for FSK4% group). Whole body, muscle proximate composition, and mineral status of Atlantic salmon postsmolts from the all-male population were only analyzed for control and high level of FSK4%. All the statistical analyses and the graphs were performed in GraphPad Prism (Version 8.4.3 (686) San Diego, California, USA). Significance was set at P < 0.05 for all statistical tests, and the value is presented as mean ± SEM.As the trial study was performed in a dose–response design, linear and nonlinear regression analyses were used to evaluate dose-dependent responses by determining the best-fit line for each dataset. In addition, one-way ANOVA was performed to assess statistically significant differences among experimental groups, and if the data were significant different, then followed up with Tukey' s multiple comparison post hoc analysis. For all datasets, Bartlett/Brown–Forsythe's test was used to assess the homogeneity of variance and Shapiro–Wilk's test was used to check the normality residuals. The ROUT test was done for the identification and removal of the outliers of the growth dataset. One of the 4% FSK tanks was removed as outlier. Tank was used as the experimental unit in growth, whole-body proximate, and mineral composition  with FSK inclusion  among the experimental groups. At the end of the 10-week trial, the mean cataract score was below 1 (0.7 ± 0.04) for all groups, and no difference was seen among the experimental groups. Moreover, there was no difference in visually assessed welfare indicators between experimental groups .During the experiment, the fish almost doubled the weight in all experimental groups . Total f Figures . In compn = 10 per tank, n = 30 per diet) was a little higher than the mixed population  at the end of the experiment, but it was still within the same range for both groups . This resulted in a 5%–7% lower weight gain, but the reduction was also not significant (P=0.15). The growth performance results for the all-male population are also reported separately as supplementary material . The protein ADC ranged between 86.9 ± 0.6% in the control group and 88 ± 0.3% in the experimental groups. Gross energy digestibility was 77.9 ± 1.2% and 80.1 ± 0.4% for the control and experimental groups, respectively. The average of the carbohydrate ADC  was calculated to be 66.6 ± 2.0% for NDF, −8.4 ± 2.2% for ADF, 44.0 ± 7.1% for ADL, 79.9 ± 1.5% for hemicellulose, and −16.5 ± 2.2% for cellulose.No difference was seen in the digestibility of macronutrients except for total fat ADC . Total fAvailability of iodine significantly increased with FSK inclusion in the diet under a segmental linear regression response with a broken point in FSK1% . The iodP=0.001, P=0.003, P=0.01, respectively) and decreased with FSK inclusion in diet .The concentration of iodine in the fish whole body increased in a dose-dependent manner with increasing iodine level in the feed −1 WW in the control group to 0.6 ± 0.0 mg kg−1 WW in the FSK4% group under a simple linear regression response . There was no effect of FSK diets on protein and ash retention.The retention of total fat, gross energy, and DM decreased with a higher FSK inclusion . The higP=0.01). Furthermore, iodine retention decreased significantly by adding 1% of FSK to the diet and followed almost a plateau pattern fitted by a segmental linear regression with a broken point in FSK1%  in the diet for Atlantic salmon [Previous studies have indicated that using high levels of macroalgae in fish feed influences the digestibility of nutrients, which in turn can be a potential cause of growth impairment . This efc salmon  and sugac salmon . One of c salmon , which cc salmon . It has c salmon , 68. In The lower dietary lipid level in the FSK4% diet compared with the other experimental diets (28% lower than the control group), likely resulted in a higher lipid digestibility and a relatively higher retention of total fat in the FSK4% group, while the whole-body fat content in this group was 14% lower than the control group. These changes were not reflected in the somatic indexes, which were contrary to the study by Granby et al. .−1 DW) [−1) [−1 (based on 880 g kg−1 DW) [−1 in farmed fish [−1 diet fed to adult Atlantic salmon  [−1 diet fed to rainbow trout (Oncorhynchus mykiss) [−1 WW in the control feed up to 138 mg kg−1 WW in the 4% FSK feed. Due to the overall low dietary energy that influenced growth performance, it is challenging to determine if the high dietary iodine level caused the growth reduction. Nevertheless, the increase in iodine AAC and improvement in iodine body status until reaching a plateau level (FSK3% containing 124 mg kg−1 WW) suggested a possible regulation of iodine uptake and deposition in the fish body. Additionally, exposure to high dietary iodine levels resulted in a decrease in iodine retention. These results indicated that fish have a mechanism to adjust their iodine metabolism in response to high dietary iodine levels. This finding is consistent with previous research which has shown that certain species of fish are capable of efficiently excreting excess metals and maintaining normal levels of concentration in their bodies [Sugar kelp contains a high level of iodine as reported by previous studies  , 70, 71.−1 DW) . In comm−1 DW) . Due to −1 DW) . The reqDW) [−1) , 74, andkg−1 DW)  which stkg−1 DW) . Howevermed fish , 76. Feealar L.) , and 2%  mykiss)  had no ar bodies .−1 WW, respectively) was around 0.3 ± 0.0 mg kg−1 WW (four-fold of control diet) and reached around 0.6 ± 0.0 mg kg−1 WW in the muscle of fish fed FSK3% and 4% (124 and 138 mg iodine kg−1 WW). However, in the study by Granby et al. [−1 WW in fish fed 1% sugar kelp (57 mg iodine kg−1 WW) to 1.2 ± 0.45 mg kg−1 WW in fish fed 4% sugar kelp (220 mg iodine kg−1 WW). It is important to note that Granby et al. [Salvelinus sp.) displayed a five-fold higher iodine concentration compared to the skinless muscles [Previous studies have shown that the dietary iodine concentrations can be reflected in the muscle iodine level , 79–81. y et al. , the musy et al.  included muscles , further−1 WW (below detection limit), and just above the detection limit (0.01 mg kg−1 WW) after the fermentation, which was consistent with the findings of Bruhn et al. [−1 DW) [The sugar kelp used in this study had a Se concentration of less than 0.008 mg kgn et al. . Althoug [−1 DW) , a high  [−1 DW) , and it  [−1 DW) , which sThe inclusion of FSK negatively affected the distribution and retention of Cu in the whole body. This may be attributed to the higher level of iodine in the fish, as both iodine deficiency and oversupply can disrupt mineral  homeostasis , 84.Overall, the incorporation of FSK in the experimental diets reduced the growth, which may be related to the overall lower energy content in these feeds since feed intake and feed utilization (FCR) were similar. The use of FSK did not influence the digestibility of macronutrients except for lipids. The retention of lipid, energy, and DM was reduced with FSK inclusion in diet, which corresponded with whole-body macronutrient composition. Apparent mineral availability (except iodine and Se) and mineral retention (except iodine and Cu) were not affected by FSK inclusion by up to 4%. The incorporation of FSK in the diets improved iodine availability. Our results indicated that up to 3% FSK supplementation in the Atlantic salmon diet has the potential to improve the muscle iodine concentration. Up to 2% FSK inclusion in the postsmolt salmon diet improved Se availability. FSK inclusion in the diet of Atlantic salmon had no influence on the welfare indices studied.


This study investigated the proteomic characteristics of cerebrospinal fluid (CSF) in patients with varicella zoster virus (VZV) meningitis to understanding the pathogenesis of central nervous system (CNS) infection by reactivated VZV.We used data‐independent acquisition model to analyze the CSF proteomic differences of 28 patients with VZV meningitis and 11 herpes zoster (HZ) patients. According to the clinical manifestations at discharge, 28 VZV meningitis patients were divided into favorable outcome group and unfavorable outcome (UO) group and their differences in CSF proteome were also analyzed.p < .01). The protein related to the nerve function and energy metabolism  were significantly downregulated (p < .05). The levels of a pro‐inflammatory factor, IL‐18, in CSF were significantly higher in patients in the UO group as compared to patients with favorable prognosis (p < .05).Compared with the HZ group, the proteins  related to inflammation and immune cell activation were significantly upregulated in the VZV meningitis group , and the cerebrospinal fluid IL‐18 levels might be a potential prognostic indicator of the outcomes of VZV meningitis. After infection, VZV stays latent in trigeminal ganglia or dorsal root ganglia. When patient's immune system is compromised, the latent VZV can be reactivated and leads to regional rash and severe pain, known as herpes zoster (HZ), which can also affect peripheral nervous system. The most common complication of HZ is postherpetic neuralgia (PHN) causing intractable chronic pain. HZ can also cause intracranial infection,VZV meningitis is one of the common complications of CNS infection caused by reactivated VZV. The pathological mechanism of VZV meningitis is multifactorial. Direct viral infection, inflammation, virus‐induced hypercoagulability, vasculitis, and so on have been implicated,Studying responses to infections at the proteome level facilitates early diagnosis, risk stratification, and prediction of outcomes, and can help to understand the pathophysiological mechanisms. But to date, few studies have been conducted on CSF proteomics in patients with VZV meningitis.22.1Twenty‐eight patients with VZV meningitis hospitalized in the Department of Neurology, Hangzhou Third People's Hospital from June 2021 to November 2022 were selected, including 21 males and seven females, with an average age of 56.5 ± 16.3 years. Eleven patients with HZ hospitalized during the same period were selected as the control group, including three males and eight females. The average age was 54.5 ± 16.3 years. The diagnosis, treatment, and follow‐up records of patients with HZ and VZV meningitis were obtained through the hospital case information system. The contents of the records include genders, ages, sites of HZ, disease courses, concomitant diseases, clinical symptoms, results of routine and biochemical examinations of CSF, lengths of stay, treatment situations, and so on.2.26/L or protein content >0.45 g/L or positive VZV DNA. Exclusion criteria: (1) Patients ≤14 years; (2) CSF test confirmed bacterial, fungal, and other microbial infections; (3) Patients with incomplete clinical data; (4) HIV‐infected patients; (5) Patients with mental illness or severe dementia who could not accurately express clinical symptoms.Patients with shingles within 2 weeks of onset were selected. Shingles was determined by dermatologists according to the diagnostic criteria.2.3The outcomes of patients with VZV meningitis were judged by their conditions at the time of discharge. The favorable outcome (FO) group includes those patients that had no symptoms of discomfort upon discharge. The unfavorable outcome (UO) was defined as death, severe disability, or any neurological sequelae affecting the patient's daily life at the time of discharge.2.4All patients underwent lumbar puncture within 48 h of admission, and 2 mL of cerebrospinal fluid (CSF) was reserved for routine examination  in 100 mM ammonium bicarbonate at 37°C for 14 h. The peptides were acidified with 0.1% formic acid (FA) and desalted by hydrophilic‐lipophilic‐balanced plates (Waters) before nanoLC‐MS/MS analysis.2.6Peptides were separated by the U3000 UPLC nano liquid phase (ThermoFisher) system equipped with a self‐packed column  (Dr. Maisch) with a flow rate of 450 nL/min. Mobile phase A: 0.1% FA, B: 0.1% FA dissolved in 98% acetonitrile. The 120‐min gradient is set as follows: 5 min 3%–8%B; 85 min 8%–24%B; 20 min 24%–38%B; 5 min 38%–80%B. It was then held at 80% B for 5 min until the next injection. The peptides were analyzed by Exploris 480 mass spectrometry working on the DIA mode that collects a full MS survey scan from 400 to 1000 Th at the resolution of 120,000 full‐width at half‐maximum (FWHM) (at m/z 200 Th) with automatic gain control (AGC) set to 100%, followed by MS2 spectra with 75 isolation windows of 8 Th with an overlap of 1 Th. Precursors co‐isolated in each window were fragmented by higher‐energy collision dissociation with normalized collision energy set to 30%. All MS2 spectra were acquired with 30,000 FWHM resolution with AGC set to standard.2.7t test (p < .05) and fold‐change (≥1 or ≤−1) was used to select differentially expressed proteins.The collected RAW files were loaded into the DIA‐NN v1.8 for spectral‐library‐free search against the human UniProt database supplemented with VZV and common contamination sequences. Peptide length was restricted to 7–30 amino acids and precursor charge was limited to <5. Other search parameters were set as follows: oxidation (M) and carbamoyl (C) were set as variable and fixed modification, respectively; protease was set to trypsin and a maximum of two missed cut sites were allowed, and the mass error was set to 7 ppm. The results were filtered by PSM and protein level 1% false discovery rate. For quantitative analysis, relative protein intensity in each sample was log2 transformed. Student's‐2.8p < .05 was considered to be statistically significant.SPSS 20.0 software was used for data processing and statistical analysis. The measurement data of normal distribution were expressed as mean ± standard deviation. T test was used to compare the mean of two samples. Data with nonnormal distribution were described by median (interquartile distance) {M (Q1–Q3)}, and comparison between the two groups was conducted by Mann–Whitney U test. Count data were expressed by frequency and percentage, and comparison between groups was performed by Chi‐square test or Fisher's exact test. 33.1p < .05).There was no difference in ages and concomitant diseases between the HZ control (without CNS infection) and VZV meningitis groups, but there were more males than females with VZV meningitis, the difference was statistically significant (p > .05). The data are shown in Table The zoster site of VZV meningitis occurred in the head and face in 20 cases 71.4%), followed by the chest and back in four cases (14.3%), neck (10.7%) in three cases, waist (3.6%) in one case. In the HZ group, zoster was mainly distributed in the head and face in six cases (54.5%), followed by chest and back in five cases (45.5%) and neck in two cases (18.2%). Among the 28 cases of VZV meningitis, there were 11 cases (39.3%) with headache, 14 cases (50.0%) with fever, five cases (17.9%) with vertigo, and RHS (Ramsay Hunt Syndrome) five cases (17.9%). In the HZ group, there were six cases (54.5%) with headache and one case (9.1%) with fever. There were no significant differences in the zosters site and clinical symptoms between the HZ control and VZV meningitis groups . The number of days of antiviral therapy in the VZV meningitis group was 13.9 ± 2.8 days, which was also longer than that in HZ group 8.1 ± 3.9 days. The proportion of patients in the VZV meningitis group receiving glucocorticoid therapy  was significantly higher than that in the HZ group . The data are shown in Table All patients received intravenous antiviral therapy and intravenous glucocorticoid for 3–5 days (40–80 mg/d Methylprednisolone). The mean hospital stay in the VZV meningitis group was 15.2 ± 3.7 days, which was significantly longer than that in the HZ group (10.8 ± 3.3 days)  mg/dL, chlorine content (122.6 ± 5.8) mmol/L in the CSF of the VZV meningitis group was significantly higher than that of the HZ group, , while the expression of proteins related to nerve function and energy metabolism  was significantly decreased (p < .05) . The interval between the onset of neurological symptoms to antiviral therapy in the UO group was significantly longer than that in the FO group (p < .05) Table According to prognostic assessment criteria, 18 patients (64.3%) with VZV meningitis had FO at discharge, while 10 patients (35.7%) had UO. Among them, the proportion of female patients with UO was higher, with statistical difference (p > .05).There were no differences in ages, lengths of stay, days of antiviral therapy, proportions of glucocorticoid therapy, CSF leukocyte counts, protein, and glucose contents between the two groups (p < .05)    was significantly increased  Figure . while t) Figure .4VZV lurks in the peripheral and CNSs, and can be reactivated when the host immune function is repressed.VZV is one of the most common pathogenic herpesviruses in CNS infection,Our study showed that inflammation is an important mechanism in the occurrence and development of VZV meningitis. In this study, we used DIA technology to detect 39 CSF samples  and identified 2502 proteins in the CSF. We found that the expression of neutrophil‐activating proteins  and proteins reflecting inflammation and immune cell activation  were significantly upregulated, while those related to nerve function and energy metabolism  were downregulated.Normally, leukocyte entrance into the CNS is limited by the blood–brain barrier and/or by the blood‐CSF barrier. Our study found that the CSF leukocytes count and neutrophil activation markers, ELANE, MPO, and PRTN3 were most increased in the VZV meningitis group. ELANE is a serine protease released by neutrophils that can cause harmful inflammatory responses. Sun et al.This study showed that the levels of proteins related to nerve function and energy metabolism decreased significantly . In tissues with high energy requirements, including kidney, brain, testis, placenta, and sperm, CKMT1B expression was significantly negatively correlated with the infiltration levels of B cells, CD8 + T cells, CD4 + T cells, macrophages, neutrophils, and dendritic cells.Currently, HSV‐1, HSV‐2, and VZV are known to be common agents of CNS infection in adults and lead to significant neurological morbidity and mortality. Clinical symptoms and disease outcome depend on the specific infectious agent and the immune status of the host.As an ideal specimen for the CNS studies, CSF can accurately and comprehensively reflect the proteomic information of brain diseases, and proteomics can identify potential driving factors and improve our understanding of the pathogenesis of VZV reactivation. Nonetheless, our study did came with limitations. First, the sample size is relatively small, particularly on the control group of 11 HZ patients, which could affect the power to reveal proteomic differences. Second, the time points for symptom onset and CSF collection are variable, which could also possibly affect the result on the proteomic expression profile. Third, the outcomes of patients with VZV meningitis were judged by their conditions at the time of discharge, which could also make the classification inaccurate, following discharge for one or 3 months may be more reliable. Fourth, our study is a retrospective study, VZV meningitis patients with incomplete clinical data were excluded, which may lead to bias in patient selection. Future prospective studies are needed to confirm our results.5Our study suggests that inflammatory response is one of the important pathophysiological mechanisms of VZV CNS infection, and the expression level of IL‐18 protein in CSF might be a potential prognostic indicator that can be used to judge the outcome of VZV meningitis patients.Huili Liu: Conceptualization; data curation; formal analysis; investigation; writing—original draft. Jun Wang: Data curation; formal analysis; investigation; writing—review and editing. Yan Zhang: Conceptualization; data curation; investigation. Jing Gu: Conceptualization; investigation. Yu Wang: Data curation; formal analysis. Yongxing Yan: Conceptualization; funding acquisition; resources; writing—review and editing. Dongli Pan: Funding acquisition; project administration; writing—review and editing. Zeyu Sun: Formal analysis; funding acquisition; project administration; writing—review and editing.The authors declare no conflict of interest.The study protocol was approved by the Ethics Committee of Hangzhou Third People's Hospital (NO: 2021KA013).


JBMR Plus published by Wiley Periodicals LLC. on behalf of American Society for Bone and Mineral Research.Fibroblast growth factor (FGF)23 is one of the major regulators of phosphate homeostasis. Hypophosphatemia can lead to muscle weakness, fatigue, and osteomalacia. In the setting of hypophosphatemia, serum FGF23 can be measured to differentiate between FGF23‐mediated and non‐FGF23‐mediated renal phosphate wasting. C‐terminal FGF23 (cFGF23) assays detect both cFGF23 and intact FGF23 (iFGF23). Circulating FGF23 is regulated by 1.25‐dihydroxy‐vitamin D, parathyroid hormone (PTH), serum phosphate, and serum calcium but also by, for example, iron status, inflammation, erythropoietin, and hypoxia‐inducible‐factor‐1‐α. We present the case of a 48‐year‐old woman with unexplained mild hypophosphatemia, very high cFGF23, and normal iFGF23. The patient proved to have an iron deficiency. Iron deficiency alters the iFGF23‐to‐cFGF23 ratio. After initiation of iron treatment, cFGF23 strongly decreased. This case report illustrates the limitation of cFGF23 assays and urges clinicians to be aware that cFGF23 concentrations do not necessarily reflect iFGF23 concentrations and that alternative causes for its elevation should be considered . © 2023 The Authors.  We present the case of a 48‐year‐old woman with unexplained mild hypophosphatemia, very high cFGF23 and normal iFGF23. The patient proved to have an iron deficiency. Iron deficiency alters the iFGF23‐to‐cFGF23 ratio. After initiation of iron treatment, cFGF23 strongly decreased. This case report illustrates the limitation of cFGF23 assays and urges clinicians to be aware that cFGF23 concentrations do not necessarily reflect iFGF23 concentrations and that alternative causes for its elevation should be considered . One of the major regulators of phosphate is fibroblast growth factor (FGF)23. Recently it has been shown that FGF23 also interacts with iron metabolism and erythropoiesis. The current case illustrates the interaction between FGF23 and iron status, and challenges the applicability of C‐terminal FGF23 (cFGF23) assays for the work‐up of hypophosphatemia in the setting of FGF23‐related disturbances.Inorganic phosphate is important for several metabolic processes, including intracellular signal transduction and energy production, and for mineralization of the skeleton by formation of hydroxyapatite. Acute severe hypophosphatemia can lead to neurological symptoms and impaired cardiac and respiratory function. Chronic hypophosphatemia is usually characterized by muscle weakness, fatigue, and the development of osteomalacia, accompanied by bone pain.1 One 2D) was slightly increased, 197 pmol/L , and intact parathyroid hormone (PTH) was 7.2 pmol/L  (Table ) was 234 RU/mL , suggesting FGF23‐related renal phosphate wasting. There were no signs of a generalized tubulopathy, for example, glucosuria or proteinuria, and renal ultrasound showed no nephrocalcinosis or nephrolithiasis. Whole‐exome sequencing was performed for renal hypophosphatemic disorders and showed no mutations in genes SLC34A1, SLC34A3, or SLC9A3R1. She was treated with oral phosphate supplementation three times daily 20 mmol, resulting in repeatedly normal serum phosphate measurements, ranging from 0.86 to 1.39 mmol/L, and she was discharged from follow‐up.A 48‐year‐old woman was referred to the nephrologist of Erasmus Medical Center in 2012 suffering from bone and joint pain, fatigue, and muscle weakness for 6 months. Her family history was negative for bone‐related conditions and her physical examination did not show signs of growth retardation or bone deformities. Her medical history included asthma; microcytic hematuria; iron deficiency, for which she had used iron supplementation in the past; and pangastritis in 2010. She used salbutamol and formoterol/beclometasone for her asthma, and omeprazole. At biochemical evaluation, kidney function, serum sodium, potassium, and magnesium were within the reference range, but her phosphate was low, 0.71 mmol/L . Albumin‐adjusted calcium was 2.25 mmol/L , 25‐hydroxy‐vitamin D (25OHD) was 107 nmol/L , 1.25‐dihydroxy‐vitamin D (1.25OH) (Table 4) was2D was 96.5 pmol/L, and intact PTH was 3.9 pmol/L . Iron status was not measured at this time but was normal earlier that year, when serum phosphate was 0.85 mmol/L. Twenty‐four‐hour urine calcium was 6.72 mmol . Celiac disease was excluded based on absence of endomysial antibodies. Despite the fact that hypophosphatemia was mild and responded well to supplementation, TMP/GFR was repeatedly decreased, suggesting renal phosphate wasting. cFGF23, measured later that year, was 697 RU/mL . Whole‐exome sequencing was repeated and showed no mutations in FGF23 and PHEX. Our differential diagnosis included tumor‐induced osteomalacia (TIO) but physical examination and CT, FDG‐PET‐CT, and 68‐Gallium‐DOTATE PET‐CT did not show signs of a causative tumor.Six years later, in 2018, she was referred to the Bone Center of our hospital with persistent incapacitating fatigue and bone, muscle, and joint pain. She had never suffered a fracture. Serum phosphate was 1.39 mmol/L while on phosphate supplementation, but after cessation serum phosphate decreased to 0.77 mmol/L. At this time, albumin‐adjusted calcium was 2.45 mmol/L , 25OHD was 79 nmol/L, 1.25(OH)In 2019, cFGF23 had increased up to 935 RU/mL, whereas serum phosphate was normal or slightly decreased without supplementation. At that time, serum 25OHD was decreased (20 nmol/L) and serum alkaline phosphatase (AF) was elevated, 177 U/L  as well as bone specific alkaline phosphatase, 48.9 μg/L . A bone biopsy showed severe osteoporosis and low bone turnover but no signs of osteomalacia. We initiated cholecalciferol after which serum 25OHD and AF normalized, but serum phosphate was still decreased, 0.77 mmol/L.In the years that followed, cFGF23 concentrations kept rising and there was no finding of TIO on imaging. Because iron deficiency can be associated with hypophosphatemia and the patient had been treated with iron supplementation for iron deficiency in the past, we measured her iron status again in 2022. At this time, her hemoglobin was 8.0 mmol/L , mean corpuscular volume was 80 fl , but her iron was 5.7 μmol/L , indicating iron deficiency. Because of the discrepancy between the very high cFGF23 and normal or slightly decreased serum phosphate, we decided to measure the C‐terminal and intact FGF23 (iFGF23) in our patient and in a healthy control in our research lab for calcium and bone metabolism because measurement of iFGF23 for clinical purposes was not available to us at that time. cFGF23 was 1260 RU/mL in the patient and 51 RU/mL in the healthy control . iFGF23 was 36 pg/mL in our patient and 47 pg/mL in the healthy control . After excluding gastrointestinal blood loss by performing gastroscopy and colonoscopy, we treated the iron deficiency with ferrous fumarate 200 mg three times daily. Serum iron increased to 18.5 μmol/L, serum phosphate was normal at 0.95 mmol/L without phosphate supplementation, and cFGF23 decreased from 935 to 158 RU/mL. The patient was pleased that the cause for the high cFGF23 concentrations was found and that she did not have a tumor. The iron supplementation improved her fatigue but the muscle pain remained. It turned out she had normal iFGF23 concentrations and increased cFGF23 due to iron deficiency. This case raises two questions: (i) Can elevated cFGF23 cause mild hypophosphatemia in the setting of iron deficiency; (ii) Is the use of cFGF23 assays to differentiate between the causes of hypophosphatemia appropriate in the setting of serum FGF23 disturbances such as iron deficiency.We describe the medical history of a 48‐year‐old female patient with bone pain, fatigue, muscle complaints, mild hypophosphatemia, and very high cFGF23 concentrations. Because cFGF23 concentrations kept rising, we conducted extensive diagnostic procedures, including advanced imaging, in search of a growing tumor causing TIO, and genetic testing.5 It t2D, PTH, and FGF23. 1.25(OH)2D increases phosphate absorption from the intestine, whereas PTH and FGF23 stimulate renal phosphate excretion. Both 1.25(OH)2D and PTH induce bone resorption, which leads to release of phosphate. FGF23 also inhibits 1α‐hydroxylase, thereby decreasing the synthesis of 1.25(OH)2D. The differential diagnosis of hypophosphatemia can be divided into three major groups: hypophosphatemia from increased renal excretion, from decreased intestinal absorption, or due to intracellular shift of phosphate. Increased renal excretion can be subdivided into FGF23‐mediated and non‐FGF23‐mediated renal phosphate wasting. Increased FGF23 concentrations can be caused by monogenetic disorders such as X‐linked hypophosphatemia or by a FGF23‐producing tumor in the setting of TIO.Serum phosphate is mainly regulated by 1.25(OH)25(OH)2D.2 The 2D, PTH, alpha‐klotho, serum phosphate, and serum calcium. Non‐classical regulators of FGF23 include iron status, inflammation, erythropoietin, hypoxia‐inducible factor 1α (HIF1α), insulin and diabetes, and leptin. The FGF23 gene encodes for a 251‐amino‐acid‐long glycoprotein that requires posttranslational O‐glycosylation in order to be stabilized and phosphorylation to be cleaved. Currently, two types of FGF23 assays exist for the quantification of circulating FGF23 concentrations in humans. The first one is the C‐terminal assay, which detects both the active full‐length FGF23 and the C‐terminal fragments that are released after cleavage, by using antibodies that target epitopes in the C terminus. The second one is the intact FGF23 assay, which only detects the presumed active full‐length FGF23. In the Netherlands, FGF23 concentrations are determined at the Amsterdam University Medical Center by a C‐terminal FGF23 (cFGF23) assay (Immutopics) .FGF23 is mainly produced by osteoblasts and osteocytes. Classical regulators of circulating FGF23 include 1.25(OH)d leptin.7 The  Two types of conditions that can lead to high cFGF23 with normal or low iFGF23 concentrations, resulting in a low i:cFGF23 ratio, have been described. On the one hand, there are conditions, including iron deficiency, inflammation, and increased erythropoietin (EPO), that stimulate FGF23 transcription and cleavage, resulting in high cFGF23 but normal iFGF23 concentrations. On the other hand, there is a condition named tumoural calcinosis, which can be caused by a mutation in N‐acetylgalactosaminyltransferase 3 (GALNT3), resulting in increased cleavage of iFGF23 with high cFGF23 and low iFGF23 concentrations.Edmonston and colleagues describe the application of an intact FGF23‐to‐C‐terminal FGF23 ratio (i:cFGF23), in combination with the concomitant serum phosphate level, in discriminating between different FGF23‐mediated syndromes.3 Two 2D concentrations and an increase in urinary phosphate excretion but no change in serum phosphate. The role for iron in FGF23 regulation in humans was illustrated by Imel and colleagues in patients with autosomal dominant hypophosphatemic rickets (ADHR), which is caused by mutations in the FGF23 gene, leading to impaired cleavage of iFGF23. In this study, low serum iron was associated with both elevated cFGF23 and iFGF23 in patients with ADHR, but only with elevated cFGF23 in healthy controls. This suggested that iron deficiency leads to increased FGF23 expression, but increased cleavage retains homeostasis in humans without ADHR. Similarly, serum iron was found to be associated only with increased cFGF23 and not with iFGF23 in a study in healthy premenopausal women. In this study, serum phosphate correlated with iFGF23 in all women but with cFGF23 in black women only. In contrast, in a Swedish population‐based cohort study of elderly men, low serum iron was found to be associated with high serum iFGF23.The interaction between FGF23 and iron status and erythropoiesis has been extensively studied. Induction of iron deficiency by a low‐iron diet in wild‐type mice resulted in increased serum intact FGF23 and C‐terminal FGF23 concentrations, with a corresponding decrease in 1.25(OH)hosphate.8 The  It has been reported that EPO affects both production and cleavage of FGF23. In human studies with administration of recombinant EPO, iFGF23 remains normal or increases only slightly, while the increase in cFGF23 is more pronounced. HIF1α is stabilized in the setting of hypoxia or iron deficiency and activates the production of EPO. Interestingly, HIF1α may also affect FGF23 directly. David and colleagues conducted a study in mice who were injected with IL‐1β to mimic a pro‐inflammatory state, leading to functional iron deficiency. These mice were found to have increased cFGF23, while iFGF23 remained unchanged. When IL‐1β‐injected MC3T3‐E1 osteoblast‐like cells were pretreated with a HIF1α inhibitor, the effects of IL‐1β on FGF23 mRNA expression and FGF23 protein were partially blocked, indicating that HIF1α may target FGF23 transcription directly.The effect of iron status on FGF23 could be mediated by erythropoietin (EPO) and HIF1α (Fig. 12 It  In iron deficiency, iFGF23 remains mostly unchanged, but it is possible that iFGF23 concentrations were increased at a certain point in time, causing the mild hypophosphatemia. However, other causes of mild hypophosphatemia should be considered, for example, hyperparathyroidism, use of alcohol, and less well‐known causes of hypophosphatemia such as obesity and Cushing's disease. After treatment of the iron deficiency, serum phosphate concentrations were normal, but the complaints of the patient only partly resolved. The muscle pain remained, which suggests that the mild hypophosphatemia was not clinically significant and that there is another cause for the complaints than hypophosphatemia. Still, her high cFGF23 concentrations led to extensive diagnostic procedures and a significant diagnostic delay, which would not have been conducted had we known iFGF23 concentrations. Alarmingly, the fact that cFGF23 does not necessarily reflect iFGF23 concentrations is not incorporated in commonly applied diagnostic algorithms of hypophosphatemia. In the Netherlands and also in other countries, hospitals only use cFGF23 assays and not iFGF23 assays, even though Hartley and colleagues found that measurement of iFGF23 is superior to cFGF23 in making a diagnosis of FGF23‐mediated hypophosphatemia.We acknowledge the limitation of our case study that the cFGF23 and iFGF23 assays used in our research lab are not meant for clinical practice. However, the iFGF23 concentration in our patient was comparable to the healthy control, and it is clear that our patient had a very low i:cFGF23 ratio, which can point to untreated iron deficiency.3 In iIn conclusion, increased cFGF23 concentrations, especially in the setting of hypophosphatemia, can be wrongly classified as a FGF23‐associated disorder, while iFGF23 concentrations may be normal, for example in the setting of iron deficiency. Our case illustrates the limitation of cFGF23 assays, and we urge clinicians to be aware that cFGF23 concentrations do not necessarily reflect iFGF23 concentrations and that other causes should be considered, including iron deficiency as in our case. Another lesson we learned is that one should be cautious to attribute general complaints like fatigue and muscle weakness to a mildly decreased serum phosphate concentration, and keep an open mind to alternative diagnoses.Ariadne Bosman: Conceptualization; formal analysis; investigation; visualization; writing – original draft; writing – review and editing. Danielle M. A. Ratsma: Conceptualization; formal analysis; investigation; writing – review and editing. Bram C.J. van der Eerden: Conceptualization; resources; supervision; writing – review and editing. M. Carola Zillikens: Conceptualization; resources; supervision; writing – review and editing.The authors have no conflicts of interest to declare.https://www.webofscience.com/api/gateway/wos/peer-review/10.1002/jbm4.10790.The peer review history for this article is available at


The immune system may influence prognosis, and lymphopenia is a frequent side effect of concurrent chemoradiotherapy (CCRT). Radical irradiation for locally advanced esophageal cancer (LA‐EC) exposes significant vascular and heart volumes. In this study, we hypothesized that lymphopenia is linked to cardiac and pericardial doses and affects patient prognosis.We identified 190 LA‐EC patients who received radical CCRT. Multivariate analysis (MVA) was performed to correlate clinical factors and dosimetric parameters with overall survival (OS). We collected lymphocyte‐related variables and ratios before and during CCRT. MVA was performed to correlate hematologic toxicity with OS. The relationship between dosimetric parameters and G4 lymphopenia was determined using logistic stepwise regression. Finally, a nomogram of G4 lymphopenia was developed and validated externally.30 (PV30) was linked to worse survival . The median OS stratified by PV30 > 55.3% and PV30 ≤ 55.3% was 24.0 months and 54.0 months, respectively (p = 0.004). G4 lymphopenia was shown to be linked with worse OS in the MVA of hematological toxicity with OS . Thirty of the 100 patients in the training set had G4 lymphopenia. Logistic stepwise regression was used to identify variables associated with G4 lymphopenia, and the final model consisted of stage‐IVA (p = 0.017), platelet‐to‐lymphocyte ratio during CCRT (p = 0.008), Heart V50 (p = 0.046), and PV30 (p = 0.048). Finally, a nomogram predicting G4 lymphocytopenia were constructed and externally validated. The ROC curve showed an AUC for internal validation of 0.775 and external validation of 0.843.Median follow‐up time for all patients was 27.5 months. On MVA for OS, higher pericardial VHigher doses of pericardial radiation might affect LA‐EC patients' prognosis by inducing G4 lymphopenia during CCRT. Further prospective studies are warranted to confirm these findings, especially in the era of immune‐checkpoint inhibitor treatment. In this study, we investigated the relationship between pericardial and cardiac radiation dosimetric parameters and lymphopenia. Since lymphocytes are extremely sensitive to radiation and the pericardium anatomically contains the heart and part of the large blood vessels, higher pericardial radiation doses are prone to cause circulating immune cell reduction. Since grade 4 (G4) lymphopenia is strongly associated with prognosis, we developed a nomogram model to predict G4. Radical concurrent chemoradiotherapy (CCRT) is the standard of care for patients with unresectable locally advanced esophageal cancer (LA‐EC). However, survival remains disappointing, with a 5‐year overall survival (OS) of only about 20%.Several studies have already been published which have explored the potential relationship between higher doses of irradiation to the heart and clinical outcomes for patients.Myelosuppression is also a common side effect of antineoplastic therapy, and lymphocytopenia has been found to be significantly linked to patient treatment outcomes. Since lymphocytes are the most radiosensitive cells in the hematopoietic system, and the heart and lungs, as specific organs of the blood pool, receive high doses of radiation that may damage circulating lymphocytes.The primary objective of the present study was to investigate the relationship between radiation parameters and the risk of G4 lymphopenia in LA‐EC patients undergoing radical CCRT. Additionally, we assessed whether G4 lymphocyte reduction during CCRT was associated with patient prognosis and attempted to establish a nomogram for predicting G4 lymphocyte nadir.This retrospective study included 190 patients with LA‐EC  who received radical CCRT at West China Hospital and Sichuan Provincial People's Hospital between February 2011 and April 2021. The inclusion criteria were as follows: new pathologically verified ESCC with no distant metastases, no history of chest radiotherapy, and conventional segmentation at doses ≥50 Gy. Age > 75 years, a history of chest radiotherapy, surgical treatment, distant metastases, and a performance status (PS) score ≥2 were excluded from the study. Ethical approval was obtained from both institutions for this study, and the requirement for informed consent was waived due to its retrospective nature.30 (volume receiving 30 Gy) ≤ 30%, MHD < 30 Gy; and to the whole lung, V20 ≤ 20%, V30 ≤ 15% and MLD < 20 Gy as recommended by NCCN panels.All patients underwent CCRT, with a radiation technique of intensity‐modulated radiation therapy (IMRT). The target volume contours were in accordance with the protocols in the study of Nishimura et al.3/mL, which was used to dichotomize the lowest lymphocytes during CCRT.We collected blood cell variables such as ALC, absolute platelet count (PLT), absolute white blood cell count (WBC), absolute neutrophil count (ANC), neutrophil‐to‐lymphocyte ratio (NLR = ANC/ALC), and platelet‐to‐lymphocyte ratio (PLR = PLT/ALC) 1 month before CCRT and weekly throughout CCRT. According to CTCAE5.0, G4 lymphopenia was defined as ALC nadir <0.2 10p‐values with two tails less than 0.05 were regarded as statistically significant. All analyses were performed using R software (version 4.2.2) and SPSS (version 26.0).We made the following assumptions; first, higher pericardial, cardiac, and pulmonary doses were linked to poorer OS. The primary outcome in the first analysis was OS, defined as CCRT completion to all‐cause death or cutoff follow‐up time. Second, we hypothesized that severe lymphopenia was associated with higher cardiac and macrovascular radiation doses thus affecting prognosis. In the second analysis, the primary endpoint was G4 lymphopenia. Kaplan–Meier curves and log‐rank tests were used to compare the results of the G4 nadir. Stepwise backward logistic regression was used to find the association of clinical, hematological, and dosimetric variables with G4 lymphopenia. The variable with the lowest Akaike information criterion (AIC) value was selected for the actual modeling due to the strong covariance between cardiac and pericardial DVH parameters. The results of multivariate analyses were used to construct a nomogram. A total of 100 from West China Hospital and 90 patients from Sichuan Provincial People's Hospital were included in the training and validation cohorts, respectively. Results of multivariate logistic regression analysis were used to construct a nomogram. Internal validation of the nomogram was performed using the area under the subject operating characteristic (ROC) curve (AUC), calibration curve (1000 bootstrap resampling), and decision curve analysis (DCA). The final prediction model was applied to the external cohort to generate the  C‐index.The baseline patient characteristics are summarized in Table 25 (PV25), and pericardial V30 (PV30) were associated with poorer OS (p < 0.05), while gender, age, smoking status, ECOG, prescribed dose, and tumor location were not significantly different. Notably, cardiac and pulmonary dosimetric parameters were not significantly associated with OS . We plotted the ROC curve of PV30 versus OS and calculated the optimal cutoff for PV30 at 55.3% .The clinical and dosimetric factors associated with OS are summarized in Table % Figure . Dichotos Figure . The med9 cells/L, 175.2 × 1012 cells /L, 119.67, and 2.275, respectively. The median ALC during CRT, PLT, PLR, and NLR during CRT were 0.59 × 109 cells/L, 156 × 1012 cells /L, 264.17, and 5.19, respectively. The median lymphocyte nadir during CRT was 0.27 × 109 cells/L (range: 0.04–0.78). At the nadir, all patients experienced grade 2 or higher lymphopenia, with 111/190 (58%) cases having grade 3 lymphopenia and 58/190 (31%) grade 4 lymphopenia. We plotted the comparative blood cell changes before and after CRT . NLR and PLR were significantly higher (p < 0.001). However, WBC and ANC were not significantly different. Table p = 0.001). The median OS was 19.0 and 54.0 months for G4 and non‐G4 groups, respectively (p = 0.001).The median baseline ALC (ALC‐b), baseline PLT (PLT‐b), baseline PLR (PLR‐b), and baseline NLR (NLR‐b) before chemoradiotherapy (CRT) were 1.43 × 105‐HV55, MPD, and PV5‐PV35 were associated with G4 lymphopenia. Next, stepwise backward regression was performed to select the variable with the lowest Akaike information criterion (AIC) value to establish the model. The final prediction models were constructed by stage‐IVA (p = 0.017), PLR (p = 0.008), HV50 (p = 0.046), and PV30 (p = 0.048).Next, we used a training group of 100 patients from West China Hospital, 30 of whom had G4 lymphopenia. Logistic univariate and multivariate analyses were performed to identify the factors associated with G4 lymphopenia  was strongly linked to G4 lymphopenia and might consequently affect prognosis in LA‐EC patients undergoing CCRT. The study also developed a nomogram for predicting the occurrence of G4 lymphopenia, which showed excellent predictive value in both internal and external validation.According to the NCCN guidelines for esophageal cancer, OARs are recommended with dose‐volume constraints of HVExtensive studies have shown that higher cardiac radiation doses were significantly associated with cardiotoxicity, which can affect clinical outcomes.10 and heart V10 and poorer survival.5 and affected prognosis.30 and PV55) were significantly associated with OS, but the mechanism was not further explored.Lymphocytes are known to be extremely sensitive to radiation, with D50 (the dose required to inactivate 50% of the cells) as low as 2 Gy.https://clinicaltrials.gov identifier: NCT 04210115 and NCT 03957590), which may become the standard treatment modality for LA‐EC in the future. We eagerly await their updates on clinical advantages, treatment‐related toxicity, and other translational work.T lymphocytes play a central role in the immune system, as CD8+ and CD4+ T‐cells can directly damage tumor cells or generate cytokines that activate effector cells, thereby improving the prognosis of EC patients.Our study had some limitations. First, due to its retrospective nature, this study was subject to selective bias. Second, the sample size in this study was relatively small as we only included patients who received radical CCRT and IMRT under strict inclusion criteria. In several similar studies, clinical factors such as chemotherapy  and RT techniques (protons/IMRT) might have impacts on treatment outcomes for these LA‐EC patients. Additionally, even though we recorded blood counts weekly, as recommended by the NCCN guidelines,In conclusion, higher pericardial radiation doses are strongly associated with G4 nadir and affect the prognosis of LA‐EC patients undergoing radical CCRT. In the era of immune checkpoint inhibitor therapy, optimization of RT regimens to conserve the immune system may be an important direction to improve prognosis.Youling Gong conceived and designed the study. Xianyan Chen, Xiaoman Tian, Xuanwei Zhang, Qianyue Deng and Min Wang collected the data. Xianyan Chen and Youling Gong analyzed and interpreted the data and drafted the article. Yong Xu, Lin Zhou, Xiaojuan Zhou, Yongmei Liu, Bingwen Zou, Rui Ao, You Lu critically revised the manuscript. All the authors approved the final submitted version.The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.The authors declare no conflicts of interest.