Aspergillus antibody testing tools are lacking. This study aimed to investigate the diagnostic laboratory findings in a retrospective CPA cohort and to evaluate the performance of a novel Aspergillus IgG lateral flow assay . During January 2016 and December 2021, suspected CPA patients were screened at the Center for Infectious Diseases at Huashan Hospital. A total of 126 CPA patients were enrolled. Aspergillus IgG was positive in 72.1% with chronic cavitary pulmonary aspergillosis, 75.0% with chronic necrotizing pulmonary aspergillosis, 41.7% with simple aspergilloma, and 30.3% with Aspergillus nodule(s). The cavitary CPA subtypes had significantly higher levels of Aspergillus IgG. Aspergillus IgG was negative in 52 patients, who were finally diagnosed by histopathology, respiratory culture, and metagenomic next-generation sequencing (mNGS). Sputum culture was positive in 39.3% (42/107) of patients and Aspergillus fumigatus was the most common species . For CPA cohort versus controls, the sensitivity and specificity of the LFA were 55.6% and 92.7%, respectively. In a subgroup analysis, the LFA was highly sensitive for A. fumigatus-associated chronic cavitary pulmonary aspergillosis . Given the complexity of the disease, a combination of serological and non-serological tests should be considered to avoid misdiagnosis of CPA. The novel LFA has a satisfactory performance and allows earlier screening and diagnosis of CPA patients.Chronic pulmonary aspergillosis (CPA) is a chronic and progressive fungal disease with high morbidity and mortality. Avoiding diagnostic delay and misdiagnosis are concerns for CPA patients. However, diagnostic practice is poorly evaluated, especially in resource-constrained areas where IMPORTANCE There are concerns on avoiding diagnostic delay and misdiagnosis for chronic pulmonary aspergillosis due to its high morbidity and mortality. A proportion of CPA patients test negative for Aspergillus IgG. An optimal diagnostic strategy for CPA requires in-depth investigation based on real-world diagnostic practice, which has been rarely discussed. We summarized the clinical and diagnostic laboratory findings of 126 CPA patients with various CPA subtypes. Aspergillus IgG was the most sensitive test for diagnosing CPA. However, it was negative in 52 patients, who were finally diagnosed by non-serological tests, including biopsy, respiratory culture, and metagenomic next-generation sequencing. We also evaluated a novel Aspergillus IgG lateral flow assay, which showed a satisfactory performance in cavitary CPA patients and was highly specific to Aspergillus fumigatus. This study gives a full picture of the diagnostic practice for CPA patients in Chinese context and calls for early diagnosis of CPA with combined approaches. Aspergillus species. In past decades, CPA has gained increasing clinical attention and is recognized as a potential public health threat. The disease burden of CPA is estimated to be over 3,000,000 cases worldwide, and approximately one-third of patients have it as a sequel to pulmonary tuberculosis (PTB) (Chronic pulmonary aspergillosis (CPA), first systemically recognized in early 1980s , is a chis (PTB) . Patientis (PTB) . OptimalAspergillus infection. Currently, Aspergillus IgG antibody testing plays a central role in CPA diagnosis, and the most widely used method is enzyme-linked immunosorbent assay (ELISA)  . The sen (ELISA) , 7. The en noted . A combien noted .Aspergillus IgG assays are often not accessible or cost-effective in many resource-constrained areas, where direct microscopy and fungal culture are alternative, low-sensitivity methods of CPA diagnosis. In recent years, immunochromatography has been used to detect Aspergillus-specific antibodies in the form of a lateral flow assay (LFA). LFA has the advantages of simplicity, speed, and cost-effectiveness over ELISA in identifying CPA patients  in our CPA cohort. The illustrative and representative examples of the LFA were shown in Globally, CPA is still under-recognized because current commercial patients . In thisA total of 126 CPA patients were enrolled in this study, including 62 proven and 64 probable cases, during January 2016 and December 2021. The geographic distribution of the CPA patients is illustrated in Aspergillus nodules , chronic necrotizing pulmonary aspergillosis , simple aspergillosis , and chronic fibrosing pulmonary aspergillosis .Demographic and clinical characteristics of CPA patients are shown in A. fumigatus  was the most common species. Bronchoscopy was performed in 51 patients and bronchoalveolar lavage fluid (BALF) samples were routinely sent for fungal culture, with a positivity rate of 15.7% (8/51), which identified A. fumigatus in 5 patients, A. niger in 2 patients, and unspecified Aspergillus in 1 patient. PCR was positive for 21 patients by metagenomic next-generation sequencing (mNGS), and 6 of these also had compatible positive sputum culture results. Both mNGS and fungal culture identified A. fumigatus in 4 patients; the Aspergillus species in the remaining 2 patients were confirmed by mNGS and included A. niger and A. flavus, respectively. A total of 62 patients had histopathological findings suggestive of non-invasive Aspergillus hyphae through percutaneous/transbronchial lung biopsy and/or surgery. Most of these proven cases were referred from other hospitals. Notably, patients presenting with Aspergillus nodule(s) were common in our cohort, and 84.8% (28/33) were pathologically confirmed. For the remaining 5 probable cases, we observed responsiveness to antifungal treatment on follow-up.Diagnostic laboratory findings are detailed in Aspergillus IgG detected by the IBL ELISA . The positivity rate was 72.1% in CCPA, 75.0% in CNPA, 41.7% in SA, and 30.3% in AN patients, as shown in Aspergillus IgG antibody than the SA and AN patients, as shown in A. fumigatus and non-A. fumigatus cases, as shown in Aspergillus IgG was negative in a total of 52 CPA patients, who were diagnosed by histopathology (n = 38), sputum fungal culture (n = 7), and mNGS (n = 7).A total of 74 patients had positive n = 27), mucormycosis (n = 9), mycobacterial lung disease (n = 5), lung cancer (n = 4), nocardiosis (n = 3), penicilliosis (n = 2), histoplasmosis (n = 2), chromomycosis (n = 1), coccidioidomycosis (n = 1), and actinomycosis (n = 1). The specificity of the Aspergillus IgG LFA was 92.7% . The diagnostic performance of the LFA is summarized in Aspergillus species, the LFA performed better for detecting A. fumigatus cases than non-A. fumigatus cases .Control sera were obtained from 55 patients with proven lung diseases, including cryptococcosis . Among the 126 patients enrolled in the cohort, positive Aspergillus IgG detected by ELISA was the only microbiological diagnostic finding in 10 patients. Aspergillus IgG antibody (P = 0.895). Agreement between the LFA and ELISA was shown in 71.5% (83/116) of the patients, with a Cohen’s kappa coefficient of 0.426 (95% CI: 0.261 to 0.591).Patients with positive Aspergillus IgG detected by the ELISA as the only microbiological evidence, while the remaining 17 had additional supportive laboratory findings, including fungal culture (n = 9), histopathology (n = 8), and mNGS (n = 3). Of the 16 patients with negative ELISA and positive LFA results, all were diagnosed by non-serological tests, including histopathology (n = 9), fungal culture (n = 5), and mNGS (n = 4).A total of 36 patients had inconsistent results. Of the 20 patients with positive ELISA/negative LFA results, 3 had A. fumigatus was the most common causative species. Aspergillus IgG has proven to be reliable for establishing and/or confirming a CPA diagnosis compared with routine non-serological tests. In terms of simplicity, speed, and cost-effectiveness, the novel Aspergillus IgG LFA is deemed superior to the traditional ELISA for screening and earlier diagnosis of the disease, especially in resource-constrained settings  detection has been recognized as a key diagnostic test for invasive pulmonary aspergillosis (IPA) in neutropenic patients, its value for CPA diagnosis is still controversial even with BALF samples  for CCPA and 75.0% (15/20) for CNPA and a specificity of 89.1% in the diseased controls. The novel Aspergillus IgG LFA, which uses colloidal gold immunochromatography, is portable and easy to use. When testing positive samples, the Aspergillus IgG antibody in the sample combines with gold-conjugated mouse anti-human IgG antibody to form an immune complex, which flows forward on the nitrocellulose membrane through the chromatography effect. In this study, we investigated its value for CPA diagnosis. The LFA showed a similar performance compared with the Aspergillus IgG ELISA, while it was more specific to A. fumigatus because it detected antibody in only 1 out of 12 patients with non-A. fumigatus infections. Because the antigen coated in the LFA test is purified thioredoxin reductase GliT from A. fumigatus, the LFA is expected to be less sensitive for detecting non-A. fumigatus cases, which are less frequently encountered in the CPA population. In contrast, the positive rate of Aspergillus IgG was not significantly different between A. fumigatus and non-A. fumigatus cases for the ELISA . The coated antigen from A. fumigatus in this assay is not disclosed. Of note, a substantial proportion of SA and AN patients were serum negative, which has also been observed in other studies using the ImmunoCAP system, a highly sensitive fluorescent enzyme immunoassay (Aspergillus nodule(s), fewer than half (10/24) had positive Aspergillus IgG antibody  criteria . The sensitivity and specificity of the assay were 91.6% and 98.0%, respectively, in a French multicenter study (Aspergillus LFA may be because it detects both IgG and IgM in addition to different antigens used (mixed antigen from A. fumigatus). However, the assay showed less satisfactory performance in an Indonesian post-tuberculosis cohort, with sensitivity and specificity of 85.0% and 72.1%, respectively . Diseased control sera were collected from patients with pathologically confirmed active lung diseases without evidence of Aspergillus infection or colonization. This study was reviewed and approved by the Huashan Hospital ethics review committee .This study was carried out at the Center for Infectious Diseases, Huashan Hospital, Fudan University. During January 2016 and December 2021, patients who had persistent or progressive radiological abnormalities on a chest CT scan and presented clinical manifestations for at least 1 month were defined as suspected cases and were classified into proven and probable CPA cases based on the diagnostic criteria described below. Demographic data clinical and diagnostic laboratory findings of these patients were collected. After establishing the CPA cohort, we evaluated the performance of an Aspergillus spp. with or without positive culture from sterile lung tissue were defined as proven cases. Aspergillus hyphae did not invade the lung parenchyma except in cases of chronic necrotizing pulmonary aspergillosis. A probable diagnosis was made if the patient had any of the following evidence of Aspergillus infection: (i) positive Aspergillus DNA results in respiratory specimens, (ii) Aspergillus growth from respiratory culture, or (iii) positive Aspergillus IgG detected by an Aspergillus IgG ELISA . Fungal culture and species identification were performed by the clinical mycology laboratory following standard operating procedures as previously described  and the European Respiratory Society (ERS) , with miescribed . Isolateeviously .Aspergillus nodule(s).CPA patients were divided into five different subtypes based on clinical and radiological characteristics according to ESCMID guidelines : chronicAspergillus IgG ELISA was performed according to the manufacturer’s instructions as described by Kim et al.  was added into the sample hole of the cassette. The result was read by the naked eye after 10 min. A red band in both the test (T) and control (C) lines indicated a positive result, while a red band only in the C line indicated a negative result. If there was no red band in the C line, the test was invalid and was repeated. In rare cases, an “equivocal” result was seen in which a very faint band appeared in the T line. This result was considered negative. Positive results ranged from weak positive (+) to strong positive (+++) based on the relative intensity of the C and T lines, see The m et al. . For theAspergillus IgG ELISA and LFA was performed using McNemar’s test. Diagnostic performance, including sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and Youden’s index of the LFA were calculated. P < 0.05 was considered statistically significant for all tests. Data were analyzed using Stata/SE version 15.0 software , R statistical software version 4.1.3  within RStudio version 1.4.1564 (RStudio), and Graph Pad Prism 9.0 .Continuous data were expressed as median and interquartile range (IQR) and were compared using a Mann–Whitney U test. For categorical variables, data were reported as numbers (percentages) and compared between groups using Pearson’s chi-squared test or Fisher’s exact test, as appropriate. Pairwise comparison of sensitivity levels between the The data that support the findings of this study are available on request from the corresponding authors. Data are not publicly available due to privacy and ethical restrictions.


Meningoencephalitis of unknown origin is a common cause of severe neurological disease in dogs. The term covers a heterogeneous group of noninfectious inflammatory diseases, with immune dysregulation widely accepted as the underlying disease mechanism. Current treatment consists of immunosuppression, with corticosteroids being the mainstay of virtually all treatment regimens. However, side effects of corticosteroids can be severe, and might be the cause of death in some patients. This retrospective, multi-centric study aimed at describing a population of Scandinavian dogs with meningoencephalitis of unknown origin in regards to reported side effects and cause of death, and to highlight possible differences in survival, when comparing corticosteroid monotherapy with other treatment regimens.(P = 0.31) in survival between dogs treated with corticosteroid monotherapy , dogs receiving a combination of corticosteroids and ciclosporin , and dogs receiving corticosteroids combined with either cytosine arabinoside, leflunomide, or a combination of 2 or more add-on drugs . Side effects were registered for 47/63 dogs. Polyphagia (n = 37/47), polyuria/polydipsia (n = 37/47), diarrhea (n = 29/47) and lethargy (n = 28/47) were most frequently reported. The most common cause for euthanasia was relapse , followed by insufficient or lack of treatment response . Side effects were the direct cause of euthanasia in 2/35 dogs (5.7%).Within the 5-year study period, 63 dogs were included. Of these, 35 (49.3%) died or were euthanized during the study period. Median survival time from time of diagnosis based on Kaplan-Meier curves for the overall population was 714 days . There was no statistically significant difference A large proportion of dogs in the overall population were euthanized due to relapse, emphasizing a need for treatment regimens aimed at specifically preventing relapse for an improved long-term survival. Side effects in dogs receiving corticosteroid monotherapy were rarely a direct cause of death, but were reported for all dogs. No statistically significant difference in survival was found when corticosteroid monotherapy was compared to other treatment regimens. Meningoencephalitis of unknown origin (MUO) is an “umbrella” term covering different histopathological subtypes of noninfectious inflammatory central nervous system (CNS) diseases affecting dogs, including necrotizing meningoencephalitis (NME), necrotizing leucoencephalitis (NLE) and granulomatous meningoencephalitis (GME). However, as a histopathological diagnosis is rarely reached ante mortem, the term MUO is commonly applied in a clinical setting . The patDifferent treatment protocols have been suggested; collectively, the purpose is suppression of the immune system, thereby suppressing the improper immune response causing either the formation of perivascular cuffs and a mixed lymphoid inflammatory process (GME) or necrosis and infiltration of lymphocytes, plasma cells and monocytes (NLE and NME) , 14. MonTreatment response to CS in dogs suffering from MUO may vary . Even thThe aim of this study was to investigate side effects to treatment, survival and cause of death for a population of Scandinavian dogs diagnosed with MUO. This study further aimed to compare CS monotherapy with combination treatment protocols in regards to survival.This retrospective cohort multicenter study included medical records from dogs presented at the University Hospital for Companion Animals (UHCA), University of Copenhagen, Denmark, and Fredrikstad Small Animal Hospital (FSAH), Fredrikstad, Norway. Initially, medical records from January 2016 to November 2021 from dogs where MUO was considered a differential diagnosis were reviewed, and inclusion and exclusion criteria were then applied.Inclusion criteria were based on the guidelines proposed by Granger et al.  with theIn regards to imaging, abnormal findings on MRI examinations, if any, had to be consistent with inflammatory disease  , 36. EveDogs, in which only CT was available were included with imaging changes considered compatible with MUO, i.e., ill-defined hypo-attenuating lesions with/without variable and ill-defined contrast enhancement . HoweverDogs were excluded if there were insufficient medical records, other neurological diseases identified explaining clinical signs, if histopathology was non-diagnostic, or if they suffered from severe concurrent systemic disease. Dogs diagnosed with meningomyelitis only were also excluded.All information from patient records regarding signalment, clinical and neurological findings, results from paraclinical examinations, registered side effects, treatment response and cause of death were noted. For cause of death, dogs were allocated to one of the following: ‘Spontaneous death’, ‘Euthanized at diagnosis’ (either by owner request or recommended by the responsible veterinarian), ‘No treatment response’ , ‘Relapse’ , ‘Side effects’ , ‘Possible side effects’ (when cause of death may indirectly be attributed to treatment), or ‘Other’ (including death with no obvious relation to disease or treatment). Changes in weight were calculated based on weight at initial presentation and the last registered weight in the patient record. Changes in body weight were investigated in relation to treatment (data missing in 1/47). For dogs where no time of death was registered, owner contact for a clinical status on their dog was attempted.Included dogs were further allocated to one of the following subgroups: Monotherapy with CS, therapy with CS + Ci, therapy with CS + CA, therapy with CS + at least two add-ons (CS + 2), and therapy with CS + Le. Dogs that died or were euthanized within the first 72 h from diagnosis were allocated to a separate group termed ‘early death’. This cut-off was set based on previous studies , showingIn order for dogs to be included, diagnostics to rule out other diseases as the cause of clinical signs needed to be performed, as deemed by the responsible clinician.CSF analysis was either performed in-house (UHCA) or sent to an external laboratory ). Analysis included a macroscopic evaluation, protein concentration measurement, manual red blood and total nucleated cell counts, and a differential cell count. Protein content was either measured semi-quantitatively using a urine multi-dipstick  or quantitatively ). At UHCA, MRI examinations were performed using a 0.2 Tesla magnet  or a 1.5 Tesla magnet (Phillips). At FSAH, a 1.5 Tesla magnet  was used for all examinations. For all MRI examinations, a gadolinium-based contrast was given at a dosage of 0.1 mmol/kg IV. CT examinations were performed using either Siemens Somatom Emotion single-slice helical CT-scanner (UHCA) or a GE Healthcare Brivo CT 385 16-slice helical CT-scanner (FSAH), and an iohexol-based contrast was used at a dosage of 300–600 mg/kg IV.Data regarding signalment, initial neurological examination, paraclinical parameters, CSF analysis, advanced imaging, side effects, and cause of death were presented descriptively. Median, mean and range were calculated when applicable for all data. Side effects were described for the overall population, and for treatment subgroups. Median survival time from the time of diagnosis (MST) based on Kaplan-Meier survival curves were evaluated for the overall population, for the two largest treatment subgroups separately (CS and CS + Ci), and the three smaller treatment subgroups  combined. Even though the combination of different treatment groups did not allow for a differentiation between their effects on survival, an overall survival could be calculated, and compared to CS, as well as CS + Ci. The three subgroups were compared using Gehan-Breslow-Wilcoxon test with the statistical significance level set at P < 0.05 . Since survivors are censored from the Kaplan-Meier survival curve steps, dogs that were lost to follow-up or still alive at the end of the study period were visualized with thick marks directly on the curve. To give an overview of which dogs fell into which group, and to allow for comparison to other studies, a summary of the population at different time intervals was made .The initial investigation of patient records yielded 103 dogs with MUO as a possible differential diagnosis. Of these, 20 were diagnosed with other diseases as the cause of clinical signs and thus excluded, and 20 were excluded due to other causes, leaving 63 dogs in the study population . Chihuahuas (n = 12), Pugs (n = 9) and Yorkshire terriers (n = 5) were the most common breeds. Mean age at presentation was 56.6 months . A summary of the signalment for the study population can be seen in Table The time from onset of clinical signs until the first neurological examination varied greatly. 37/63 dogs (58.7%) were presented for examination within seven days of clinical onset; 8/63 dogs (12.7%) between 8 and 14 days, and 9/63 dogs (14.3%) between 15 and 30 days of clinical onset. 9/63 dogs (14.3%) had had clinical signs for more than 30 days at the time of presentation. For 2 of these dogs (3.2%), clinical signs had been present for more than six months.On initial neurological examination, 15/63 dogs (23.8%) were described as alert. 40/63 dogs (63.5%) were deemed depressed, 1/63 dogs (1.6%) was stuporous, and 3/63 dogs (4.8%) presented in a comatose state. The mental state of the remaining 4/63 dogs (6.3%) could not be evaluated, as they presented in either status epilepticus  or were in a medically induced sedated state . Notably, a history of seizure activity was given in more than a third of the included dogs .40/63 dogs (63.5%) presented with abnormal movement, with 37/63 (58.7%) presenting with ataxia, and 2/63 (3.2%) presenting with paresis/plegia. Proprioceptive placing reactions were affected on one or more legs in 39/63 dogs (61.9%). Of these 39 dogs, 23/63 (36.5%) had proprioceptive placing deficits on one side. On cranial nerve examination, the most commonly reported finding was reduced menace response, which was noted in 31/63 dogs (49.2%). Findings on neurological examinations are summarized in Table A clinical neurolocalization was noted for 56/63 dogs (88.9%), with 34/63 dogs (54.0%) being characterized as having a multifocal localization. 22/63 dogs (34.9%) had signs compatible with a focal neurolocalization: Cerebrum  or brain stem . For 7/63 dogs (11.1%), localization was not specified.Angiostrongylus vasorum . Other performed tests included investigation for antibodies against Anaplasma phagocytophilum and/or platys (not differentiated by the test), Borrelia burgdorferi, Ehrlichia canis, Ehrlichia ewingii, and Dirofilaria immitis, which were performed in 5/63 dogs , investigation of serum antibodies against Leptospira (1/63 dogs), PCR for tick borne encephalitis virus and/or the agent causing Lyme disease (2/63), and serum antibody measurements for Toxoplasma gondii (IgG and IgM) and/or Neospora caninum (2/63). All tests for infectious diseases were negative, apart from a snaptest  performed in one dog, which was positive for Anaplasma spp. This test result was not followed up with PCR analysis, as Anaplasma spp. was deemed unlikely as the cause of the clinical signs, and multifocal lesions explaining signs were identified on MRI examination of the brain. The dog responded to treatment with CS + Ci and lived 24 months after initial diagnosis.For all dogs, as a minimum, routine hematology and blood biochemistry were performed. Samples were either analyzed at place of inclusion, at the referring veterinarian, or at an external laboratory. For 48/63 dogs, analysis of canine C-reactive protein (CRP) was available. 12/48 dogs (25.0%) had a CRP above the local reference range . Tests for infectious diseases were performed at the clinician’s discretion. The most frequently performed test (16/63 dogs) was for antibodies against MRI examination was performed in 34 dogs. In 3/34 (8.8%) the MRI study was deemed normal. For the dogs where MRI was available for review, and deemed abnormal (30 dogs), all had hyperintense lesions on both T2-weighted and FLAIR images. In 26/30 dogs (86.7%) enhancement of identified lesion(s) or a patchy diffuse enhancement were noted post contrast. Multifocal lesions were most commonly reported , followed by diffuse lesions  and focal lesions . Lesions were most often localized to cerebrum , followed by lesions in the brain stem , spinal cord , thalamus  and cerebellum . In 5/30 dogs (16.6%), imaging findings consistent with cerebellar herniation were reported, with one of these dogs also having findings consistent with caudal transtentorial herniation. In 2/30 dogs (6.7%), signs of elevated intracranial pressure were noted. In one dog, MRI was performed at another animal hospital. Images were therefore not available for review, but the imaging report specified two findings; one lesion in the caudal medulla oblongata, and one lesion related to the right olfactory recess. Both lesions were consistent with an inflammatory process with necrotic changes.CT examinations were available for 27 dogs. In 6/27 (22.2%) dogs, either multifocal lesions  or focal lesions  were noted. In all six dogs, contrast uptake was noted, and in two, a rim-like contrast enhancement was seen. In 3/27 dogs (11.1%), lesions could not be classified as focal or multifocal, but mass effect was noted. In 19/27 dogs (70.4%), no signs of inflammatory disease were noted. Signs of cerebellar herniation were reported in 1/27 dogs (3.7%).Results of a complete or partial CSF analysis were available for 45/63 dogs. Reasons for missing analyses were clinical contraindications for CSF tap (n = 10), marked blood contamination (n = 4), death or euthanasia before CSF tap could be performed (n = 1), and causes not specified (n = 3).An exact total nucleated cell count (TNCC) was available for 30/45 dogs. For five other dogs, pleocytosis was noted, but the TNCC was not specified. The median TNCC was 94 cells/µL . The TNCC was > 5 cells/µL in 32/35 dogs (91.4%). For 3/35 dogs (8.6%), TNCC was normal (TNCC ≤ 5/µL).Cytological evaluation was available for 45 dogs. Of these, the three dogs that had a normal TNCC also had a normal cytological evaluation. The remaining 42/45 dogs (93.3%) had a pleocytosis. 31/45 dogs had a pleocytosis with predominance of monocytes; of these, 14/45 dogs (31.1%) had a predomination of small mononuclear cells, 5/45 (11.1%) had a predominance of large mononuclear cells, and 12/45 (26.7%) had a pleocytosis with almost equal amounts (40–60% of each) of small and large mononuclear cells. A mixed pleocytosis, with a neutrophil count ≥ 25%, was reported in 6/45 dogs (13.3%). For five dogs, cytological evaluation was reported as abnormal without further specification.Total protein concentration measurement was available for 34 dogs. For 8/34 dogs (23.5%), the protein count was less than 30 mg/dL, and therefore deemed normal. For 26/34 dogs (76.5%), total protein concentration was above 30 mg/dL. Of these, 12/34 dogs (35.3%) had a protein count from > 30–100 mg/dL, 10/34 dogs (29.4%) had an abnormal protein concentration between > 100–300 mg/dL, 3/34 dogs (8.8%) had a total protein count between > 300–2000 mg/dL, and 1/34 dog (2.9%) had a protein count > 2000 mg/dL. This particular dog was the same dog that had the highest TNCC overall in the study (4000 cells/µL).Treatment was registered as follows: CS: n = 26, CS + Ci: n = 15, CS + CA: n = 5, CS + 2: n = 5 and CS + Le: n = 3. Of these, at least one follow-up visit was registered for all but seven dogs in the CS monotherapy group, leaving 19 dogs in this group for assessment of side effects. A total of 16 dogs were excluded from investigation of side effects, due to either early death (n = 9) or a lack of follow-up visits registered (n = 7), leaving 47 dogs in total. For an overview of medications given initially, see Table The most commonly reported side effects were polyphagia 37/47, 78.7%) and PU/PD . Gastrointestinal signs such as diarrhea  and vomiting , were also commonly noted. A weight gain of more than 10% was seen in 24/46 dogs (52.2%), whereas weight loss of more than 10% was only seen in 3/46 dogs (6.5%). Only slight differences in side effects were seen between treatment subgroups. 1/5 dogs in the CS + 2 group developed gingival hyperplasia. 5/47 dogs developed corneal ulcers during treatment, two in the CS group, two in the CS + Ci group and one in the CS + 2-group. Side effects are summarized in Table 7, 78.7% Overall, 35/63 dogs (49.3%) died within the 5-year study period. At the one-year mark, 38.1% 24/63 dogs) were dead, 44.4% (28/63 dogs) were alive, and 17.5% (11/63 dogs) were lost to follow-up. In the CS group, 13 dogs (50.0%) died during the study period, and in the CS + Ci-group, 8 dogs (53.3%) died. See Fig. 4/63 dogsKaplan-Meier curves for the overall population n = 63), as well as the three separate subgroups , can be seen in Fig. , as wellThe most frequently reported cause of death in general was relapse of disease , followed by a lack of response to treatment . Of the 35 dogs that died during the study period, 9/35 (25.7%) died or were euthanized within the first 72 h from diagnosis. Of these, five were euthanized due to a lack of initial response to treatment, three were euthanized at diagnosis, and one died spontaneously. Overall, only 2/35 dogs (5.7%) were euthanized due to causes directly related to side effects from medication, one in the CS group, and one in the CS + Le group. For the dog in the CS group, fatigue, vomiting, PU/PD, poor coat quality, muscle atrophy, panting, and a weight loss of 14.3% of initial body weight were described. The dog also further presented with a corneal ulcer and skin wounds with slow healing and in the end developed severe gastrointestinal signs. The dog was euthanized after 99 days of treatment based on the recommendation of the veterinarian responsible for its treatment. For the dog in the CS + Le group, PU/PD, polyphagia and lethargy were reported, and the owner elected euthanasia after a treatment period of 469 days. Cause of death in five dogs was not reported as directly related to MUO or treatment. However, of these, the cause of euthanasia was pancreatitis in one dog, and a gastrointestinal foreign body in the other; both dogs were in the CS group, and euthanasia could potentially be related to treatment with corticosteroids, as both could potentially be secondary to polyphagia - cause of death was therefore classified as possibly related to treatment. Causes of death are summarized in Table This study aimed to investigate cause of death and side effects in a Scandinavian population of dogs with MUO, as well as to compare survival between dogs receiving different treatment regimens. The study population closely resembles those of previous studies with regard to age and weight but included a larger proportion of Chihuahuas and Yorkshire terriers , 26, lik(P = 0.31). However, the limited sample size of all three investigated groups must be kept in mind, and a potential bias towards more severely affected animals in the two subgroups with dogs receiving combination therapy should be considered. In the current study, dogs with more severe clinical signs or an insufficient response to treatment were more likely to receive combination therapy; this is especially true for the dogs included at the UHCA (41/63 dogs), since CS monotherapy in general was the treatment of choice during the five-year study period. On the other hand, dogs receiving combination therapy may be representative of more dedicated owners, as these dogs may need more clinical check-ups and are more costly; this could potentially bias data towards longer survival in this group. A clinical trial with a randomized allocation of dogs to treatment groups with or without add-ons (case control) at the time of diagnosis could be useful. However, this approach may not be ethically acceptable. Alternatively, already existing retrospective raw data could be pooled from multiple locations with sufficient knowledge and diagnostic equipment. This would allow for a larger study population, as well as representation of different treatment regimens since preferred treatment regimens likely differ between different animal hospitals – strict alignment of inclusion and exclusion criteria, as well as standardization of data registration would, however, need to be applied.The MST in the total study population was 714 days (range 0-1678 days). There was no statistically significant difference when comparing survival for dogs treated with CS monotherapy , CS + Ci combination therapy , or dogs receiving other combination therapies in the current study population  Direct comparison to available retrospective studies of MUO survival in regards to treatment regimens is challenging due to differences in study set-ups and inclusion and exclusion criteria. Further, Kaplan-Meier survival curves, which are used in a variety of studies to calculate survival in regards to treatment of MUO , 41–47, In a study by Paušová et al. , 182 dogThe combination of CS + Ci for treating MUO was investigated in a retrospective study by Brady et al. , where aCytosine arabinoside have been investigated for treating MUO in several studies , 45, 46 Little is published regarding treatment of MUO with Le. In Gregory et al.  Le and iOther immunomodulatory drugs that have been investigated in the attempt to treat dogs with MUO includes MMF , 23, 42 When looking at the primary cause of death in the current population, the main cause of death or euthanasia was relapse , followed by a lack of response to treatment . It is important to keep in mind that the decision to euthanize is often complex, and several factors may affect the decision of the owner, as well as recommendations to euthanize by the responsible veterinarian. However, relapse in dogs suffering from MUO has also previously been described to correlate with a shorter survival time . PreventNot surprisingly, the nature of the observed side effects was similar in all treatment groups and characteristic for CS, as CS was used in all treatment regimens. These included polyphagia , gastrointestinal signs , and PU/PD . Due to the retrospective nature of this study, it was not possible to grade side effects objectively, nor to assess if side effects decreased as CS was tapered, or if tapering was done faster for the dogs receiving add-on drugs. A prospective study investigating possible correlations between CS dosage and side effects is warranted, as has been done for dogs suffering from SRMA . InteresMany unspecific side effects were reported during treatment with CS, and for some dogs, it may therefore be difficult to discriminate side effects from ordinary disease events that might have occurred anyway. For instance, corneal ulcers were seen in five dogs; however, they were all brachycephalic breeds (four pugs and one Chihuahua), which are known to have abnormal cranial conformation and buphthalmos, predisposing to corneal ulcers . It is aDue to its retrospective nature, this study has several limitations. Side effects were not recorded in a systematic manner in the medical records and may be underreported. In addition, recording the exact dosage at which specific side effects were seen was not done, and treatment protocols were not standardized, leading to different dosages and time of initiation of add-on drugs, as well as the timing of check-ups. Since data collection went back five years, a group of dogs were lost to follow-up despite attempts to contact owners, and clinical status for these dogs were therefore unknown.The fact that dogs were recruited from two different hospitals allowed inclusion of a larger population; however, it also led to differences in methods of CSF analysis, and the use of different types of MRI and CT machines, as well as differences in preferred treatment protocols. We did also include dogs with a CT examination, but no MRI examination, which may have led to inclusion of dogs with an unclear diagnosis. Larger, multicentric studies might be necessary in order to include sufficient cases, but care should be taken to reduce the methodological differences between places.limitation to this study was the lack of histopathological confirmation of the presumed diagnosis of MUO - however, as brain biopsies are rarely performed ante mortem, studies including only histologically confirmed MUO cases are usually strongly biased towards a short MST. However, we acknowledge the risk of misdiagnosis in the present study of dogs with presumed MUO, and that this may affect study data. For example, there is a risk that dogs with neoplastic lesions may have been included, affecting outcome negatively. Due to the lack of specific biomarkers for MUO, however, even prospective studies may be challenging in this matter.When investigating treatment of MUO, a lack of knowledge of the underlying disease mechanisms poses a great challenge. Current treatment protocols aim at general immune suppression. Investigations of the underlying pathophysiology and identification of specific drug targets might aid in better treatment options with less side effects, as also suggested by Jeffery & Granger .The reported side effects to treatment were rarely a direct cause of death in the current study – however, side effects were reported for all dogs. Median survival time (MST) for dogs with MUO receiving corticosteroid monotherapy did not statistically differ from other treatment groups, but a possible bias towards more severe cases in the combination groups needs to be taken into account when interpreting results. Larger prospective studies, or, alternatively, retrospective studies, are needed to reach a conclusion.Relapse was the number one cause of euthanasia, followed by a lack of response to treatment regardless of treatment group, indicating that the available treatment options are suboptimal. Clinical trials prospectively examining treatment alternatives are warranted if these can be conducted in an ethically acceptable manner. Further investigations into the underlying disease mechanisms are warranted.


The expanding population of older adults with obesity is a public health challenge, in part, because of the increased risk of fractures despite normal or high bone mineral density. Potential factors predisposing to fractures in this group include sarcopenia associated with obesity and impaired bone quality. We aimed to determine the contribution of sarcopenic obesity (SO) indices to bone strength as assessed by microfinite element analysis (μFEA) of high-resolution peripheral quantitative computed tomography (HR-pQCT).2) participated in lifestyle intervention trials at our medical center. All underwent baseline measurements of bone strength (failure load and stiffness) using μFEA from HR-pQCT of the distal radius and tibia. In addition, SO indices [appendicular lean mass/weight (ALM/W) and percent body fat (FM%)] by dual-energy X-ray absorptiometry and handgrip strength (HGS) by dynamometry were assessed. SO was diagnosed and staged based on the 2022 ESPEN and EASO expert consensus statement.One-hundred eighty-nine older (age ≥ 65 years) adults with obesity (BMI ≥ 30 kg/mp < 0.001). In contrast, FM% was negatively correlated explaining 22% to 31% of the variance in failure load and stiffness at the distal radius and tibia . The associations of SO indices with failure load and stiffness remained significant after controlling for age, sex, race/ethnicity, diabetes, and 25-OH vitamin D  (p < 0.001 to 0.011). SO was diagnosed in 75/189 (40%) participants with 66/75 (88%) having functional or metabolic complications (stage II). Participants with SO had lower failure load and stiffness at the distal radius than participants with no SO (both p < 0.05).Both ALM/W and HGS were positively correlated explaining 28% to 36% of the variance in failure load and stiffness at the distal radius and tibia (all These findings demonstrate that lower muscle mass and strength and higher fat mass may impair bone quality. Therefore, interventions that focus on preserving muscle mass and strength while reducing fat mass may be important to decrease fracture risk when older adults with obesity undertake lifestyle intervention therapy. In o2] . Potenti years an2] , which h2] . In addi2] , the mus2] , while f2]  within t2] . However2] .Common tools for diagnosing osteoporosis such as dual-energy X-ray absorptiometry (DXA) that measures areal BMD (aBMD) can be subject to various scanning artifacts  , 15. MorIn this population of older adults with obesity, we hypothesized that ALM/W and HGS would positively contribute while FM% would negatively contribute to failure load and stiffness at the distal radius and tibia. We also hypothesized that those meeting the criteria for SO  would ha2), were sedentary (regular exercise <1 h/week), and had had a stable body weight ( ± 2 kg) and stable medication use for 6 months prior to enrolment. Persons who had severe cardiopulmonary disease , musculoskeletal impairments that precluded exercise training, cognitive impairments , and osteoporosis (T-score −2.5 and below or history of fragility fractures) or who used drugs that affect bone metabolism  or had secondary osteoporosis  were excluded from participation. The current study was approved by the Institutional Review Board of Baylor College of Medicine and the Research and Development Committee of the MEDVAMC.The current study is a secondary analysis of baseline data from older adults with obesity consecutively recruited to two lifestyle intervention trials (diet-induced weight loss and exercise training) at the Michael E DeBakey VA Medical Center (MEDVAMC) in Houston, TX (NCT03329963 and NCT02367105). Participants were recruited from the community through advertisements. Persons were eligible for inclusion in the trials if they were 65 years of age or older, were obese (BMI ≥ 30 kg/mth rib. Blood pressure (BP) was measured with a sphygmomanometer cuff of the appropriate size after the subject rested for approximately 5 min. Serum lipoprotein levels were measured by using an automated enzymatic/colorimetric assay . Plasma glucose was determined by using the glucose oxidase method . 25-Hydroxy vitamin D (25-OH vitamin D) was measured using the immunochemiluminometric assay .Body weight was measured in the morning without shoes following a 12-h fast. Height was measured to the nearest 0.5 cm and BMI was calculated. Waist circumference was measured horizontally at the midpoint between the highest point of the iliac crest and the lowest portion of the 12Lean mass and fat mass of the total body, arms (both right and left), and legs (both right and left) were measured by using dual-energy X-ray absorptiometry . Appendicular body composition was calculated by summing the right and left arms and legs. Areal BMD (aBMD) of the total body, hip, and lumbar spine was also measured by using DXA. The CV at our center for body composition is 1.5% , and forHGS was measured by using a JAMAR handheld dynamometer  following a standard protocol. Briefly, the test was performed in the seated position, with the shoulder adducted and neutrally rotated, the elbow flexed to 90°, and the forearm and wrist in a neutral position. Participants were instructed to squeeze the handle as hard as possible for approximately 3 s. This was repeated three times alternating between right and left hands and 30 s rest between trials. The best of the six grip strength measurements was used for analysis.3 for trabecular and 450 mg HA/cm3 for cortical. Bone microarchitecture was assessed in the trabecular and cortical regions by using voxel-based measurements. Segmentation between cortical and trabecular bone was done when necessary. μFEA element analysis of the radius and tibia, represented by failure load and stiffness, was performed creating μFEA models that accurately describe the actual bone microarchitecture in detail, automatically converting each voxel of bone tissue into equally sized brick elements, as previously described  at the non-dominant distal radius and tibia as previously described by our group . In brieescribed . Corticaescribed . The μFEescribed . The faiescribed . Stiffneescribed .The diagnosis of SO was based on the 2022 European Society for Clinical Nutrition and Metabolism (ESPEN) and the European Association for the Study of Obesity (EASO) international expert consensus statement . AccordiR = −0.93 to 0.65; p < 0.001) and the combined multivariable models regressing ALM/W, FM%, and HGS on fracture load and stiffness adjusted for confounders resulted in VIF >5.0, each SO index was studied separately by multivariable regression models. Standardized beta values (β) and standard errors for standardized β are presented. Statistical significance of the p-values in the multivariable regression models was controlled for multiple testing with the Benjamini–Hochberg procedure  was used for all statistical analyses. Descriptive estimates of body composition and SO indices were stratified by sex. Bivariate and multivariable linear regression analyses assessed the associations between SO indices  and bone strength (failure load and stiffness) at the distal radius and tibia. Since there were no significant SO indices × sex interaction effects, sex was entered as a covariate. In addition to sex, age, race/ethnicity, diabetes, and 25-OH vitamin D were entered in the full model due to their established effects on body composition and bone strength , 33. Forrocedure . The proParticipants were older adults with obesity Table 1.As expected, men had larger whole body and appendicular lean mass than women, while women had larger appendicular fat mass than men Table 2.Each SO index  was associated with bone strength at the distal radius and tibia in bivariate regression analyses Table 4.β values. ALM/W was positively correlated with failure load and stiffness at the distal radius and ulna (R2 = 0.301 to 0.448). HGS was also positively correlated with failure load and stiffness at the distal radius and ulna (R2 = 0.346 to 0.472). Conversely, FM% was negatively correlated with failure load and stiffness at the distal radius and ulna (R2 = 0.299 to 0.432).Adjustments for potential confounders  accounted for an additional 3% to 8% and 9% to 12% of the variance in the SO indices–bone strength relationships at the distal radius and tibia, respectively Table 4.p = 0.02 for between-group comparisons). Cardiometabolic syndrome was present in 57/75 (76%) participants with SO and in 85/114 (75%) participants with no SO (p = 0.48). Importantly, failure load and stiffness at the distal radius were lower in participants with SO vs. those with no SO (both p < 0.05) with a trend for also lower failure load and stiffness at the distal tibia. There were no significant differences in aBMD between participants with SO and no SO  met the criteria for SO, whereas 114 (60%) did not meet the criteria Table 5.In this population of community-living older adults with obesity, we have shown that indices of SO are significantly associated with bone strength as assessed by μFEA of HR-pQCT at the distal radius and tibia. Specifically, both ALM/W and HGS were positively associated with failure load and stiffness at the distal radius and tibia. On the other hand, FM% was negatively associated with failure load and stiffness at the distal radius and ulna. These associations remained significant after controlling for confounders such as age, sex, race/ethnicity, diabetes, and 25-OH vitamin D. These data suggest that fracture risk in older adults with obesity is likely related to decreased muscle mass and strength, as well as to increased fat mass, which may lead to impaired bone quality.To the best of our knowledge, the present study is among the first to correlate multiple indices of SO with bone strength as determined by μFEA of HR-pQCT specifically in older adults with obesity. SO is a geriatric syndrome with a multifactorial etiology, characterized by excess fat mass and sarcopenia that negatively influence patient-centered outcomes . AlthougThe positive association between muscle mass and bone strength can be explained in several ways. One possible explanation for this relationship is the mechanostat hypothesis which explains the adaptation of bone to mechanical loads acting on it . ContracOur findings suggest that lower muscle mass and strength and higher fat mass may compromise bone quality. These findings may help guide clinicians in the treatment of older adults with obesity. Calorie restriction (CR) is effective for reducing fat mass but may also accelerate the age-related decrease in muscle and bone mass . CombiniBecause of different definitions and approaches to diagnose SO, prior studies have reported wide ranges of prevalence  . The pre3-creatine dilution) which may not be routinely available in clinical practice. Accordingly, the ESPEN and EASO consensus statement endorses DXA as an adequate compromise between the precision and accuracy of measurements and the availability of instruments for clinical implementation of SO diagnosis  may be important determinants of bone quality in older adults with obesity. Therefore, interventions that focus on preserving muscle mass and strength while reducing fat mass may be important to maintain bone quality and decrease fracture risk when older adults with obesity undertake lifestyle intervention therapy.dennis.villareal@bcm.edu.The datasets presented in this article are not readily available because restrictions apply to the availability of some or all data generated or analyzed during this study to preserve patient confidentiality or because they were used under license. The corresponding author will on request detail the restrictions and any conditions under which access to some data may be provided. Requests to access the datasets should be directed to The studies involving humans were approved by Baylor College of Medicine Institutional Review Board. The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study.GG: Investigation, Writing – review & editing. AP: Investigation, Supervision, Writing – review & editing, Project administration. YB: Methodology, Supervision, Writing – review & editing, Investigation. AC: Writing – review & editing, Investigation. MS-E: Writing – review & editing, Methodology, Project administration, Supervision. RA-V: Investigation, Writing – review & editing, Methodology, Conceptualization, Supervision. NN: Investigation, Writing – review & editing. CQ: Formal Analysis, Investigation, Methodology, Writing – original draft, Writing – review & editing. DV: Conceptualization, Formal Analysis, Funding acquisition, Investigation, Methodology, Project administration, Supervision, Writing – original draft, Writing – review & editing.


The prognosis of patients with mucinous breast cancer (MuBC) is affected by several factors, but the low incidence of MuBC makes it difficult to conduct extensive and in-depth studies. This study was designed to establish a prognostic model and verify its accuracy in patients with MuBC after chemotherapy and surgery to help develop personalized treatment strategies.Patients with MuBC who underwent chemotherapy and surgery from 2004 to 2015 were identified in the Surveillance, Epidemiology, and End Results (SEER) database. The prognostic factors of patients with MuBC were investigated using a Cox proportional hazards regression analysis. Based on the identified factors, a nomogram was constructed to forecast the overall survival (OS) of patients at 3, 5, and 10 years. Internal (from SEER) and external  verification queues were used to verify the nomogram and demonstrate the predictive capacity of this model.The study comprised 1668 MuBC patients from the SEER database and 107 from the YNCC. The nomogram included four characteristics: age, anatomical stage, surgical method, and radiotherapy. The concordance indices in the training, internal verification, and external verification queues were 0.680, 0.768, and 0.864, respectively. The calibration curves for the nomogram showed excellent agreement between the predictions and observations. This nomogram has good clinical application value according to the decision curve analysis.The prognosis of patients with MuBC who have undergone chemotherapy and surgery can be forecasted using this nomogram, which would be beneficial to help create individualized treatment plans for the affected patients. Breast cancer (BC) has high incidence and mortality rates. In 2020, the incidence of BC was the highest worldwide, and was also the primary cause of cancer-related deaths in women . MucinouMuBC has a higher positivity rate for hormone receptors (HR), less lymph node metastasis, lower anatomical stage, and less human epidermal growth factor receptor 2 (HER-2) overexpression than invasive ductal carcinoma (IDC) . There aA nomogram can integrate various prognostic factors into a statistical model that then generates the numerical probability of a clinical event and is widely used to estimate cancer prognosis and survival . PresentIn this study, patient information was gathered from two sources. First, we searched the SEER database for MuBC  patient information from 2004 to 2015. The following exclusion criteria were used: (1) male; (2) patients with other malignancies in addition to BC; (3) patients with bilateral breast cancer; (4) patients treated without chemotherapy and surgery; (5) patients who lacked vital clinical information ; and (6) patients with missing or unknown survival time. Finally, 1668 qualified patients were enrolled in our research and separated into a training queue (n = 1169) and an internal verification queue (n = 499) in a 7:3 ratio. Furthermore, we retrospectively collected data from 107 eligible patients with MuBC at the YNCC between January 2008 and September 2021 for external verification.Demographic information of patients collected from the SEER and YNCC databases included age at diagnosis, estrogen receptor (ER) status, PR status, HER-2 status, anatomical stage, historic stage, radiotherapy, and surgical method. Patient survival information was obtained by reviewing medical records and telephone follow-ups, and the overall survival (OS) was calculated. OS is defined as the period from histological diagnosis to death (regardless of the cause of death) or the final follow-up. Patients were divided into anatomical stages I, II, III, and IV according to the American Joint Committee on Cancer (AJCC) TNM staging criteria (8th edition). MuBC subtypes were classified according to ER, PR, and HER-2 status, and included HR+/HER2+, HR+/HER2−, HR-/HER2+, and HR-/HER2−. X-tile software may transform continuous variables into categorical variables by determining the most appropriate cut-off values . A cut-oP < 0.05) determined by univariate analysis were incorporated into the multivariable Cox proportional hazards regression analysis to determine independent prognostic factors for MuBC [Percentages and frequencies were used to express the categorical variables. A survival curve was generated using the Kaplan–Meier method, and a log-rank test was used to assess differences in survival among the groups. The variables associated with OS were identified using univariate Cox proportional hazards regression analysis. Significant variables  and R (version 4.1.1) were used for all statistical analyses. And the symbol "*" indicates: P < 0.05.The test level of this study was α = 0.05, and all A total of 1668 MuBC patients from the SEER database and 107 MuBC patients from the YNCC were included in this study. Of these, 1169 (70%) patients from the SEER database were randomly selected for the training queue. Clinicopathological features of the patients are presented in Table According to the survival information obtained from the SEER database, the median survival time was 99.00 (1–179) months in the total queue, 97.00 (1–179) months in the training queue and 101.00 (9–179) months in the internal validation queue. The survival rates were 85.30% (1423/1668), 85.60% (1001/1169) and 84.60% (422/499) in the total queue, training queue and internal validation queue, respectively. By follow-up, the median survival time of the YNCC queue was 87.00 (13–191) months and survival rate was 93.50% (100/107).P < 0.05). These factors were incorporated into a multivariable analysis to investigate independent prognostic factors for MuBC. The results indicated that age and anatomical stage were independent factors affecting MuBC prognosis. Age > 52 years  was associated with poor prognosis and was considered a risk factor. Compared with stage I patients, those with stages II , III  and IV  had worse prognoses. Surgical method and radiotherapy were important prognostic factors for MuBC (Table P < 0.001), advanced stage (P < 0.001), and lack of radiotherapy (P = 0.052) were significantly associated with a poor prognosis. Breast-conserving surgery and mastectomy did not result in different survival rates, but both were superior to patients who underwent modified radical mastectomy (P < 0.001).A Cox proportional hazards regression model was used to investigate the prognostic factors of patients with MuBC in the training queue. Univariate analysis showed that age, anatomical stage, T stage, N stage, M stage, surgical method, radiotherapy, and historic stage were prognostic factors for OS . The calibration curves showed some difference between the predictions and observations, which was likely caused by the short sample size of the YNCC cohort. The DCA graph could not be obtained due to data reasons .P < 0.001), internal verification queue (P < 0.001), and external verification queue (P = 0.001), significant disparities in the OS between the high- and low-risk groups were observed, with the OS of the high-risk group being significantly shorter  [Several prognostic variables for MuBC were identified in this study, and the survival nomogram of MuBC patients after chemotherapy and surgery were constructed and validated, which accurately predicted the OS of patients at 3, 5, and 10 years. Molecular phenotype is an independent prognostic factor for MuBC ( < 0.05) . Our stu < 0.05) . Our stuP = 0.0003) [The AJCC classifies BCstages based on T, N, and distant metastasis (M) to help clinicians clarify the extent of the disease, determine treatment plans, and assess prognosis . Komenak 0.0003) . Lymph n 0.0003) , 21. Our 0.0003) . We beliSurgical treatment is the local treatment strategy for all patients with non-metastatic operable BC . DiffereIt is well known that MuBC has a good prognosis with low rates of local recurrence and distant metastasis. Case reports show that patients with MuBC who are treated surgically can achieve a good prognosis, with disease-free survival of up to 30 years, even without other adjuvant therapy –29. The Some limitations apply to our study. First, this was a retrospective study, which had deficiencies in the completeness and accuracy of information. Second, this study only analyzed patients with complete information, which resulted in a selection bias. Third, there was no subtype classification of MuBC, such as pure or mixed MuBC. Finally, hormone-related endocrine therapy and gene-related targeted therapy have received increasing attention in the treatment of malignant tumors –32. But


The study aimed to investigate the safety, effect, and risk factors of endovascular recanalization of symptomatic non-acute occlusion of the vertebrobasilar artery (SNOVA).Patients with SNOVA were retrospectively enrolled and treated with endovascular recanalization. The clinical data, endovascular treatment, peri-procedural complications, and follow-up outcomes were analyzed.p < 0.0001) better than that at discharge. Stroke occurred in 9 patients (11.4%) in 1 year. In-stent restenosis occurred in 19 (25.33%) patients. Significant (p < 0.05) independent risk factors were blunt occlusion for successful recanalization, duration to recanalization and blunt occlusion for peri-procedural complications, and post-dilatation for both in-stent restenosis and 1-year stroke or death events.A total of 88 patients were enrolled, with an interval to recanalization of 2–89 days (median 23) and an mRS of 2–5 (median 3 and IQR 1). Occlusion was in the intracranial vertebral artery in 68 (77.27%) patients and basilar artery in 20 (22.73%), with an occlusion length of 4.5–43.7 mm (mean 18.3 ± 8.8). Endovascular recanalization was successful in 81 (92.0%) patients. Post-dilatation was performed in 23 (28.4%) patients. After stenting, the residual stenosis was 10%–40% (mean 20.2% ± 7.6%). Peri-procedural complications occurred in 17 (19.3%) patients, with a mortality rate of 5.7%. In total, 79 (95.18%) patients underwent follow-up 5–29 (mean 16.9 ± 5.5) months later, with an mRS score of 0–6 (median 1 and IQR 1) at follow-up, being significantly (Endovascular recanalization of symptomatic non-acute occlusion of the vertebrobasilar artery is feasible even for a long occlusion segment, with a high recanalization rate, a low complication rate, and a good prognosis. Blunt occlusion and duration from the onset to recanalization may affect successful recanalization and peri-procedural complications while post-dilatation may affect in-stent restenosis and prognosis. The intracranial vertebral artery and the basilar artery are the common locations of atherosclerotic occlusion , and aftThis retrospective one-center study was performed after being approved by the ethics committee of our hospital, and all patients had given signed informed consent to participate. Between July 2017 and June 2022, patients with symptomatic non-acute occlusion of the intracranial vertebral artery and basilar artery treated with endovascular recanalization were enrolled. The inclusion criteria were consecutive patients with symptomatic non-acute occlusion of the intracranial vertebral and basilar artery; confirmation of intracranial arterial occlusion by digital subtraction cerebral angiography; 0 in the thrombolysis of the cerebral infarction (TICI) grade; occlusion duration ranging from 24 h to 3 months after symptom onset; MRI-confirmed cerebral infarction in the vertebrobasilar artery distribution area involving the pons, cerebellum, cerebral peduncle, occipital lobe, and thalamus; and repeated transient ischemic attacks or strokes in the posterior circulation even after the best medical treatment. The exclusion criteria were patients with non-atherosclerotic occlusion of the intracranial vertebral and basilar artery; severe calcification in the atherosclerotic lesions confirmed on medical imaging; allergies to heparin, aspirin, clopidogrel, metal implants, or narcotic drugs; intolerance to general anesthesia, comorbidities of malignant tumor or severe liver and kidney dysfunction; combined intracranial aneurysms or anterior circulation diseases; and an expected life span of less than 1 year .Hypertension, diabetes mellitus, and hyperlipidemia were diagnosed according to the recommended criteria in the guidelines , 17. AlcBefore endovascular recanalization, clopidogrel  combined with aspirin  was administered for ≥3 days for all patients. All endovascular procedures were performed under general anesthesia. The Seldinger technique was used to puncture the right femoral artery before inserting a 6F sheath. Thereafter, unfractionated heparin at 50 IU/kg was injected intravenously for systemic heparinization. During the recanalization procedure, 1,000 IU of unfractionated heparin was added intravenously every hour. After a 6-F guiding catheter was placed at the vertebral artery proximal to the occluded location, an Excelsior SL-10 microcatheter  was carefully passed through the occluded segment under the guidance of a 0.014 inch (0.036 cm) micro-guide wire with a length of 200 cm . Thereafter, an ASAHI 300 cm micro-guide wire was exchanged and introduced into the relevant artery before the selection of a Gateway balloon with a diameter of 80% of the normal vessel diameter  for pre-dilatation. An appropriate Enterprise stent  was chosen and placed at the occluded segment with the stent of size 3–5 mm longer than either the proximal or distal end of the occluded segment. If the available stent was not long enough to completely cover the occluded segment, a self-expandable or balloon-expandable stent was used to overlap the previously deployed stent and cover the occluded segment. If the residual stenosis was >50% after stenting, post-stenting dilatation was performed with a balloon.After recanalization, the forward blood flow was evaluated according to the TICI grading system, and TICI ≥2b was defined as successful recanalization of blood vessels. The patient was instructed to stay in bed with monitoring of neurological function. Blood pressure was strictly controlled, with the systolic pressure being maintained at 110–130 mmHg within 24–48 h after recanalization. Head CT was rechecked within 24 h. Usually, the patient was discharged from the hospital 5 days after recanalization. Clopidogrel  and aspirin  were routinely administered for 6 months before when aspirin  alone was taken orally for a long time.Follow-up was conducted by telephone and outpatient or inpatient reexamination. The clinical follow-up time was 1 week, 6 months, and 1 year after the procedure, and all neurological and non-neurological complications were assessed. The patients were scored with the mRS before and after recanalization and during follow-up. Digital subtraction angiography was performed 6 months after recanalization, and restenosis was defined as a stenosis of ≥50% in the stent .The following parameters were evaluated, including the patient’s age, sex, past history, smoking, alcohol abuse, location of occlusion, length and angle of occlusion that was measured after stenting , types oFigures 2In measuring the length of the occluded segment, a 5F 115 cm intermediate catheter was first navigated to the beginning of the intracranial segment of the vertebral artery before performing angiography . At the end of angiography (20 s), the occlusion position displayed was the proximal end of the occlusion, and the compensatory arterial segment displayed at the distal end was the distal end of the occlusion. Postoperative 3-dimensional rotational angiography was performed to accurately measure the length of the proximal and distal end of the occlusion which was determined before the operation.t-test as the median and interquartile range (IQR) if not in the normal distribution and also tested with the chi-square test. Classification data were expressed in frequency and percentage terms and Wilcoxon rank sum test was used. Univariate logistic regression analysis was used to analyze risk factors for successful recanalization, residual stenosis, peri-procedural complications, poor follow-up outcomes (mRS), in-stent restenosis, and 1-year stroke or death events, and parameters with a p-value of ≤0.1 were used in the multivariate logistic analysis for independent risk factors. The odds ratio (OR) and 95% confidence interval (CI) were calculated. Continuous measurement data in the logistic analysis were used in the receiver operating character (ROC) curve analysis. The statistically significant p-value was set at <0.05.Statistical analysis was conducted with the SPSS 17.0 software . Continuous measurement data were presented as mean ± standard deviation if in the normal distribution and tested with the paired p < 0.0001). The arterial angle formed between the segments proximal and distal to the occlusion was 56–176° (mean 148.8° ± 19.4°), and the mRS was 2–5  at admission.A total of 88 patients were enrolled, including 72 male and 16 female patients aged 37–82 years (mean 58.5 ± 9.0) ; Table 1Endovascular recanalization was successful in 81 (92.0%) patients, resulting in a TICI grade 3 reperfusion in all patients . The timp < 0.0001) better than that at admission.Post-dilatation after stenting was performed in 23 (28.4% or 23/81) patients, and the residual stenosis was 10–40% (mean 20.2% ± 7.6%). At discharge, the mRS was 1–6 , which is significantly  patients but failed in five (7.4%). Among the failed patients, two patients successfully underwent Enterprise stent angioplasty of the contralateral inferior vertebral artery for remedy while the other three patients were treated with medications conservatively. Among 63 patients with successful recanalization, the arteries for major blood supply in 62 (98.4%) patients were recanalized, with 33 (52.4%) patients being treated with the Enterprise stent angioplasty, 29 (46.0%) treated with the Enterprise stent combined with a balloon-expandable stent, and one (1.6%) with balloon-expandable stents. Overlapped stents were used in 29 (46.0%) patients while a long Enterprise stent (28 mm or 37 mm) was used in 44 (69.8%) patients. Peri-procedural complications took place in 11 (16.2%) patients including five (7.4%) cases with unsuccessful recanalization. Overall, five (7.4%) patients experienced arterial dissection, and the dissection in three patients resulted in failed recanalization, with the dissection in the other two patients being treated with stenting. Among four (5.88%) patients with intraprocedural thrombosis, one (1.5%) patient with distal thrombosis in the basilar artery died, and the other three (4.41%) patients improved after thrombolysis.In 20 patients with basilar artery occlusion, recanalization was successful in 18 (90%) patients. In total, two (10%) patients failed, including one with dissection and one with bleeding who both died during the peri-procedural period. All 18 patients were recanalized using the Enterprise stent angioplasty. Complications occurred in six (30%) patients including two patients with unsuccessful recanalization. One patient experienced acute thrombosis which was successfully treated with intra-arterial thrombolysis and thrombectomy, one patient had dissection at the procedure and died 15 days later, and one patient experienced pontine hemorrhage and died 1 week later. The death rate was 20% (4/20).Peri-procedural complications occurred in 17 (19.3%) patients, including 11 (16.2%) patients in recanalizing the vertebral artery and six (30%) in recanalizing the basilar artery . A totalp = 0.67) or successful recanalization . Patients with blunt occlusion experienced a significantly (p < 0.05) longer duration for the micro-guide wire to pass through the occlusion than those with sharp  or flame-like occlusion  (p = 0.039). In seven patients without successful recanalization, the failure occurred in blunt occlusion in six (85.71%) patients, but in sharp occlusion, failure occurred only in only one (14.29%) patient. Patients with flame-like occlusion were all successfully recanalized.The occlusion length was not significantly different between patients with and without complications  . The totp < 0.0001) better than that at discharge. Patients with the vertebral artery occlusion had a significantly (p = 0.033) better mRS than patients with the basilar artery occlusion (1.60 ± 0.19 vs. 2.47 ± 0.35). Stroke occurred in nine patients at 1 year resulting in a 1-year stroke rate of 11.4% (9/79). Among 75  patients with angiographic follow-up, in-stent restenosis occurred in 19 (25.33% or 19/75) patients.Among 83 (94.32%) patients who were alive after recanalization, 79 (95.18% or 79/83) with successful recanalization underwent follow-up 5–29 (mean 16.9 ± 5.5) months later , with anp = 0.015, OR 7.67, 95% CI 1.52–38.65) and blunt occlusion  were risk factors for failing recanalization, whereas diabetes mellitus , duration to recanalization , and blunt occlusion  were risk factors for peri-procedural complications  risk factor for poor follow-up mRS outcomes (mRS > 2), whereas post-dilatation was a risk factor for in-stent restenosis  and 1-year stroke or death events . In multivariate logistic regression analysis, significant (p < 0.05) independent risk factors were blunt occlusion for failing recanalization, duration to recanalization, and blunt occlusion for peri-procedural complications, and blunt occlusion was the only independent risk factor for both the in-stent restenosis and 1-year stroke or death events  failed. The blunt occlusion had a long occlusive time with the formation of a thick strong fibrous cap which may indicate the beginning of atherosclerosis. Among 22 patients with blunt occlusion, six (27.27%) failed to be opened, accounting for 85.71% of all seven patients without successful recanalization. This may indicate that patients with blunt occlusion may not be easily recanalized because the recanalized patients with this type of occlusion also experienced significantly longer surgical time and longer duration for the guide wire to pass through the occlusion than those with sharp occlusion. Moreover, in one patient with blunt occlusion of the intracranial vertebral artery, recanalization initially failed but was successful by reverse opening p = 0.57), and the occlusion length was not a risk factor for successful recanalization or peri-procedural complications. In one case with intracranial vertebral artery occlusion, the occlusion length was 23.5 mm even though the recanalization was not successful. However, the occlusive length is an important reference for the use of long opening tools . It is speculated that the vertebral artery occlusion that is difficult to open is not caused by a long segment of atherosclerosis. The atherosclerosis is short in most patients, but the long segment of occlusion is probably caused by secondary thrombi which can be easily opened and compressed by a stent. This is probably because fewer side and perforating branches in the intracranial vertebral artery result in blood flow retardation proximal to the occlusion and subsequent thrombus formation, which leads to longer segments of occlusion in the vertebral artery.In recanalizing VBAO, the occlusion length was not particularly important such as the occlusion shape. Although the occlusion was significantly longer in the intracranial vertebral artery than in the basilar artery in our study, the success recanalization rate was not significantly different between these two locations  patients while a long Enterprise stent (28 mm or 37 mm) was used in 44 (69.8%) patients. The reason is that the vertebral artery occlusion is long with many local thrombi. We prefer to use a closed-cell long stent (such as the Enterprise stent), which is beneficial for subsequent operations. In the use of closed-cell self-expanding stents, we should try not to use overlapped stents in order to reduce the time and complexity of the operation and subsequent occurrence of complications. Thus, a long stent is often used. In the need of overlapped or telescoped stents, it is better to place a balloon-expandable stent at the proximal end of a deployed self-expandable stent, which is beneficial to maintaining the opening and adherence of the overlapped parts of the stent. This is also helpful for endovascular devices to pass through the overlapped stents in case of complications and symptomatic restenosis. In our study, 29 (46.0%) patients were treated with the Enterprise stent combined with a balloon-expandable stent.In recanalizing chronic occlusion of intracranial arteries, the most common complications are dissection and perforation , leadingp < 0.0001). Patients with intracranial vertebral artery occlusion tend to have relatively mild brainstem infarction, which is often caused by low perfusion, and recanalization at the vertebral artery can increase brainstem blood perfusion without causing dissection of the basilar artery and subsequent brainstem injury. Moreover, the basilar artery has rich side and perforating branches, which may be easily injured by endovascular recanalizing operation. This may account for the better outcomes of recanalizing the vertebral artery occlusion, and it may also indicate that endovascular recanalization is safer and more effective in the vertebral artery than in the basilar artery. These rich arterial branches in the basilar artery may necessitate the appropriate selection of the indications for endovascular recanalization and careful operation during the recanalization procedure.Our study found that patients with intracranial vertebral artery occlusion had better complications and follow-up outcomes. In addition to the abovementioned complications, there was only one death in the vertebral artery but four deaths in the basilar artery occlusion in the peri-procedural period though the difference was not significant. Moreover, the 6-month mRS scores were also significantly better in patients with vertebral artery occlusion than those with basilar artery occlusion. Nonetheless, patients with vertebral artery occlusion had a significantly greater occlusion length than those with basilar artery occlusion (21.11 ± 7.9 mm vs. 8.88 ± 3 0.50 mm, In our study, in-stent restenosis occurred in 25.3%  of patieSome limitations existed in this study, including the retrospective and one-center study design, only Chinese patients enrolled, a small cohort of patients, no comparison with symptomatic non-acute occlusion in the anterior circulation, and no randomization, which may all affect the generalization of the outcomes. In the future, large controlled, randomized, controlled studies with multiple medical centers, and races involved will be needed to confirm the findings of this study.In conclusion, endovascular recanalization for symptomatic non-acute occlusion of the vertebrobasilar artery is feasible even for a long occlusion, with a high recanalization rate, a low complication rate, and a good prognosis. Blunt occlusion and duration from onset to recanalization may affect successful recanalization, peri-procedural complications, and poor outcomes, while post-dilatation may affect in-stent restenosis and prognosis. Considering it is a procedure with a high risk of adverse effects, patients should be counseled accordingly.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding authors.The studies involving human participants were reviewed and approved by the Ethics Committee of Henan Provincial People’s Hospital. The patients/participants provided their written informed consent to participate in this study.TL and LZ: study design. ZZ, LW, MG, ZM, YL, JQ, and BG: data collection. ZZ and BG: data analysis. ZZ: supervision. ZZ, LW, MG, ZM, YL, JQ, and BG: validation. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Bacteroidota, Desulfobacterota, Firmicutes, and Proteobacteria increased while those of Acidobacteriota, Chloroflexi, and Planctomycetota decreased. Moreover, milk vetch rotation increased the relative abundance of phosphorus-related gene K01083 (bpp), which was significantly correlated with soil phosphorus content and crop yield. Network analysis showed that taxa of Vicinamibacterales were positively correlated with total phosphorus and available phosphorus, which was a potential taxon contributing to the availability of soil phosphorus stock. Our results indicated that milk vetch rotation could enrich key taxa with latent phosphate-solubilizing ability, increase the content of soil available phosphorus, and finally enhance crop yield. This could provide scientific guidance for better crop production.Legume crop rotation is often adopted in rice cultivation to improve soil productivity. However, little is known about the role of microbes under legume rotation in affecting soil productivity. To elucidate this, a long-term paddy cropping experiment was set up to study the relationship between crop yield, soil chemical properties, and key microbial taxa under a double-rice and milk vetch rotation. Milk vetch rotation significantly improved soil chemical properties compared to no fertilization treatment, and soil phosphorus was a major factor correlated with crop yield. Long-term legume rotation increased soil bacterial alpha diversity and changed soil bacterial community. After milk vetch rotation, the relative abundances of  Ensuring sustainable rice production is imperative for global food security . Increasvia changing the activity of certain enzymes , Jiangxi Province in China. This site has a subtropical monsoon climate with mean temperature of 17.6°C and mean precipitation of 1795 mm annually. The soil of the site is paddy soil developed on quaternary red clay. Double-cropped rice was cultivated on the site for about 30 years, and all agronomic practices were very similar between treatments except for cropping regime.2O5, and 84 kg K2O per ha per year); and (3) NPKGM, double-rice and milk vetch rotation with mineral fertilization . Milk vetch  was planted after harvest of the later rice crop and plowed during flowering stage, where 5,000 kg of fresh milk vetch  ha−1 year−1 was applied.Three cropping and fertilization regimes with three replications were applied: (1) CK, double-rice with no fertilization; (2) NPK, double-rice with mineral fertilization  were collected using an earth-boring auger in a ‘X’ pattern and merged into one soil sample for each plot after rice harvest. Each soil sample was sieved through 2 mm and divided into two parts: one part was air-dried for physiochemical assay, and the other was stored at −40°C for DNA extraction.2.2.Soil pH was determined using a pH meter in a 1:2.5 of soil and water suspension. The total nitrogen (TN) content in soil was determined using the Kjeldahl method . The ava2.3.® High-Fidelity PCR Master Mix (New England Biolabs), 0.2 μM of forward and reverse primers, and about 10 ng of template DNA. Thermal cycling consisted of initial denaturation at 98°C for 1 min, followed by 30 cycles of denaturation at 98°C for 10 s, annealing at 50°C for 30 s, and elongation at 72°C for 30 s, with a final step of 72°C for 5 min. The samples after PCR amplification were sequenced on an Illumina NovaSeq platform, and 250-bp paired-end reads were generated. The raw reads were denoised, dereplicated, and clustered using a VSEARCH pipeline. High-quality sequences of 84,811–141,764 reads per sample were obtained, and we finally obtained 12,913 operational taxonomic units (OTUs). A trained Naïve Bayes classifier based on the Silva 138 database was deployed to classify the OTU sequences . The hyper-variable region (V4–V5) of prokaryotic 16S rRNA was amplified using the primers 515F (5′-GTGCCAGCMGCCGC GGTAA-3′) and 907R (5′-CCGTCAATTCCTTTGAGTTT-3′) with barcodes. All PCR reactions were carried out with 15 μL of Phusionequences . After fgcd; K01083, bpp; and K06137, pqqC) and taxa contributing up to 80% of their abundance were analyzed.Bacterial community gene abundances were predicted using Phylogenetic Investigation of Communities by Reconstruction of Unobserved States , and a f2.4.rcorr’ of the R package ‘Hmisc.’ The bacterial Shannon and richness indices were calculated using functions ‘diversity’ and ‘estimateR’ of the R package ‘vegan,’ respectively. Constrained principal coordinate analysis (CPCoA) was adopted to display the discrepancies of bacterial community structure between treatments using function ‘capscale’ of the R package ‘vegan.’ A network containing TP, AP, rice yield, and OTUs contributing to the previously selected phosphorus-related genes was constructed based on the Pearson correlations with p < 0.001   .Pearson correlation analysis showed associations between rice yield and soil chemical properties . Among a3.2.Bacteroidota, Desulfobacterota, Firmicutes, and Proteobacteria but reduced those of Acidobacteriota, Chloroflexi, and Planctomycetota  > K06137 (pqqC) > K01083 (bpp) .The predicted absolute abundances of the enriched genes were in the following order in all treatments: K00117 (83 (bpp) . The abuBryobacterales, Solibacterales, Vicinamibacterales, Rhizobiales, and Pedosphaerales were the top five taxa contributing most to K00117 abundance. Xanthomonadales, Flavobacterales, Streptomycetales, Gemmatimonadales, and Myxococcales were the top five taxa contributing most to K01083. Acidobacteriales, Rhizobiales, Nitrospirales, Burkholderiales, and Caulobacterales were the top five taxa contributing most to K06137. Bryobacterales, Solibacterales, and Pedosphaerales contributed more K00117 genes in CK, while Vicinamibacterales and Rhizobiales contributed more K00117 in NPKGM. Gemmatimonadales and Myxococcales contributed more K01083 genes in NPK, while Xanthomonadales, Flavobacterales, and Streptomycetales contributed more K01083 in NPKGM. Acidobacteriales and Rhizobiales contributed more K06137 genes in CK, while Nitrospirales contributed more K06137 genes in NPK. Burkholderiales and Caulobacterales contributed more K06137 in NPKGM.3.4.Gemmatales were negatively correlated with TP, AP, and crop yield, while nodes belonging to Vicinamibacterales were positively correlated with TP and AP. From the order Polyangiales, OTU 6233 had a positive correlation with TP and AP, while OTU 8505 was negatively correlated with AP. The OTU 2853 was positively correlated with AP, while another Acidobacteriales (OTU 7944) was negatively correlated with AP  and K06137 (pqqC), known as genes that encode enzymes to mediate the resolving of soil phosphate, had non-significant or even negative correlations with soil AP and TP in CK or NPK in our study. Only K01083 (bpp) was positively correlated with AP and TP in NPKGM. A possible mechanism was the phosphate released from soil minerals being easily leached from red paddy soil under high precipitation , the Key Research and Development Program of Jiangxi Province (20212BBF63007), the Central Guidance for Local Science and Technology Development Projects (20231ZDD02003), China Agriculture Research Systems of MOF and MARA (CARS-22), and the Jiangxi Modern Agricultural Innovation Project (JXXTCXQN202008).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.The reviewer YJ declared a shared affiliation with the authors MX, XM, ML, MW, and ZL to the handling editor at the time of review.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Diagnosis and treatment of diffuse malignant peritoneal mesothelioma (DMPM) are still challenging. The aim of the present study was to explore the correlation between CD74, CD10, Ki-67 and clinicopathological parameters, and identify independent prognostic factors of DMPM.Seventy patients with pathologically proven DMPM were retrospectively reviewed. The expression of CD74, CD10 and Ki-67 in peritoneal tissues was detected by immunohistochemical analysis using standard avidin biotin complex (ABC) immunostaining technique. Kaplan-Meier survival analysis and multivariate Cox regression analyses were performed to assess prognostic factors. The nomogram based on the Cox hazards regression model was established. C-index and calibration curve were performed to evaluate the accuracy of nomogram models.r = -0.278), Ki-67(r = -0.251) and TNM stage(r = -0.313). All patients were effectively followed up in the survival analysis. Univariate analysis revealed that PCI, TNM stage, treatment, Ki-67, CD74 and ECOG PS were associated with DMPM prognosis. CD74 , Ki-67,TNM stage , ECOG PS, systemic chemotherapy  and intraperitoneal chemotherapy  were independent predictors by multivariate Cox analysis. The C‑index of the nomogram for predicting overall survival (OS) was 0.81. The OS calibration curve showed good agreement between nomogram-predicted and observed survival.The median age of DMPM was 62.34 years, and the male-to-female ratio was 1: 1.80. CD74 expression was identified in 52 (74.29%) of 70 specimens, CD10 in 34 (48.57%) specimens, and higher Ki-67 in 33(47.14%) specimens. CD74 was negatively associated with asbestos exposure(CD74, Ki-67, TNM stage, ECOG PS and treatment were independent factors affecting prognosis of DMPM. Reasonable chemotherapy treatment might improve the prognosis of patients. The proposed nomogram was a visual tool to effectively predict the OS of DMPM patients. Diffuse malignant peritoneal mesothelioma (DMPM) is a rare aggressive tumor originating from the mesothelial cells lining the peritoneal cavity . The lacThe standard for determining the degree of peritoneal carcinomatosis is peritoneal carcinomatosis index (PCI). PCI is an important indicator for predicting survival, higher PCI is significantly associated with poorer OS and progression free survival (PFS) .Cluster of differentiation (CD) proteins play important roles in tumor development by regulating tumor formation, proliferation and metastasis. It has found that the expression of CD74 is linked to some forms of tumors, such as non-small cell lung cancer  and pancCD10 immuno-expression is reported in several nonhematopoietic neoplasms, such as endometrial stromal sarcoma  and renaKi-67 is a nuclear protein that is detected at every stage of the cell cycle of proliferating cells but is not expressed in G0 phase cells . Ki‑67 iThe patients with DMPM were screened according to the guidelines for pathologic diagnosis of malignant mesothelioma . InclusiOf the 126 patients diagnosed with DMPM during January 2013 and December 2018, 56 patients were excluded according to the criteria  staging system proposed in 2011 , which wThe primary antibodies used for immunohistochemistry were shown in Table Immunohistochemical sections were examined by two independent investigators in a blinded manner, any discrepancies were resolved through re-examination and discussion until a consensus was reached.The standard avidin–biotin-complex peroxidase (ABC) technique was used for immunohistochemical detection. Paraffin-embedded DMPM tissues were cut into 4 μm-thick sections and mounted on glass slides. Sections with the larger number of fixed cells were used for immunohistochemical staining. Each section was deparaffinized, rehydrated and incubated with 0.3% hydrogen peroxide in methanol for 30 min at 25 °C. After washing with PBS, the section was heated in 10 mmol/L citrate buffer (PH = 6) for 10 min. Sections were reacted with antibody against CD74, CD10, and Ki-67. Appropriate controls were used for every case with each staining run. The same protocol was followed for negative controls with omission of the primary antibody. Histologic sections of lymph node, endometrial stroma and breast cancer as mentioned in the antibody datasheets were used as positive controls for CD74, CD10, and Ki-67 staining respectively.The intensity score  and distribution score  for immunostaining were summed into a total score (range 0–6). The positivity intensity was graded according to the total score . Grade 0 was defined as negative, and grades 1+, 2+ and 3+ were defined as positive. According to the positive cell rate of Ki-67 in per 500 tumor cells, we divided Ki-67 into low Ki-67 group (≤ 15%) and high Ki-67 groups (> 15%).After the first treatment, we conducted regular follow-up evaluation for all patients every 3 months through telephone interviews or short massage platform. The last follow-up time and vital status were recorded. OS was defined as the duration from the date of diagnosis to the end point (in months). The end point was defined as patient’s death or December 31, 2019 in this study. Patients who were still alive or lost at the last visit were censored at the date of last follow-up.P values less than 0.05 were considered statistically significant. Statistical analyses were performed using SPSS 22.0  and R version 4.1.3 software (http://www.r-project.org/). Extension packages, including “survival”, “nomogramEx”, “rms” and “survminer” were used.Descriptive statistics for demographic and clinicopathological data were performed. Categorical variables were presented as numbers (percentages), continuous variables were presented as mean ± standard deviation. Correlations between parameters were tested by Spearman rank correlations (Spearman’s rho). Kaplan-Meier analysis was used to calculate the overall cumulative probability of survival, and the log-rank test was used to assess differences in survival. Univariate analysis was performed to assess the association between prognostic parameters and survival. Cox Proportional hazards regression model (stepwise backward method) was used for the multivariate analysis to evaluate the prognostic value of related factors. The clinically significant variables calculated from the Cox proportional hazards model were integrated into a nomogram. The nomogram was formulated to predict the prognosis of DMPM, forest plot of hazard ratio was used to illustrate the results. The predictive accuracy of the model was estimated using the concordance index (C‑index). The calibration curve was used to evaluate the consistency between the predicted survival rate and the actual survival rate. Two tailed Clinical and pathologic features of patients were summarized in Table P = 0.008, r = -0.313), asbestos exposure  and Ki-67  , CD10 was positive in peritoneal tissue of 34 DMPM patients , and higher Ki-67(> 0.15) was in peritoneal tissue of 33 DMPM patients . To assess OS, 12 potential prognostic parameters  were included in the univariate analysis. As shown in Fig. P values < 0.05 in the univariate analysis were included in the model for the multivariate analysis to identify independent influencing factors. All 70 patients were followed up successfully with complete information. At the time of last follow-up, five patients were still alive. Multivariate analysis showed that CD74, TNM stage , Ki-67, ECOG PS and treatment protocols, including systemic chemotherapy and intraperitoneal chemotherapy  were independent prognostic factors of DMPM  is a tumor originating from peritoneal epithelium and mesothelium, it is easily missed or misdiagnosed because of low incidence. The incidence rate of MPM in the United States was 1–2/100 million , but wasP < 0.001) [Epidemiological studies have confirmed that the occurrence and development of tumors are inseparable from inflammation . Continu< 0.001) . Moreove< 0.001) . The con< 0.001) . Our res< 0.001) . This inCD10 is a zinc dependent metalloproteinase expressed on cell surface, it can degrade bioactive peptides in the extracellular matrix. CD10 staining was observed in 54% of diffuse malignant mesotheliomas, slightly more than half of which(55%) showed high immuno-expression . In the PCI is one of the indicators to evaluate the extent of peritoneal mesothelioma lesions. Silja reported that PCI had a significant association with survival and significant prognostic value in patients with MPM . PCI sumECOG PS is an important factor determining prognosis in a number of malignant conditions, including mesothelioma patients . PatientThe expression level of Ki-67 represents the proliferation status of tissues and can be used to determine the malignancy of tumors. DMPM Ki-67 has been demonstrated to be an important prognostic marker . Our stuIn general, epithelial DMPM has a better prognosis than other types . The incCurrently, there is no standard therapy for DMPM, the optimal treatment of DMPM remains controversial. CRS and HIPEC are internationally recommended for the treatment of DMPM, they can effectively improve survival . PemetreIt is always hard to construct a model with universal applicability and high accuracy, and establishment of a survival prediction nomogram of rare tumors based on a single mechanism may be another option . In presOur study has several limitations that may affect prognosis. First, it is a retrospective and single-center study with a small clinical database, institutional heterogeneity and number of patients may affect the results. Second, the lack of CRS/HIPEC information in the present study may affect the prognosis of the DMPM, and these important factors should be considered in future studies. Finally, the factors affecting the prognosis of DMPM are varied and complicated, the indexes we choose are relatively limited and biased to some extent, which may affect the prognosis of the disease. Therefore, comprehensive research on large samples, multi-center, multi-indicators and multi discipline may improve the evaluative accuracy.In conclusion, we determined that CD74, Ki-67, TNM stage, ECOG PS and tumor‑directed treatment were associated with DMPM prognosis. Reasonable chemotherapy treatment may improve the prognosis of DMPM patients. The nomogram is a visual tool to effectively predict OS in DMPM patients, but needs to be tested in prospective clinical trials.