Tribonema aequale that possesses the ability to grow rapidly and synthesize significant amounts of EPA. A series of studies were conducted in a glass column photobioreactor under laboratory culture conditions and in pilot-scale open raceway ponds outdoors. The emphasis was placed on the specific nutrient requirements and the key operational parameters in raceway ponds such as culture depth and mixing regimes. When optimized, T. aequale cells contained 2.9% of EPA (w/w) and reached a very high biomass concentration of 9.8 g L−1 in the glass column photobioreactor. The cellular EPA content was increased further to 3.5% and the areal biomass and EPA productivities of 16.2 g m−2 d−1 and 542.5 mg m−2 d−1, respectively, were obtained from the outdoor pilot-scale open raceway ponds, which were the record high figures reported thus far from microalgae-based EPA production. It was also observed that T. aequale was highly resistant to microbial contamination and easy for harvesting and dewatering, which provide two additional competitive advantages of this filamentous microalga over the unicellular counterparts for potential commercial production of EPA and other derived co-products.It has long been explored to use EPA-rich unicellular microalgae as a fish oil alternative for production of the high-value omega-3 fatty acid eicosapentaenoic acid . However, none of the efforts have ever reached commercial success. This study reported a filamentous yellow-green microalga  Arabidopsis thaliana and Camelina sativa seed oil which contain 3.0% and 3.3% of EPA in their fatty acid profiles [2) into desirable products including PLA and/or EPA. For example, Isochrysis galbana [Thalassiosira pseudonana [Nannochloropsis spp. [Phaeodactylum tricornutum [Halamphora coffeaeformis [Tetraselmis spp. [Nitzschia laevis [Monodus subterraneus [Scenedesmus obliquus [Eustigmatos spp. [Palmitoleic acid PLA, C16:1) is a ω-7 fatty acid that may play a role in preventing chronic metabolic diseases such as insulin resistance, nonalcoholic fatty acid liver disease (NAFLD), obesity, coronary heart disease, and atherosclerosis 6:1 is a . Fish oiprofiles . Convers galbana , Thalasscornutum , Halamphaeformis , Tetrasemis spp. , Nitzscha laevis , and Monerraneus  produce obliquus , Eustigmtos spp. , and somtos spp.  produce tos spp. . The secTribonema spp., in the class Xanthophyceae, have drawn great attention because they possess the ability to produce both PLA and EPA, are resistant to grazers or predators, and have low cell harvesting costs owing to the filamentous nature of these species [Tribonema spp. have so far devoted to PLA production in an autotrophic or a heterotrophic culture mode, and little attention was paid to EPA [In recent years, the freshwater filamentous microalgae  species . As far d to EPA ,20. It wd to EPA ,22. Metad to EPA . If allod to EPA , magnesiArthrospira platensis, Chlorella spp., Dunaliella salina, and Haematococcus pluvialis [Tribonema spp. Are mostly performed at laboratory scale, and a very few attempts were made to grow filamentous Tribonema spp. In ORP [Open raceway ponds (ORP) have been used for mass culture of several microalgae of commercial interest, such as luvialis ,26,27. Cluvialis ,29. Thus. In ORP .Arthrospira platensis, which achieved areal productivity of 21.22 g m−2 d−1, while another conventional pond with a lower depth only had a productivity of 11.05 g m−2 d−1 [Tetraselmis suecica was 8.37 g m−2 d−1 in 15 cm depth ORP, which was comparable with 8.9 g m−2 d−1 in 5 cm depth ORP [To obtain high productivity of microalgal biomass or desirable products in ORP, an appropriate depth of culture suspension is critically important. The areal productivity generally increases with the increase in culture depth. Compared with 20 cm depth ponds, for example, the areal biomass productivity at a depth of 40 cm increased nearly 2-fold . Moreove m−2 d−1 . On the epth ORP .Chlorella biomass productivity was observed for both continuous and daytime-only mixing [Different culture mixing regimes, namely continuous culture mixing versus culture mixing provided only during the daylight period, may not only affect energy consumption associated with the operation of the paddlewheel but also influence the occurrence and development of predatory protozoa and zooplankton that prey on microalgae. It was reported that no difference in y mixing . Howevery mixing ,36.Tribonema strain grown under both laboratory and outdoor open conditions with different reactors or scales. For this purpose, optimization of the nutrient requirement for algal growth is also a part of this work. In batch culture, the algal growth and EPA production are also monitored as a part of this work.A series of exploratory studies are needed for a potential EPA-producing algal strain, thus the present work aimed at the maximization of EPA production by a Spirulina spp. [3, NaCl, and K2SO4, which create a high-salt and high-alkali environment. These three chemicals were discarded from Zarrouk’s medium and named modified Zarrouk’s medium, or M-zarrouk. BG11 medium and M-zarrouk medium, which differs from M-zarrouk medium mainly in phosphorus and magnesium concentrations , were used. Cultivation of T. aequale was subsequently carried out in five kinds of culture medium, including BG11, M-Zarrouk, BG11 medium with excess phosphorus (BG11 + P), BG11 medium with excess magnesium (BG11 + Mg), and BG11 medium with excess phosphorus as well as magnesium. CO(NH2)2, K2HPO4·3H2O, and MgSO4·7H2O were used as nitrogen, phosphorus, and magnesium sources, respectively. The nutrient composition of each medium was listed in Zarrouk’s medium was the synthetic culture medium originally formulated for the cultivation of ina spp. , which cT. aequale grew rapidly in BG11 and a biomass concentration of 7.5 g L−1 was observed after 12 days of cultivation. However, a 30% increase in biomass yield (9.81 g L−1) was obtained in T. aequale culture maintained in the M-Zarrouk medium. The addition of 0.2 g L−1 MgSO4 to the BG11 medium significantly increased biomass yield as compared to that from the BG11 cultures, whereas the addition of 0.655 g L−1 K2HPO4 to the BG11 medium resulted in the reduction in growth. Interestingly, when the BG11 medium was spiked with both 0.2 g L−1 MgSO4 and 0.655 g L−1 K2HPO4, the final biomass yield of T. aequale cultures was as high as that with the M-Zarrouk medium  after 12 days of cultivation in BG11 and BG11 spiked with 0.6 g L−1 K2HPO4 (BG11 + P). It decreased to 26 ± 0.5%, 18 ± 0.2%, and 12 ± 0.1% in BG11 + Mg, BG11 + P + Mg, and M-Zarrouk, respectively. As the productivity of TFA is a function of cellular TFA content and biomass concentration, the maximum TFA productivity of 200 mg L−1 d−1 was obtained from the BG11 + P cultures. A slightly lower TFA productivity (180 mg L−1 d−1) was observed in the BG11 cultures, but much lower TFA productivities of 120, 150, and 90 mg L−1 d−1 were measured in BG11 + Mg, BG11 + P + Mg, and M-Zarrouk cultures, respectively  content of ectively A.T. aequale grown in the different culture media exhibited quite different trends from that of TFA. The highest EPA content  and the proportion of EPA (15.3%) in TFA were obtained in the BG11 + P + Mg cultures, followed by the M-Zarrouk and BG11 + Mg cultures. The lowest EPA content and proportion of EPA in TFA occurred in the BG11 and BG11 + P cultures. As a result, the highest EPA productivity of 22.63 mg L−1 d−1 was obtained in the BG11 + P + Mg cultures  for the production of biomass, EPA, and PLA. Each ORP had a culture surface area of 0.56 m2 and a culture volume of 133 L. The only difference between 1/2BG11 and BG11 was that the former contained a half of the amount of CO(NH4)2 used in the BG11. It revealed that the alga grew well in these media, resulting in more or less the same final biomass concentration of 1.1 ± 0.03 g L−1 and biomass productivity of 15.5 ± 0.73 g m−2 d−1. However, the EPA and PLA contents in the algal cells grown in 1/2BG11 were significantly higher than that in the BG11 and BG11 + P + Mg cultures, resulting in the highest productivities of EPA (542.5 ± 27.1 mg L−1 d−1) and PLA (570.4 ± 34.6 mg L−1 d−1)  .T. aequale cultivated in ORP with the three different culture media were further analyzed and the results are shown in The fatty acid profiles of −2 s−1 with a maximum photon flux density of 2320 μmol m−2 s−1  from the 10 cm ORP.The alga grew well in the S-ORP of various culture depths and yet the shallower the culture depth the more rapid growth occurred on a per-volume basis. The final biomass concentrations were 1.32, 1.09, 0.97, and 0.84 g Lectively C. When bectively D, howeveT. aequale grown in the 15, 20, and 25 cm S-ORP for 10 days were 12.27%, 12.53%, and 12.40%, which was slightly higher than that (11.56% DW) achieved in the 10 cm S-ORP. The highest areal TFA productivity of 1291.07 ± 16.96 mg m−2 d−1 was obtained in the 20 cm S-ORP, which also yielded the highest areal productivities of EPA (348.02 ± 4.93 mg m−2 d−1) and PLA (449.20 ± 5.19 mg m−2 d−1) (The TFA contents in m−2 d−1) .A comparison was made between the cultures with continuous culture mixing and the ones with mixing occurred only during the daylight period with regard to the production of algal biomass, EPA, and PLA.2) under outdoor environmental conditions. It revealed that , 4.50% (w/w), and 11.89% (w/w), respectively, which were 6.95%, 18.11%, and 11.64% higher than those obtained with the periodic mixing regime , which were more or less the same as those measured in S-ORP and M-ORP  outdoors. During the cultivation period, the average and maximum solar irradiances were 924.23 μmol mectively A, and thectively B. The vand M-ORP . These rT. aequale cultures in this study began with monoalgal inocula without any noticeable contamination by protozoa and zooplanktons, these microorganisms occurred in the cultures in just a few days. Yet, the number and phylogenetic diversity of the contaminated microbial species or strains increased as the cultures were maintained for a longer period. A total of 18 species/strains of microbial contaminants in T. aequale cultures were observed under a light microscope, and these microbes were classified into three groups, flagellates/ciliates, amoeba, and rotifers  largely from Bacillariophyceae and Eustigamtophyceae have been subjected to investigation. However, none of these unicellular microalgae with adopted mass culture technologies has made commercial success, due mainly to microbial contamination in mass culture of microalgae, resulting in unsustainable cultures with severe reduction in productivity of microalgal biomass or desirable product. Another reason was a projected high cost associated with harvesting and dewatering of the unicellular microalgal cells by centrifugation. It was estimated that harvesting of unicellular microalgae from culture broth may account for 20–30% of the total cost of microalgal biomass production [Microalgae have long been regarded as a natural source of EPA and PLA, and a number of unicellular microalgae  and a higher EPA productivity of 344.5 mg m−2 d−1 than that obtained from the cultures maintained at 10–15 cm depth   . It seemProper mixing of culture suspension by means of a paddle wheel in an ORP is a prerequisite for improved microalgal photosynthesis and thus biomass productivity. However, it might be case by case whether or not culture mixing would be necessary at night when light is not available. An apparent reason for lowering the mixing rate or stopping mixing at night is to reduce energy consumption and thus operational costs . AnotherT. aequale produced over 30% more biomass in M-Zarrouk culture medium than in BG11 medium , that are at roughly the same latitude  during the same time of the year [−2 d−1 obtained from T. aequale culture was among the highest figures reported for microalgae-based EPA production in an ORP setting, but somewhat lower than that  obtained from the cultivation of Nannochloropsis sp. in a 500 L flat panel photobioreactor  from July through September 2019. Three sizes of ORPs were employed, and they were small-size ORP (S-ORP) with an illuminated surface area of 0.5652 m2, medium-size ORP measuring a surface area of 5.2 m2, and large-size ORP of 52 m2 .All the culture experiments started with 0), then washed twice with distilled water and dried at 85 °C overnight, and then weighed (DW1). Algal biomass concentration of culture was calculated as Equation (1):Biomass concentration was measured in terms of cell dry weight (DW). A certain volume of culture (v) was filtered through a pre-weighed 0.45 μm cellulose acetate membrane filter , was used to stain the organism [w/v). A small volume  of the Nile Red staining solution was added to 1 mL of culture sample with appropriate dilution and incubated in a 45 °C water bath for five minutes. The sample was then cooled down to room temperature (ca. 25 °C), and then another 5 μL of the Nile Red staining solution was added and kept in the water bath for another five minutes. The cells stained with Nile Red were observed and photographed with a fluorescent microscope  equipped with a 100/1.40 oil immersion objective and fluorescence light source components (U-HGLGPS). The excitation wavelength ranged from 505 to 566 nm.Morphological characteristics of organism . Brieflyv/v), 200 μL chloroform, and methanol  solutions in the presence of 25 μL of tridecanoic acid (10 mg ml−1). Tridecanoic acid, an odd-chain fatty acid that does not naturally occur in microalgae, was transesterified together with the sample to quantify total fatty acid methyl esters (FAMEs) by gas chromatography  and was used as an internal standard. Extraction and transesterification of fatty acids took place at 85°C for 1 h. The FAMEs were analyzed by an Agilent 7890B + 5977A GC-MS . The capillary column was HP-88 (60 m × 0.25 mm × 0.2 μm). The initial temperature of the oven was 50 °C and maintained for 2 min, heated at a rate of 25 °C min−1 to 175 °C, and maintained for 5 min, then heated again at a rate of 7 °C min−1 to 210 °C and maintained for 1 min. The injector temperature was kept at 250 °C in split (20:1) mode for an injection volume of 1 μL. The auxiliary heater, electron ionization (EI) source, and MS Quadrupole temperatures were 250 °C, 230 °C, and 150 °C, respectively. Helium was used as the carrying gas at a flow rate of 1 mL min−1.Fatty acid profiles of the organism were determined by a protocol described by Van Wychen, et al.  with modObservations and photomicrography of protozoa and rotifers were made with a differential interference contrast microscope . Species identifications and classification were based on the morphology of the organisms.The EPA, PLA, or TFA content (% DW) were calculated according to Equation (2):biomassVP or EPA/PLA/TFAVP) were calculated as Equations (3) and (4).fW and iW represented the final and the initial biomass concentration in the culture, respectively, and T was the cultivation time.Volumetric biomass, EPA, PLA, or TFA productivity (biomassAP), and productivity of EPA, PLA, or TFA (EPA/PLA/TFAAP) of cultures in ORP were calculated according to Vadlamani et al. [The areal biomass productivity  and reached a biomass concentration of 9.8 g L−1 in a glass column photobioreactor under laboratory conditions. The very high cellular EPA content of 3.5%, and the high areal biomass and EPA productivities of 16.2 g m−2 d−1 and 542.5 mg m−2 d−1, respectively, were obtained from an outdoor open raceway pond study. The high yield potential of biomass and EPA production and high resistance to microbial grazers as well as easy biomass harvesting could make T. aequale an ideal organism for commercial EPA production.The yellow-green filamentous microalga


Agents targeting the programmed cell death‐1 pathway have demonstrated encouraging activity across multiple solid tumor types. The dose expansion phase of this phase I study evaluated the safety, tolerability, and antitumor activity of durvalumab monotherapy, and durvalumab plus tremelimumab  combination therapy, in patients from Asia with biliary tract cancer (BTC), esophageal squamous cell carcinoma (ESCC), or head and neck squamous cell carcinoma (HNSCC).Patients with advanced BTC, ESCC, or HNSCC with disease progression during or following ≥1 platinum‐based therapy received durvalumab monotherapy (10 mg/kg every 2 weeks) or durvalumab plus tremelimumab . The primary objective was safety and tolerability. Secondary objectives included antitumor activity.Durvalumab monotherapy was assessed in 116 patients  of whom, 42, 42, and 32 had BTC, ESCC, or HNSCC, respectively. Grade ≥3 treatment‐related adverse events (TRAEs) were reported in 19.0%, 9.5%, and 25.0% of patients with BTC, ESCC, and HNSCC, respectively. Objective response rate (ORR) was 4.8%, 7.1%, and 9.4% in BTC, ESCC, and HNSCC. Durvalumab plus tremelimumab was evaluated in 124 patients  of whom 65 had BTC and 59 had ESCC. Grade ≥3 TRAEs were reported in 23.1% and 13.6% of patients with BTC and ESCC. ORR was 10.8% and 20.3% in BTC and ESCC. There were two complete responses and 10 partial responses in ESCC, and seven partial responses in BTC.In general, durvalumab monotherapy and durvalumab plus tremelimumab combination therapy displayed acceptable safety profiles consistent with published literature, and also demonstrated clinical benefit, in patients from Asia with BTC, ESCC, or HNSCC with disease progression on ≥1 prior treatment. ClinicalTrials.gov Identifier: NCT01938612. Durvalumab monotherapy and durvalumab plus tremelimumab both showed manageable toxicity profiles and clinical benefit in Asian patients with BTC, ESCC, and HNSCC who had progressed on previous systemic chemotherapy. In the preliminary dose escalation study of durvalumab monotherapy, the maximum tolerated dose was not reached, and dose‐limiting toxicities were not identified.22.1This was a phase I, open‐label, multicenter study (NCT01938612). During the previously published dose‐escalation phase, durvalumab monotherapy was evaluated in patients with advanced solid tumors.For the study expansion phase, two regimens were selected. The first cohort of patients received durvalumab monotherapy: 10 mg/kg Q2W, via intravenous (IV) infusion, for either a maximum of 12 months or until confirmed progressive disease or unacceptable toxicity, initiation of an alternative cancer therapy, or withdrawal of consent. This dosing schedule for the durvalumab monotherapy cohort was determined by previous safety and efficacy data available for this dose. The durvalumab monotherapy cohort included three groups of patients with BTC, ESCC, or HNSCC. Approximately 20 to 60 patients were to be enrolled in each group. The second cohort received durvalumab and tremelimumab  followed by durvalumab 20 mg/kg Q4W IV for either a maximum of 12 months or until confirmed progressive disease or unacceptable toxicity, initiation of an alternative cancer therapy, or withdrawal of consent. The dosing rationale for the combination cohort was based on data from another durvalumab plus tremelimumab study in solid tumors.The protocol for this study (Study 2) was reviewed and approved by the appropriate review committees for each institution within which this work was undertaken. This study conforms to the provisions of the Declaration of Helsinki . All patients were required to provide written informed consent prior to any study procedures.2.2Adults at least 20 years of age, with histologically or cytologically confirmed advanced or metastatic BTC, ESCC, or HNSCC, were eligible for enrollment. Patients with BTC or ESCC must have experienced disease progression during or following ≥1 platinum‐based therapy for unresectable disease for locally advanced, R/M disease. Patients with ESCC that demonstrated disease progression within 24 weeks of completing platinum‐containing therapy were enrolled. Patients with HNSCC must have demonstrated disease progression during or following ≥1 platinum‐based therapy for R/M disease. Eligible patients had ≥1 measurable lesion by Response Evaluation Criteria In Solid Tumors (RECIST) v1.1, an Eastern Cooperative Oncology Group (ECOG) performance status (PS) of 0 or 1, a minimum life expectancy of 16 weeks, and adequate organ and marrow function. Patients in the durvalumab monotherapy cohort were required to provide an unstained archived tumor tissue sample, as well as a recent tumor biopsy (taken following completion of the most recent therapy). The durvalumab plus tremelimumab cohort were to provide a fresh tumor biopsy at screening (preferred) or, alternatively, an archived tumor tissue sample obtained less than 3 years prior to screening. For both cohorts, the lesions for biopsy should not have been a RECIST target lesion, unless no others were suitable. If a RECIST target lesion was used, the longest diameter had to be ≥2 cm.Exclusion criteria included treatment with any immunotherapy  or investigational anticancer treatment within 4 weeks prior to the initial dose of the study drug (within 6 weeks if mAb therapy). Other exclusion factors included: current or previous use of immunosuppressants within 28 days of the initial study drug dose ; receipt of live attenuated vaccination within 30 days either preceding study enrollment or following receipt of study drug; major surgery within 30 days prior to the initial study drug dose, or undergoing recovery from previous surgery; and unresolved toxicity from previous anticancer treatment or any previous Grade ≥ 3 immune‐mediated adverse events (imAEs) while receiving immunotherapy. For the combination cohort, patients who experienced Grade ≥ 3 interstitial lung disease following any therapy were not enrolled. Additional exclusions included: any symptomatic or untreated central nervous system metastases requiring concurrent treatment; other invasive malignancy within 5 years preceding study enrollment; uncontrolled intercurrent illness; active or prior documented autoimmune disease within the last 2 years; history of primary immunodeficiency; or any condition that the investigators considered likely to interfere with assessment of the study drug.2.3The primary objective was to assess the safety and tolerability of durvalumab (±tremelimumab) in patients with advanced BTC, ESCC, or HNSCC. Secondary objectives were to evaluate the immunogenicity and antitumor activity of durvalumab (±tremelimumab). Analyses were performed by tumor type, with summary statistics reported for patient demographics and baseline characteristics, as well as safety data. PD‐L1 expression was measured from either archival or fresh (obtained at screening) tumor samples using the VENTANA PD‐L1 (SP263) Assay . PD‐L1 positive and high‐expression cutoffs evaluated were ≥1% tumor cells (TC ≥ 1%) or ≥25% tumor cells (TC ≥ 25%), respectively, at any staining intensity exceeding background.National Cancer Institute Common Terminology Criteria for Adverse Events v4.03 in the safety analysis. AEs were assessed by investigators; AE, serious AE (SAE), and concomitant treatment assessments were performed at regular intervals postdosing. AEs of special interest (AESI) were reviewed and imAEs were reported. An imAE was defined as an AESI that required treatment with systemic steroids or other immunosuppressants and/or endocrine therapy, was compatible with an immune‐mediated mechanism of action, and had no apparent alternative etiology. Serologic, immunologic, and histologic data, as required, were used to aid the characterization of imAEs. Antidrug antibody (ADA) analyses were conducted for durvalumab and tremelimumab; the ADA set included patients in all cohorts with available ADA data who received ≥ 1 dose of durvalumab.All patients who received ≥1 dose of durvalumab per protocol were included in the safety analysis set. Survival data were analyzed using the full analysis set, and included every patient that received a dose of durvalumab before the data cutoff. Response data were assessed using the response evaluable set, and included every patient in the full analysis set that had measurable disease at baseline, as determined by the study investigators at a baseline disease assessment. AEs were analyzed using 33.1The study data cutoff was March 31, 2018. Summary baseline demographics are reported in Table 3.1.1n = 8, 19.0%), gall bladder , intrahepatic , or bile duct . There were 42 patients with ESCC and 32 patients with HNSCC  or former  smokers  than 1  . Of these, 116 patients received treatment. There were 42 patients with BTC, with tumor locations recorded as extrahepatic , gall bladder , intrahepatic , ampulla of Vater , and unknown . There were 59 patients with ESCC  were from Japan, 28 (22.6%) were from the Republic of Korea, and 27 (21.8%) were from Taiwan  or former  smokers. An ECOG PS of 0 was recorded in 58 (46.8%) patients  plus tremelimumab (1 mg/kg Q4W), and 124 patients received treatment. The BTC group comprised 65 patients with tumor locations recorded as extrahepatic  can be found in Table 3.2.1n = 3 [2.6%]) and nausea (BTC and HNSCC), decreased appetite (ESCC and HNSCC), hyponatremia (BTC and ESCC), lymphocyte count decreased (BTC and HNSCC), and anemia (BTC) (n = 2 [1.7%] each). Treatment‐related SAEs were reported in 4 (9.5%) patients with BTC, 2 (4.8%) patients with ESCC, and 7 (21.9%) patients with HNSCC. Five patients in the monotherapy cohort discontinued therapy due to TRAEs . In the monotherapy cohort, TRAEs with the outcome of death occurred in three patients; one in the ESCC group  and two in the HNSCC group . Overall, there were 18 (15.5%) imAEs reported  (Table Median (range) treatment duration for durvalumab monotherapy was 9.86 1.3–52.1) weeks. Median (range) treatment duration was 11.8 (1.9–52.1) weeks in the BTC group, 9.8 (1.9–52.1) weeks in the ESCC group, and 8.9 (1.3–42.1) weeks in the HNSCC group. Overall, 103 (88.8%) patients experienced an AE and 31 (26.7%) were Grade ≥ 3 Table . Treatme.1 weeks.]) Table . Treatme3.2.2n = 4 [3.2%]), and decreased appetite, hyponatremia, and pruritus (≥2 patients BTC only) (n = 2 [1.6%] each). Treatment‐related SAEs were reported in 9 (13.8%) patients with BTC and 8 (13.6%) patients with ESCC. Eight patients in the combination cohort discontinued because of TRAEs . TRAEs with the outcome of death occurred in one patient with BTC who experienced drug‐induced liver injury. Overall, there were 32 (25.8%) imAEs reported  (Table The median (range) duration of exposure was 12.2 (0.7–53.9) weeks in the durvalumab plus tremelimumab cohort. Median (range) treatment duration was 11.9 (3.9–53.9) weeks for patients with BTC, and 15.7 (0.7–52.1) weeks for patients with ESCC. Overall, 117 (94.4%) patients experienced any AE and 61 (49.2%) were Grade ≥ 3 Table . TRAEs w]) Table . Treatme3.33.3.1n = 6 [14.3%]; ESCC, n = 2 [4.9%]; HNSCC, n = 1 [3.4%]). In the BTC group, 2 (4.8%) patients were transient positive. None of the patients who were ADA positive post baseline had neutralizing antibodies (nAb) post baseline  patient with BTC and 1 (3.4%) with HNSCC. Neither of these patients showed evidence of ADA post baseline. Post baseline, there were 9 (8.0%) patients who were ADA positive to durvalumab . There were no transient positive results. The one (2.2%) patient with ESCC who was ADA positive post baseline was also nAb positive  patients with BTC had positive ADA to durvalumab at baseline. None of the patients with ESCC were ADA positive at baseline. Post baseline, there were four (3.9%) patients who were ADA positive , there were three (5.4%) patients in the BTC group who were ADA positive at baseline and none in the ESCC group. Post baseline, there were 16 (17%) patients who were ADA positive . In addition, of those who were ADA positive post baseline, 10 (20.8%) patients in the BTC group and five (10.9%) patients in the ESCC group were nAb positive post baseline  was 4.8% (2 of 42 patients) for patients with BTC, 7.1% (3 out of 42 patients) for patients with ESCC, and 9.4% (3 out of 32 patients) for patients with HNSCC  was 1.5 months  in patients with BTC, 1.4 months (95% CI 1.3–1.6) in patients with ESCC, and 1.4 months (95% CI 1.1–1.6) in patients with HNSCC. Median overall survival (OS) was 8.1 months (95% CI 5.6–10.1) in patients with BTC, 5.2 months (95% CI 4.0–10.0) in patients with ESCC, and 4.5 months (95% CI 2.1–10.9) in patients with HNSCC Figure . OS at 13.4.2For patients with BTC, the ORR (investigator assessed) was 10.8% (7 of 65 patients) and 20.3% (12 of 59 patients) in patients with ESCC. Complete response was experienced by two patients with ESCC, with 10 partial responses. In the BTC group, there were seven partial responses and no complete responses (Table Median PFS was 1.6 months (95% CI 1.4–2.8) in patients with BTC and 2.9 months (95% CI 1.6–3.5) in patients with ESCC. Median OS was 10.1 months (95% CI 6.5–11.6) in the BTC group and 7.9 months (95% CI 5.9–11.5) in the ESCC group Figure . OS at 14In this phase I study, durvalumab monotherapy and durvalumab plus tremelimumab both displayed an acceptable safety profile and preliminary clinical activity in patients with BTC, ESCC, or HNSCC.In general, the safety profile for durvalumab monotherapy was consistent with published literature. However, the percentage of TRAEs Grade ≥ 3 in the HNSCC group in this study (25%) appeared higher than previously reported in the literatureThe incidence of positive ADA or nAb to durvalumab in this study was low and in line with previously reported data.As with other checkpoint inhibitors such as pembrolizumab and nivolumab,PD‐L1 expression on tumor cells and immune cells has been linked to greater clinical benefit from anti‐PD‐1‐ and anti‐PD‐L1‐targeted therapies across multiple tumor types. In the durvalumab monotherapy cohort in this study, ORR was numerically higher for all cancer types in patients with PD‐L1 expression using both PD‐L1 cutoffs (TC ≥ 25% and TC ≥ 1%) than in patients with PD‐L1 low or no expression (TC < 25% or TC < 1%), with the exception of BTC, which showed a higher ORR using the PD‐L1 cutoff of TC ≥ 25% but not with TC ≥ 1%. For the durvalumab plus tremelimumab cohort, the ORR was also higher in patients with PD‐L1 expression (TC ≥ 25% and TC ≥ 1%) in patients with ESCC. This trend was not confirmed in patients with BTC, since TC ≥ 1% was not associated with a higher ORR and none of the patients met the criteria for PD‐L1 TC ≥ 25%. The general trend for a higher ORR in PD‐L1 positive (TC ≥ 25% and TC ≥ 1%) patients with ESCC and HNSCC is consistent with published reports regardless of tumor type; however, any potential trends should be interpreted with caution due to the limited sample size in the PD‐L1 evaluable population.In conclusion, the safety profiles and the preliminary efficacy of durvalumab in combination with tremelimumab revealed through this study support further clinical development of anti‐PD‐1/PD‐L1 and anti‐CTLA‐4 combinations in additional tumor types. Durvalumab monotherapy and durvalumab plus tremelimumab both demonstrated acceptable toxicity profiles and clinical benefit in patients from Asia with BTC, ESCC, and HNSCC who had progressed on previous systemic chemotherapy.Yuichiro Doki: Personal fees from Ono pharmaceutical company, outside the submitted work. Makoto Ueno: Grants and personal fees from AstraZeneca, during the conduct of the study. Grants and/or personal fees from Astellas Pharma, Daiichi Sankyo, Dainippon Sumitomo Pharma, Eisai, Incyte, Merck Serono, MSD, Nipro Corporation, Ono Pharmaceutical, Taiho Pharmaceutical, Teijin Pharma, and Yakult Honsha, outside the submitted work. Chih‐Hung Hsu: Grants from AstraZeneca, during the conduct of the study; Grants and/or personal fees from Beigene, Bristol‐Myers Squibb, Genentech, Merck Serono, Merck Sharp & Dohme, Ono Pharmaceutical, and Roche, outside the submitted work. Do‐Youn Oh: Nothing to disclose. Keunchil Park: Personal fees and other support from AstraZeneca, outside the submitted work. Noboru Yamamoto: Grants from Astellas, Bayer, Boehringer Ingelheim, Bristol‐Myers Squibb, Chugai, Daiichi‐Sankyo, Eisai, Kyowa‐Hakko Kirin, Lilly, Novartis, Ono Pharmaceutical, Pfizer, Quintiles, Takeda and Taiho. Personal fees from AstraZeneca, Boehringer Ingelheim, Bristol‐Myers Squibb, Chugai, Cimic, Eisai, Lilly, Ono Pharmaceutical, Otsuka, Pfizer, Sysmex, and Takeda. Grants from Chiome Bioscience Inc., GSK, Janssen Pharma, Merck, MSD, and Sumitomo Dainippon, outside the submitted work. Tatsuya Ioka: Grants from AstraZeneca, during the conduct of the study. Grants from Dainippon Sumitomo, Eisai, Incyte, Shire, Taiho, and Takara Bio; Personal fees from Chugai, Servier, Taiho, and Yakult Honsha, outside the submitted work. Hiroki Hara: Grants and/or personal fees from Astellas, AstraZeneca, Beigene, Boehringer Ingelheim, Bristol‐Myers Squibb, Chugai, Daiichi Sankyo, Dainippon Sumitomo Pharma, Eisai, Incyte, Kyowa Hakko Kirin, Lilly, LSK BioPharma, Merck Biopharma, MSD, Ono Pharmaceutical, Pfizer, Sanofi, Taiho, Takeda, and Yakult Honsha, outside the submitted work. Manabu Hayama: Is an employee of AstraZeneca. Masahiro Nii: Is an employee of AstraZeneca. Keiko Komuro: Is an employee of AstraZeneca. Mariko Sugimoto: Is an employee of AstraZeneca. Makoto Tahara: Grants and personal fees from AstraZeneca, during the conduct of the study. Grants and/or personal fees from Amgen, Bayer, Bristol‐Myers Squibb, Celgene, Eisai, LOXO, Merck Serono, MSD, Novartis, Ono Pharmaceutical, Pfizer, and Rakuten Medical, outside the submitted work.Yuichiro Doki: Resources, investigation, and writing– original draft, review and editing. Makoto Ueno: Resources, investigation, and writing– original draft, review and editing. Chih‐Hung Hsu: Resources, investigation, and writing– original draft, review and editing. Do‐Youn Oh: Resources, investigation, and writing– original draft, review and editing. Keunchil Park: Resources, investigation, and writing– original draft, review and editing. Noboru Yamamoto: Resources, investigation, and writing– original draft, review and editing. Tatsuya Ioka: Resources, investigation, and writing– original draft, review and editing. Hiroki Hara: Resources, investigation, and writing– original draft, review and editing. Manabu Hayama: Data curation and writing– original draft, review and editing. Masahiro Nii: Conceptualization, methodology, data curation, formal analysis, and writing– original draft, review and editing. Keiko Komuro: Data curation and writing– original draft, review and editing. Mariko Sugimoto: Data curation and writing– original draft, review and editing. Makoto Tahara: Resources, investigation, and writing– original draft, review and editing.The protocol for this study (Study 2) was reviewed and approved by the appropriate review committees for each institution within which this work was undertaken. This study conforms to the provisions of the Declaration of Helsinki . All patients were required to provide written informed consent prior to any study procedures.Table S1Table S2Click here for additional data file.


Wistar rats had their molar extracted, and were organized into nine treatment groups: negative control group (NCG), treated with saline solution and blood-clot in the alveolus; positive control group (PCG), with blood-clot in the alveolus; BG, β-tricalcium phosphate-based biomaterial; DG, 10% doxycycline gel; aG, antimicrobial photodynamic therapy; and DBG, aBG, aDG, and aDBG, using combination therapy. After 28 days, the lowest bone volume (BV/TV) was reported in PCG (42.17% ± 2.65), and the highest in aDBG (69.85% ± 6.25) (p < 0.05). The higher values of daily mineral apposition rate were recorded in aDBG (2.64 ± 0.48) and DBG (2.30 ± 0.37) (p < 0.001). Moreover, aDBG presented with the highest neoformed bone area (82.44% ± 2.69) (p < 0.05). Non-vital bone was reported only in the PCG (37.94 ± 18.70%). Owing to the key role of the biomaterial, the combination approach (aDBG) was the most effective in preventing MRONJ following tooth extraction.Surgical trauma in those under a prolonged use of bisphosphonates, can lead to mediation-related osteonecrosis of the jaw (MRONJ). This study aimed to evaluate the preventive therapies for MRONJ. Following four cycles of zoledronic acid administration,  However, the prolonged use of these medications is a risk factor for medication-related osteonecrosis of the jaw (MRONJ)7. The prevalence of MRONJ is higher with the use of high bisphosphonate (BP) doses, especially of nitrogen-containing bisphosphonates (N-BPs), as they are more resistant to enzymatic degradation9.Anti-resorptive drugs (ARs), including anti-RANKL antibodies inhibit osteoclastic activity, and are thus commonly used to prevent hypercalcemia or bone metastases in patients with cancer9, MRONJ is defined as an area of exposure in the maxillofacial region, persistent for > 8 weeks, that can be diagnosed in patients undergoing to antiresorptive therapy alone or in combination with immune modulators or antiangiogenic medications, without prior exposure to radiotherapy in the head and neck region14. Despite its advantages and benefits in the treatment of skeletal disorders, prolonged use of BPs can lead to the development of MRONJ16.According to a recent by the American Association of Oral and Maxillofacial Surgeons (AAOMS) position paper17. Numerous published reports have described the success of antimicrobial photodynamic therapy (aPDT) in clinically treating this condition22.There is currently no universal protocol for the treatment of MRONJ. Treatment can range from conservative therapy to surgical intervention, depending on the disease stage. Several patients undergo conservative treatment, including oral health maintenance, infection removal, and antibiotic use25. Recent in vitro studies have demonstrated the use of calcium phosphate ceramics in the reduction or elimination of zoledronic acid (ZOL) toxicity in fibroblasts26. In antimicrobial photodynamic therapy (aPDT), a low-power laser biostimulates the osteoblasts, even during ZOL treatment27, and reduces the number of bacterial colonies with a photosensitizing agent24. Moreover, satisfactory results were observed with doxycycline-loaded collagen sponge placed over the alveolar bone, in rats undergoing antiresorptive drug treatment28.Thus, studies exploring the prevention of MRONJ have emergedThus, this study aimed to comparatively evaluate the preventive effects of locally applied β-tricalcium phosphate (BTCP) ceramics, aPDT, and doxycycline, alone or in combination, on the development of MRONJ, in the alveolar bone of rats undergoing ZOL therapy.29.This study was approved by the Ethics Committee on Animal Experimentation (CEUA) of São Paulo State University (UNESP), School of Dentistry, Araçatuba, Brazil (#0810-2018), following the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines, and in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health Rattus norvegicus albinus) , obtained from the UNESP facility were group-housed in cages , and acclimatized to a temperature of 24 ± 2 °C, with controlled light cycle (12/12 h), and free access to water and feed. To determine the sample size, a power test was performed at a significance level of 5% (with a standard deviation of 2%), α = 0.05, and 80% power. Thus, eight rats (n = 8) were required per group.Seventy-two 3-month-old male Wistar rats  applications of 0.035 mg/kg of ZOL, dissolved in 0.1 mL of vehicle (0.9% NaCl), through the tail vein, at 15-day intervals. One week after the fourth ZOL application, the mandibular right first molars of the animals were extracted, and locally treated. ZOL injections were continued until euthanasia.All animals were treated with Zoledronate (ZOL) , except for the animals in the negative control group (NCG), which received only 0.1 mL of saline solution (0.9% NaCl), using the same application protocol. Following an experimental model proposed by Curra et al.23 on the 52nd day of ZOL treatment initiation (corresponding to 7 days after the fourth ZOL application). The surgical procedure was performed under anesthesia via an intraperitoneal injection (IP) of 90 mg/kg of ketamine hydrochloride  and 10 mg/kg of xylazine hydrochloride . After sedation, antisepsis was performed with degerming and topical povidone-iodine  application, followed by sterile draping. The animals were placed in the dorsal decubitus position on a customized operating table, and syndesmotomy was carefully performed using a delicate detacher , followed by luxation and extraction of the tooth. The animals were randomly divided into nine treatment groups (www.randomization.com) by a blinded assessor, unrelated to the study.Negative Control group (NCG): Treated systemically with NaCl and locally left empty, except for the blood clots in the alveolus.Positive control group (PCG): Treated systemically with ZOL and locally left empty, except for the blood clots in the alveolus.Biomaterial group (BG): The alveolus in the mandibular right first molar was filled with approximately 0.03 cc of BTCP paste .Doxycycline group (DG): The alveolus in the mandibular right first molar was filled with approximately 0.03 cc of 10% doxycycline gel.aPDT group (aG): The alveolus in the mandibular right first molar was irrigated for 1 min with 1 mL of neutral methylene blue photosensitizer, followed by a diode laser of indium gallium aluminum phosphide at the red wavelength (660 ± 10 nm), with a spot size of 0.0283 mm, 35 mW, in continuous mode, with a 2.1 J/point for 60 s, according to the model proposed by Ervolino et al.24.Doxycycline + biomaterial group (DBG): The alveolus in the mandibular right first molar was filled with approximately 0.03 cc of mixture in a proportion of 50% each  .aPDT + biomaterial group (aBG): The alveolus in the mandibular right first molar received aPDT (as described above) and was subsequently filled with approximately 0.03 cc of BTCP paste .aPDT + doxycycline group (aDG): The alveolus in the mandibular right first molar received aPDT (as described above) and was subsequently filled with approximately 0.03 cc of 10% doxycycline gel.aPDT + doxycycline + biomaterial group (aDBG): The alveolus in the mandibular right first molar received aPDT (as described above) and was subsequently filled with approximately 0.03 cc of mixture in a proportion of 50% each  .All animals had the mandibular right first molars extractedPost-extraction, the soft tissue was detached from the extraction site, and sutured with 4–0 polyglactin 910 , to assist in the clot maintenance, treatment as previously randomized, and primary closure of the alveolus.33.Fifteen days after molar extraction, the fluorochrome calcein was administered intramuscularly (20 mg/kg) to each animal, and repeated after 10 days, using alizarin red (30 mg/kg)All animals were euthanized 80 days after ZOL application (28 days after molar extraction) with a lethal dose of sodium thiopental  and IV administration of 2% injectable lidocaine hydrochloride . The right hemimandibles of all animals were collected and analyzed for the following; Fig. The hemimandibles of the animals from the nine experimental groups were fixed in a 10% buffered formalin solution  for 24 h, followed by washing under running water for 24 h. After fixation, the samples were stored in 70% alcohol for microcomputed tomography (micro-CT) analysis.34. The samples were scanned in 6-µm thick Sects. (90 kV and 111 μA), with a 0.5 mm Al + 0.038 mm Cu filter, and a 0.5-mm rotation step, at a pixel size of 2,016 × 1,344 µm, and acquisition time of 52 min. The distal root of the mandibular first molar was established as the region of interest (ROI), and a circular ROI size of 200 was used for all the specimens. The X-ray projection images of the samples were stored and reconstructed, and the area of interest was determined using the NRecon software , with the following modes: smoothing, 1; correction of artifact rings, 8; correction, 24% beam hardening, and image conversion, ranging from 0.0 to 0.14. The images were reconstructed in three planes , using the DataViewer software . The percentage of bone volume (BV/TV), trabecular bone thickness (TbTh), separation and number of trabeculae (TbSp and TbN), and total porosity (PoTot) were evaluated using the CT-Analyser software , and subsequently reconstructed in three dimensions (3D), using the CTVox software .Micro-CT analysis was performed according to the methodology described by Bouxsein et al. (2010), using a SkyScan microtomograph After micro-CT analysis, the hemimandibles of the nine experimental groups were dehydrated using increasing concentrations of alcohol , and subsequently immersed at different concentrations  of Technovit® light-curing resin , in a mixture with 100% alcohol solution. The samples were stabilized on a glass plate with composite resin, such that the long axis of the distal alveolus of the mandibular first molar was parallel to the ground. Subsequently, the Technovit resin was incorporated, light-cured, Exakt microtome-cut , calcified, and ground using an Exakt system sander, until the center of the mesial root of the mandibular right second molar was located . Longitudinal sections of approximately 50-μm thickness, were obtained in the alveolar region .35.These sections were captured using a Leica CTR 4000 CS SPE confocal laser microscope  with a 10 × at the Confocal Fluorescence Microscopy Laboratory of the São Paulo State University (UNESP), School of Dentistry, Araraquara, Brazil. Thus, images of the fluorochromes calcein and alizarin red (old/new bone) were obtained separately, and finally reconstructed with an overlap, to assess bone turnover through the mineral apposition rate (MAR). The area of fluorochrome precipitation  was measured using ImageJ software . MAR was determined using the value obtained on tracing five measurements extending from the outer margin of calcein towards the outer margin of alizarin, and dividing it by 10, which represents the time interval between the two injectionsAfter confocal microscopy analysis, all the slides were stained with Stevenel’s blue and acid fuchsin. Images of the sections, obtained at 1.6 × and 40 × magnifications, were analyzed under an optical light microscope , and captured using an attached digital camera , with 1.3 MP resolution. Histomorphometric analyses were performed using the ImageLab 2000 analysis software . The measurements obtained in micrometers (µm), were presented after conversion as percentage of the neoformed bone area (%NBA), connective tissue, and necrotic bone area .Data were analyzed using SigmaPlot . After performing a normality test (Shapiro–Wilk test) to determine the data distribution, a one-way analysis of variance test (ANOVA) was performed to verify the difference between the means of the groups, followed by Tukey’s post hoc test for multiple comparisons.p = 0.193). Notably, all treatment groups exhibited a higher BV/TV than that of PCG . However, no difference was observed when compared to BG, DG, aDG, and DBG . The aDG and DBG presented with similar BV/TV values , significantly different from that of PCG, aG, and aBG .The NCG had an average BV/TV of 54.01% ± 2.60, whereas the PCG had an average BV/TV of 42.17% ± 2.65 (p = 0.193), which was significantly different from that of aDBG, aDG, and DBG , followed by aG with 55.31% ± 1.67, which was also significantly different when compared to aDBG, aDG, and DBG  . A significant difference (p = 0.043) was noted between DG (0.163 ± 0.015 mm) and aG (0.111 ± 0.0070 mm)  was observed between aDG (5.53 ± 5.3 mm) and PCG (3.68 ± 3.48 mm)  Fig. D.In contrast to BV/TV, the aDBG, aDG, and DBG, which presented with the highest bone volume, in this analysis, demonstrated the lowest TbSp ; however, all groups remained within the general average , except for DBG . All groups had higher MAR values, when compared to NCG and PCG (p < 0.05) , when compared to the other groups  and 25  days after molar extraction. It stands out that, a green line  marked the older bone, whereas a red line  marked the new bone. Concomitant observation of the images helped differentiate the amount of old and new bone  allowed bone neoformation in the middle and apical thirds, with foci of vital bone in the connective tissue Fig. D,E, wherThe combination therapies proposed in this study  were effective in the bone-healing process. Bone neoformation was noted in the apical and middle thirds of the alveolus, in addition to the cervical-third portion, in the DBG and aDBG. aBG Fig. F and aDGThe dynamics of the tissues present within the distal alveolus of the extracted tooth were quantified and organized into 1) %NBA, (2) connective tissue, and (3) necrotic bone  Fig. ; their q %NBA, (2p < 0.001) , followed by the DBG (60.82% ± 4.37). The combination therapies aBG and aDG showed satisfactory results for %NBA , which were different from those of the PCG (p < 0.001). Furthermore, the %NBA of aDG was superior to that of aG (p = 0.037). Among the isolated treatment groups, the BG showed the best results (57.13% ± 5.89), with significant differences from that of aG (p = 0.022) and PCG (p = 0.001). The DG and aG showed %NBA of 41.64% ± 11.46 and 35.48% ± 6.58, respectively, which were both superior to that of the PCG .The NCG had a %NBA of 47.16% ± 4.03, whereas the PCG had the lowest %NBA  Fig. . The aDBp = 0.688) . For the DBG, aDG, and aBG, the percentages of connective tissue were 39.17% ± 4.37, 44.10% ± 13.78, and 47.92% ± 10.70, respectively (p > 0.05). The BG had the lowest mean connective tissue among the isolated treatment groups (42.86% ± 5.89), followed by the DG (58.35% ± 11.46) and aG (64.52% ± 6.58) (p > 0.05).The NCG presented with 51.36% ± 4.03 of connective tissue, whereas the PCG presented with a smaller amount  Fig. . In contNecrotic bone was not observed in the samples from any of the groups, except for those from PCG (37.94% ± 18.70).In this study, we hypothesized that local therapies would help prevent MRONJ, thus favoring the bone-healing process. The therapies used here, either alone or in combination, facilitated the formation of vital and organized bone tissue Figs.  and 7. T37. It is noteworthy that BG exhibited the highest response among the therapies applied in isolation . It is believed that the osteoconductive potential of the BTCP-based biomaterial could induce and guide bone formation, providing good results as demonstrated in this research. Furthermore, the aDBG group conglomerated the beneficial effects of each therapy, as demonstrated by MAR.Precipitation analysis (MAR) represents the amount of bone formed after calcium precipitation in the collagen-bone matrix; owing to the fluorochromes' property of binding to calcium, the amount of fluorescence represents the amount of bone formedion Fig. , and amo16. Moreover, some BPs, such as ZOL, have antiangiogenic effects that decrease the expression of endothelial growth factors in the circulation, leading to areas of ischemia, facilitating MRONJ development39. Unfortunately, in this study, the vascularization potential was not assessed; however, it was demonstrated that the use of BPs resulted in the formation of non-vital bone fragments . However, in the micro-CT analysis using the NRecon tool in the PCG, areas of bone irregularities, sequestration, and absence of bone within the alveolus were observed, as demonstrated in previously published studies on animals treated with ZOL61.Animal models have been used to demonstrate the repercussions of BPs on bone, such as an increase in bone density and a decrease in the spaces between the trabeculae, causing a decrease in the nutritive channels of the bone tissue26, thus favoring alveolar bone healing, as observed in the present study in BG, DBG, and aBG, as %NBA  reduces the bacterial load71, thus preventing MRONJ, as observed in this study in aG  prevented MRONJ  could be a limiting factor; it is similar to that used for preventing osteoporosis79, unlike the high-dose concentrations used in cancer treatment24. In addition, it can be noted that the choice for male rats could be a limitation in this research, since in translational clinical application, women are more likely to develop osteoporosis, owing to menopause and estrogen deficiency. Further, they pose a higher risk of developing osteonecrosis with the use of antiresorptive drugs to prevent bone loss. Therefore, future studies are necessary to evaluate different concentrations of ZOL, as well as methodologies for the evaluation of demineralized tissues such as immunohistochemical analysis and real-time polymerase chain reaction.The mechanisms of alveolar bone healing are similar among speciesThis research reveals that MRONJ induced by the ZOL protocol (0.035 mg/kg) is counteracted by the use of beta tricalcium phosphate, either alone or in combination, by guiding alveolar bone healing. While the use of doxycycline and aPDT demonstrated positive effects on MRONJ prevention, the combination of all three therapies proved satisfactory.


Biliary tract cancers (BTCs), encompassing cholangiocarcinoma (CCA), gallbladder (GBC), and ampulla of Vater cancers (AVC), are common hepatobiliary cancer after hepatocellular carcinoma with a high mortality rate. As there is no effective chemopreventive agent to prevent BTCs, this study aimed to explore the role of statins on the risk of BTCs.PubMed, Embase, and Cochrane Library from inception until 24 April 2020 were searched according to the Meta‐Analyses of Observational Studies in Epidemiology (MOOSE) guidelines. The adjusted risk ratios (aRRs) of BTCs and individual cancer were pooled using a random‐effects model.Eight observational studies (3 cohort and 5 case–control studies) were included with 10,485,231 patients. The median age was 68.0 years (IQR: 67.0–71.5) and 48.3% were male. Statins were associated with a lower risk of all BTCs . The pooled aRR for CCA was 0.60 (95% CI: 0.38–0.94) and GBC was 0.78 (95% CI: 0.68–0.90). There was only one study on AVC with aRR of 0.96 (95% CI: 0.66–1.41). The pooled aRR for lipophilic and hydrophilic statins was 0.78 (95% CI: 0.69–0.88) and 0.70 (95% CI: 0.61–0.80), respectively. The effects were attenuated in studies that adjusted for aspirin and/or non‐steroidal anti‐inflammatory drugs  and metformin .Statins, both lipophilic and hydrophobic, were associated with a lower risk of BTCs, particularly CCA and GBC. Statins associate with lower risk of biliary tract cancers: a systematic review and meta‐analysis. The inclusion criteria included2.3For the eligible studies, items including first authors, publication year, country, study design, inclusion/exclusion criteria, exposure of interest, outcome of interest, sample size, age, sex, follow‐up time, and variables considered in the propensity score (PS) analysis or multivariable analysis were recorded. The PS represents the probability of prescribing statins that is dependent on other covariates . PS matching ensures the balance of measured confounding factors between statins and control groups, so that any difference in the outcome (BTCs) would ideally be due to the effect of statin use only. We also recorded data of adjusted effect estimates (aHR/aOR/adjusted relative risk) derived by PS matching or multivariable analysis. The quality of observational studies was assessed by two authors (MYWY and WSW) independently using the Newcastle‐Ottawa scale (NOS) with the highest score of nine points. The risk of bias was categorized into 3 groups: Low risk (7–9 points), moderate risk (4–6 points), and high risk (<4 points).2.4All statistical analyses were performed using the R version 3.2.3  statistical software. Continuous variables were expressed as median (interquartile range [IQR]) or mean (±1 standard deviation [SD]). Comparisons of outcome were expressed as adjusted risk ratio (aRR) with 95% confidence interval (95% CI) using the random‐effects model, and were presented as Forest plot. Studies that did not report adjusted effect estimates were excluded from the primary analysis since striking imbalance in baseline characteristics likely existed between statin and statin non‐users. As statins users more often have cardiovascular risk factors including DM, smoking, alcohol use which are also known risk factors for BTCs,p‐value < 0.1 indicating significant heterogeneity. We calculate I2 statistics to measure the proportion of total variation in study estimates attributed to heterogeneity. I2 values of <25%, 25%–75%, and >75% indicate low, moderate, and high heterogeneity, respectively.We used Cochran Q test to detect heterogeneity among studies, with a The effect estimate of dose of statins on the BTC was also calculated. There were two studiesThe pooled effect estimate from unadjusted RR was also reported. Subgroup analysis was performed according to the class of statins (lipophilic vs. hydrophilic), cancer type, ethnicity (East vs. West), study design (case–control study vs cohort study), presence of adjustment for aspirin and/or non‐steroidal anti‐inflammatory drugs (NSAIDs), metformin, smoking, alcohol use, DM, and PSC. Lipophilic statins that were investigated in various studies included atorvastatin, simvastatin, lovastatin, fluvastatin, while hydrophilic statins included rosuvastatin and pravastatin. Statin users often had underlying cardiovascular diseases and risk factors necessitating concomitant aspirin and/or metformin use, and these two drugs have been reported to be associated with lower BTC risk.3Figure 3.1A total of 10,485,231 subjects were included in the primary analyses. The median age of patients was 68.0 years (IQR: 67.0–71.5) and 48.3% were male. The characteristics of comparison group are shown in Table p < 0.001; I2 = 92.46%). Funnel plot did not suggest publication bias (Egger's test: p = 0.626)   Figure . There w) Figure . Five st) Figure .3.2p = 0.007, I2 = 61.15%). Three studies were on atorvastatin with an aRR of 0.77 (95% CI: 0.61–0.98), three on simvastatin with an aRR of 0.85 (95% CI: 0.65–1.13), one on lovastatin with an aRR of 0.69 (95% CI: 0.58–0.83), and one on fluvastatin with an aRR of 0.77 (95% CI: 0.61–0.96) . There were two studies on rosuvastatin with an aRR of 0.79 (95% CI: 0.53–1.18), and two on pravastatin with an aRR of 0.77 (95% CI: 0.55–1.07)  Table . For hyd6) Table . There w7) Table .3.3There were two studies that reported a dose–response relationship between statins and BTCs.3.43.4.1p < 0.001, I2 = 93.52%) . There were two studies on eCCA, with aRR being 0.37 (95% CI: 0.13–1.02) and there was no significant heterogeneity .Five studies on CCA were included. The pooled aRR was 0.60 (95% CI: 0.38–0.94), with significant heterogeneity among the studies  Figure . There w3.4.2p = 0.353, I2 = 16.71%) . There was no significant heterogeneity between the studies  Figure .3.4.3There was only one study investigating AVC, reporting an aRR of 0.96 (95% CI: 0.66–1.41).3.4.4p = 0.282, I2 = 11.22%)   and there was no significant heterogeneity  Figure . For the) Figure .3.4.5p < 0.001, I2 = 90.96%)  with pooled aRR of 0.65 (95% CI: 0.48–0.88). There was significant heterogeneity among the studies  Figure . There w3.4.6Three studies adjusted for aspirin and/or non‐aspirin NSAID use, with a pooled aRR of 0.80 (95% CI: 0.72–0.89) Figure A, while Two studies adjusted for metformin use, with a pooled aRR of 0.80 (95% CI = 0.72–0.90) Figure . The remThere were five studies that adjusted for smoking with a pooled aRR of 0.62 (95% CI: 0.34–1.11) Figure . For theFour studies adjusted for alcohol use showed a pooled aRR of 0.63 (95% CI: 0.30–1.30) Figure , while tSix studies adjusted for DM with a pooled aRR of 0.64 (95% CI: 0.40–1.03) Figure . For theTwo studies adjusted for PSC, with a pooled aRR of 0.75 (95% CI: 0.57–0.99) Figure . The rem4In this meta‐analysis of eight studies involving more than 10 million patients, we found that statins use was associated with a 33% lower risk of BTCs, including CCA and GBC. Both lipophilic and hydrophobic statins were associated with lower risks of BTCs.While the main indications of statins are hyperlipidemia and prevention of major vascular events such as stroke or acute coronary syndrome, statins were also found to possess anti‐inflammatory and immunomodulatory effectsThe chemopreventive effects of statins can be explained by the inhibitory effects of statins on cell proliferation via multiple mechanisms. First, statins suppress cell migration and invasion by inhibiting post‐translational modification of G‐proteins that are involved in growth factor signal cascades, cell membrane trafficking, and cell adhesion, such as Ras, Rac, and Rho.Pooling adjusted effect estimates is important in assessing the potential association between statins and cancer development. One may argue that the observed beneficial effect of statins is due to healthy user bias. However, the median age of included subjects in this meta‐analysis was 68.0 years and older individuals (in whom BTCs more likely to occur) generally have more comorbidities that may increase cancer risk, hence potentially negating the concern of healthy user bias.In‐vitro studies suggest a differential chemopreventive effect between hydrophilic and hydrophobic statins—lipophilic statins are associated with cytotoxicity in cancer cellsn = 3). Moreover, observational studies with adjustment for different variables makes it unreliable to compare the effect of individual statins with each other.Furthermore, the mechanisms of each type of statin vary. For instance, simvastatin acts on dysregulating the cholesterol biosynthesis pathway, while fluvastatin depends on reactive oxygen species formation. Nevertheless, we found that both lipophilic and hydrophilic statins were associated with a lower risk of BTCs . Although network meta‐analysis can characterize the comparative efficacy of various statins by indirect comparison, it may not be ideal in this scenario because the scanty number of studies on individual statin effect , ethnicity (West vs. East), and presence of adjustment for certain variables . The pooled effect estimate was similar for cohort and case–control study design (0.67 and 0.60 respectively), although it was of borderline significance for case–control study design. While the pooled effect estimate was 0.65 and 0.80 for studies conducted in West and East respectively, more studies are warranted from the Asian population as there was only one study from Taiwan. The study from Taiwan was the only one that adjusted for parasitic infection. Importantly, the protective effective was lower in studies which adjusted for aspirin (20% vs. 38% risk reduction) and metformin (20% vs. 37% risk reduction). Aspirin reduces CCA risk by suppressing bile duct inflammation and cancer growth factor pathways through COX‐2 inhibition,A few limitations of this study should be acknowledged. First, there are so far no randomized controlled trials (RCTs) investigating the chemopreventive role of statins in BTCs development. All included studies are observational studies, hence raising the possibility of unmeasured/residual confounding. In particular, case–controls studies generally suffer more biases than cohort studies, including selection bias, recall bias, and interviewer bias. Second, a significant majority of the study subjects were aged ≥60 years, and therefore whether statins are protective among younger subjects requires further investigation. Third, a number of risk factors for BTCs, such as cholelithiasis, PSC, bile duct cysts or parasitic infections,5Our meta‐analysis showed that statins were associated with a 33% lower risk of BTCs, in particular CCA and GBC. The beneficial effect was observed for both lipophilic and hydrophilic statins, and was consistent with cohort study or case–control study. Future prospective RCTs are warranted to confirm the chemopreventive effect of statins on BTCs development.Ka Shing Cheung was involved with study concept and design, analysis and interpretation of data, drafting of manuscript, and approval of the final version of the manuscript. Matthew YW Yeung and Wing Sum Wong was involved with literature search, analysis and interpretation of data, and drafting of manuscript. Bofei Li was involved with analysis and interpretation of data and drafting of manuscript. WK Seto was involved in interpretation of data and critical revision of the manuscript. Wai K Leung was involved with the study concept and design; analysis and interpretation of data; critical revision of the manuscript for important intellectual content; study supervision; and approval of the final version of the manuscript.KSC has received speaker fee from AstraZeneca and honorarium for attending advisory board for Janssen and AstraZeneca. WK Seto has received speaker fee from AstraZeneca and Mylan, honorarium for attending advisory board for CSL Behring, and speaker fee and honorarium for attending advisory board for AbbVie and Gilead. WKL has received speaker fee from Eisai, Ferring, Ipsen, and honorarium for attending advisory board for Janssen and Pfizer.Ethics approval was not required as this was a meta‐analysis of published articles.Patient consent was not required as this was a meta‐analysis of published articles.Appendix S1Click here for additional data file.


This retrospective study compared the survival between elderly and non‐elderly patients.A total of 5545 treatment‐naive patients with hepatocellular carcinoma (HCC) who visited 7 different hospitals from January 2000 to December 2018 were included. Patients ≥80 years old were defined as elderly patients. We divided the patients into three groups based on the timing of the initial treatment: Early, middle, and late periods defined as 2000 to 2005, 2006 to 2012, and 2013 to 2018, respectively.p < 0.001). In both elderly and non‐elderly patients, the median albumin‐bilirubin score significantly improved over time and the diagnosis of HCC was made slightly earlier over time. The median overall survival (OS) in elderly patients was 52.8, 42.0, and 45.6 months in the early, middle, and late period, respectively, without a significant improvement (p = 0.17) whereas the OS in non‐elderly patients was significantly improved (p < 0.001). The percentage of elderly patients receiving curative treatments did not significantly increase (p = 0.43), while that of non‐elderly patients did (p = 0.017). Non‐liver‐related death in elderly patients significantly differed among periods (p = 0.023), while liver‐related death did not (p = 0.050). Liver‐ and non‐liver‐related death in non‐elderly patients significantly differed among periods .There were 132 (8.9%), 405 (17.5%), and 388 (22.2%) elderly patients in the early, middle, and late period, respectively, showing a significant increase over time (Survival in elderly patients was not improved despite an improvement in their liver function. Curative treatments should be conducted when appropriate after evaluating each elderly patient. For instance, in the United States of America, the number of people ≥65 years old markedly increased from 39.6 million in 2009 to 54.1 million in 2019, with an additional 19.6 years of average life expectancy.It is well known that cancer incidence increases with age,The present study compared the survival between elderly and non‐elderly patients based on a 19‐year experience.22.1In this multicenter retrospective study, we reviewed the medical records of 5545 patients with HCC who visited seven different hospitals  from January 2000 to December 2018. We collected the baseline patient information, including their demographic features, underlying liver diseases, serum biochemistry, tumor extent, and initial treatment. Patients were diagnosed based on the pathological findings or typical radiological features of contrast‐enhanced computed tomography (CT), magnetic resonance imaging (MRI), or ultrasonography (US).2.2The underlying liver diseases in all HCC patients were determined as hepatitis B virus (HBV), hepatitis C virus (HCV), HBV + HCV, alcohol‐related, or others. Cases with seropositivity for hepatitis B surface antigen (HBsAg) and positivity for anti‐HCV antibody were attributed to HBV and HCV infection, respectively. Cases with seropositivity for both HBsAg and anti‐HCV antibodies were classified as HBV + HCV, and cases with seronegativity for both markers and with habitual significant alcohol intake were attributed to alcohol‐related liver disease.2.310 bilirubin [μmol/L] × 0.66) + .Before the initial treatment, The severity of the liver function was evaluated by the Child‐Pugh classification, albumin‐bilirubin (ALBI) score,2.42‐test and Fisher's exact when appropriate. Continuous values were described as the median (interquartile range) and compared using the Mann–Whitney U test or Kruskal–Wallis test. The overall survival (OS) was defined as the period between the starting date of initial treatment and the date of death or last visit. We generated the Kaplan–Meier curve and compared values using the log‐rank test. A post‐hoc analysis was conducted using the Bonferroni method if significant differences were observed. The Cox proportional hazard model was used to investigate the factors associated with the OS. Gray's test was used to perform the competing risk survival analysis. The Fine‐Gray model was applied to assess the factors relevant to non‐liver‐related death. Because there is a high correlation between BCLC stage and TNM stage, and between Child‐Pugh classification and ALBI grade, we built two models to avoid multicollinearity. p values < 0.05 were considered to indicate statistical significance. All statistical analyses were performed using the EZR software program, ver. 1.53 , which is a graphical user interface for R .The categorical values were described as numbers (percentage) and compared using the χ33.1p < 0.001), and there were 132 (8.9%), 405 (17.5%), and 388 (22.2%) elderly patients in the early, middle, and late periods, respectively, showing a significant increase in the proportion of elderly patients over time (p < 0.001). The proportion of male patients was roughly consistent over time. The median body mass index, the percentage of obesity and diabetes mellitus (DM) is increasing . The percentage of HCV significantly decreased over time, while the proportion of alcohol‐related and other causes significantly increased over time (p < 0.001). Regarding the liver function, the percentage of patients with Child‐Pugh class A significantly increased over time, going from 68.8% in the early period to 70.8% in the middle period to 76.3% in the late period (p < 0.001). The median ALBI score also significantly improved over time (p < 0.001), resulting in an increase in the proportion of mALBI grade 1 and a decrease in the proportions of mALBI grade 2a, 2b, and 3 (p < 0.001). While the difference was quite small, the HCC stage began to be diagnosed significantly earlier, and the serum level of α‐fetoprotein (AFP) decreased significantly over time. Regarding the initial treatments, the proportion receiving curative treatment showed an increasing trend.Of the 5545 patients, 1485 (26.8%), 2315 (41.7%), and 1745 (31.5%) underwent initial treatment in the early, middle, and late period, respectively. The characteristics of overall patients are shown in Table p < 0.001). The post‐hoc analysis showed significant differences between the early and late periods (p < 0.001) and between the middle and the late period (p = 0.008). The 5‐year OS rates in early, middle, and, late period were 47.1% (95% CI 44.2%–49.8%), 50.3% (95% CI 48.0%–52.5%), and 53.9% (95% CI 50.8–56.9%), respectively  patients were dead, and the remaining patients were recorded as having been lost to follow‐up or still alive. The median OS in the overall patients in the early, middle, and late period was 54.0 months , 61.2 months (95% CI 56.4–66.0), and 70.8 months (95% CI 62.4–79.2), respectively, showing statistical significance  in the early period to 23.0% (n = 93) in the middle period to 14.9% (n = 58) in the late period. What is particularly striking in this table is that the proportion receiving a curative treatment did not significantly increase over time (p = 0.43). The median OS in the early, middle, and late periods was 52.8 months (95% CI 33.6–64.8), 42.0 months (95% CI 37.2–50.4), and 45.6 months (95% CI 42.0–61.2), respectively. The 5‐year OS rates in early, middle, and late periods were 47.2% (95% CI 37.3%–56.5%), 37.6% (32.0%–43.3%), and 42.9% (95% CI 35.3%–50.4%), respectively. Surprisingly, there were no significant differences in OS in the elderly patients among the periods . A post‐hoc analysis revealed that the extent of significance differed between the early and late periods (p < 0.001) and between the middle and the late period (p = 0.020). The 5‐year OS rates in the early, middle, and late periods were 47.1% (95% CI 44.1%–50.0%), 52.6% (95% CI 50.1%–55.0%), 56.2% (95% CI 52.8–59.5%), respectively  and 460 (8.3%), respectively, while the cause of death of 897 (16.2%) patients was not recorded. Among 1600 patients who were judged to liver‐related death, 1190 (21.5%), 370 (6.7%), and 40 (0.7%) patients were dead mainly due to HCC, liver failure, and bleeding events, respectively. The 5‐year cumulative incidence of the liver‐related, non‐liver‐related, and unknown death was 41.2% (95% CI 39.4%–43.0%), 11.5% (95% CI 10.4%–12.7%), and 22.3% (95% CI 20.8%–23.8%), respectively Figure . The cump = 0.050; Figure p = 0.023), respectively, with a post‐hoc analysis showing a significant difference between the early and late periods  in the early period, 36.1% (95% CI 30.1%–42.1%) in the middle period, and 48.2% (95% CI 39.6%–56.2%) in the late period. Interestingly, a significant difference was not observed (p < 0.001), respectively, with a remarkable absolute increasing. There were significant differences between the early and middle periods (p = 0.016), between the early and late periods (p < 0.001), and between the middle and late periods (p < 0.001) in the post‐hoc analysis  but only a marginal absolute increase. The post‐hoc analysis showed significant differences between the early and middle periods (p = 0.005) and between the early and late periods , 40.0% (37.1%–42.8%), and 58.8%  ide analog therapy for HBV and direct‐acting antivirals therapies for HCV. Viral infection accounts for the majority of underlying liver disease in the present cohort and these therapies are high efficacy with minimal adverse events, resulting in the improvement of liver function.n = 992]; data was not shown), indicating that many Japanese physician frequently encounter HCC patients ≥75 years in clinical settings. Indeed, the patients aged 75–79 years improved the OS in our analysis  score due to the lack of serum creatinine value. A further study to evaluate the utility in MELD score will be conducted.In conclusion, the percentage of elderly patients is increasing, and the prognosis of elderly patients was not shown to have improved over time. Personalized treatment implemented after evaluating each individual case and considering age‐related comorbidities is desirable, especially in elderly patients.Takeshi Hatanaka, Satoru Kakizaki, and Takashi Kumada conceived the study, and participated in its design and coordination. Takeshi Hatanaka, Satoru Kakizaki, Atsushi Hiraoka, Kazuya Kariyama, Kunihiko Tsuji, Toru Ishikawa, Hidenori Toyoda, Satorshi Yasuda, Atsushi Naganuma, Takaaki Tanaka, Michikata Imai, and Kazuhiro Nouso performed data curation. Takeshi Hatanaka performed statistical analyses and interpretation. Toshifumi Tada, Koichi Takaguchi, Akemi Tsutsui, Ei Itobayashi, Noritomo Shimada, Hiroshi Shibata, Takuya Nagano, Shinichiro Nakamura, Hisashi Kosaka, and Masaki Kaibori draft the text. All authors have read and approved the final version of the manuscript.There are no funding sources.Takeshi Hatanaka received lecture fee from Eisai. Atsushi Hiraoka received lecture fee from Eli Lilly, and Chugai. Toshifumi Tada received lecture fee from Abbvie and Eisai. Takashi Kumada received lecture fee from Eisai. None of the other authors have potential conflicts of interest to declare.The entire study protocol was approved by the Institutional Ethics Committee of Ehime Prefectural Central Hospital (No. 27–34). All procedures were done in accordance with the Declaration of Helsinki. The need for written informed consent was waived because of the retrospective nature of the study.Table S1Table S2Click here for additional data file.Figure S1Click here for additional data file.Figure S2Click here for additional data file.Figure S3Click here for additional data file.


Anesthetic management for patients of pseudomyxoma peritonei (PMP) is challenging. This case report describes a patient of PMP with high intra-abdominal pressure. Intubation was performed in lateral position; the intraabdominal pressure was relieved slowly to prevent significant hemodynamic changes. Additionally, positive pressure ventilation was performed to reduce the risk of re-expansion pulmonary edema. During the operation, transfusion and infusion therapy was performed with target-mediated fluid therapy according to stroke volume variation (SVV) and cardiac index (CI) and blood gas analysis. Pseudomyxoma peritonei (PMP) is a rare clinical syndrome. Cytoreductive surgery (CRS) combined with hyperthermic intraperitoneal chemotherapy (HIPEC) is a safe and effective treatment for PMP . The sur2: 31.1 mmHg, PaO2: 83.6 mmHg, lactate: 1.74 mmol/L, potassium: 4.74 mmol/L, sodium: 132 mmol/L, calcium: 1.14 mmol/L, FiO2: 21%, and SpO2: 96.7%.A 62-year-old woman  was admitted to the hospital because of a 3-month history of increased abdominal circumference, shortness of breath and disable of lay down. The patient was diagnosed as PMP by pathology of ascites. We have got the patient's written informed consent to publish the case report. The patient had a history of hypertension for 11 years, paroxysmal atrial fibrillation for about one month. The blood pressure was controlled by nifedipine. Before the operation, the patient feels dyspnea and must have the passive position of left lateral and reverse Trendelenburg. The abdominal circumference was high to 115 cm . Chest c2, and bispectral index were monitored. A radial artery catheter was placed, intra-arterial blood pressure (IBP), CI, and SVV by using FloTrac vigileo system were monitored. Arterial blood pressure was 148/82 mmHg, heart rate was 97 beats /minute, and SpO2 was 96%. Arterial blood gas analysis showed the following: pH: 7.34, PaCO2: 34 mmHg, PaO2: 80mmHg, lactate: 0.9 mmol/L, potassium: 4.6 mmol/L, sodium: 138 mmol/L, calcium: 1.14 mmol/L, hemoglobin (Hb) 9.6 g/dl, FiO2: 21%, and SpO2: 94.9%. Before anesthesia, 40 μg of dexmedetomidine was infused within 10 min and gastric contents were suction through a gastric tube. The patient was preoxygenated with 100% oxygen at 6 L/minute for 5–10 min. Anesthesia induction was performed with 20 mg of etomidate, 20 μg of sufentanil, and 50 mg of rocuronium. A 7.0 endotracheal tube was inserted with the help of a video assisted device at the first position, and ventilation was performed. The tidal volume was set at 6 to 8 ml/kg, the respiratory rate was 13 to 15 breaths/minute, and the inhalation: expiration ratio was 1:2. Propofol, remifentanil, and sevoflurane were used to maintain anesthesia. Maintenance anesthesia was performed by static inhalation compound anesthesia with propofol 4–8 mg/kg/h, remifentanil 4–8 ug/kg/h, and inhaled sevoflurane 1%–2%, and the depth of anesthesia was adjusted according to the BIS value  to maintain the BIS at 40–60. After the intubation, the patient was slowly put in the supine position and there were no significant changes in blood pressure and heart rate. Then a right internal jugular venous catheter was placed guided by ultrasound and the central venous pressure and oropharyngeal temperature was monitored.The patient was in the passive position of left lateral and reverse Trendelenburg when she was in the operation room. The patient's electrocardiogram, blood pressure, SpO2O at the beginning of the operation to 21 cmH2O, the CI increased from 1.8 L/min.m2to 2.2 L/min.m2, SVV decreased from 13 to 2, central venous pressure (CVP) decreased from 23.3 cm H2O to 15.7 cmH2O. There were no significant changes in blood pressure and heart rate. After ascites drainage, we performed blood gas analysis before the start of the main steps of CRS. Arterial blood gas analysis showed the following: pH: 7.25, PaCO2: 42 mmHg, PaO2: 205 mmHg, lactate: 0.9 mmol/L, potassium: 4.8 mmol/L, sodium: 135 mmol/L, calcium: 1.18 mmol/L, Hb 8.2 g/dl, bases excess (BE) −7.8,FiO2: 60%, and SpO2: 97.0%. The respiratory rate was adjusted to 10 to 12 breaths/minute, the positive end-expiratory pressure was set at 5 cmH2O, FiO2 was adjusted from 1.0 to 0.4.The surgeon created a small incision for paracentesis. 11,000 ml of ascitic fluid was drained in 30 min . In thisCRS was continued for about 3.5 h and about 5 kg of diseased or metastasized tissues were removed . During HIPEC was performed after CRS in a closed-loop manner: after the latex drainage tubes were put in the pelvic and bilateral upper abdomen, the skin was closed and the latex drainage tubes were fixed by continuous suture. The circulation was controlled at the speed of 800 ∼1,000 ml/min. The temperature was set to 41 °C. Mitomycin (MMC) 30 mg and Cisplatin (DDP) 60 mg were dissolved in 4,000 ml of normal saline. HIPEC was continued for 60 min. In this period, the oropharyngeal temperature was increased to 37°C. Torsemide 5 mg was given during this period and another 800 ml of urine was excreted.2and 5–16. Lac was decreased from 0.9 mmol/L at the beginning to 0.6 mmol/L at the end of the surgery. Hb was decreased from 9.6 g/dl at the beginning to 8.5 g/dl at the end of the surgery.The patient was transferred to intensive care unit (ICU) after the operation. x-Ray of Chest revealed the elevated diaphragm was descended at a normal position . These increase the risk of aspiration and rapid desoxysaturation during anesthesia induction, so the airway was carefully evaluated before surgery and prepared for a difficult airway if necessary. To prevent the chance of aspiration, we performed full attraction to the gastric contents before the anesthesia induction. Because of the passive position, we performed the anesthesia induction and intubation at this position after 5–10 min of preoxygen. The patient was slowly put in the supine position and the hemodynamics changes were monitored for avoiding the rapid decrease in return blood volume caused by the huge abdominal pressure compressing the inferior vena cava after rapid supine position, and the decrease will result in great hemodynamic fluctuations. In the operation, we created a small incision for paracentesis to avoid a significant hemodynamic change due to the rapid decrease of intraabdominal pressure and it was proved effective in this patient.Low intravascular volume is very common during CRS due to ascites drainage and blood loss. A large amount of fluid transfusion will be needed. The study of Schmidt showed that all patients were supplemented with 4900m1on average of crystal fluid and 2,500 ml on average of colloidal fluid. Treatment with open fluid to supplement the intraoperative loss of body fluids is at risk of volume overload, tissue edema, and severe abdominal complications , 4. AfteHigh intra-abdominal pressure (IAP) at the level of 18–22 mmHg during HIPEC is feasible and safe in patients diagnosed with PMP . But theCytoreductive surgery combined with hyperthermic intraperitoneal chemotherapy operation is complicated, with a huge incision, completely opened peritoneal cavity, and large fluid loss, especially in HIPEC stage, which can induce significant changes in hemodynamics, high temperature and coagulation dysfunction . Anesthe


Pica and rumination disorder are known as feeding disorder diagnoses in childhood, but little is known about their occurrence in adulthood. This study aimed to assess prevalence rates of one-time and recurrent pica and rumination behaviours (PB and RB) in adults, including sociodemographic subgroups, and to examine associations with other eating disorder and general psychopathology.N = 2403) completed measures on PB and RB, symptoms of avoidant/restrictive food intake disorder (ARFID), body image and symptoms of depression and anxiety.The representative population sample , and less positive body image. However, there were no differences regarding age and body mass index.Any PB and RB were reported in 5.33 and 5.49%, respectively, while recurrent PB or RB occurred in 1.08 and 0.71%, respectively. Co-occurrence was high, with 35.29% of recurrent PB in RB, and 23.08% vice versa. Prevalence rates of recurrent PB or RB did not differ by gender, weight status, educational or migration history from those without recurrent behaviours. Adults with Our findings highlight the clinical significance of PB and RB in adults regarding both prevalence and associations with other psychopathological symptoms. In particular, associations with body image need to be investigated further, as in contrast to other eating disorders, body image disturbance is not yet represented in the diagnostic criteria for pica and rumination disorder. In sum, the findings highlight the need for clinical attention for these disorders and related behaviours in adults. PB has been associated with intellectual disabilities . Of those not participating (n = 2392), n = 738 households refused, 723 households were not reachable, nine households were unoccupied, in 15 households no individual met inclusion criteria, 715 target persons refused, 111 target persons were not reachable (after a maximum of four attempts) and of 81 target households/persons reasons for non-participation are unknown. As the study aimed to examine PB and RB in adults, we excluded participants aged <18 years (n = 86), yielding a sample of N = 2424. Of these participants, data on PB and/or RB were missing for n = 21, resulting in a final sample of N = 2403.Recruitment procedures have been extensively described by Hilbert et al. . ParticiA trained research assistant met participants at their home, informed them about the study procedure and obtained informed consent. The research assistant then asked participants to complete self-report assessments. The Ethics Committee of the University of Leipzig approved the study protocol (No. 297/16-ek). The study followed the ethical guidelines of the International Code of Marketing and Social Research Practice by the International Chamber of Commerce and the European Society for Opinion and Marketing Research.Demographic and anthropometric data: Participants provided information regarding their age, gender , nationality (German v. other) and highest educational attainment . Moreover, they reported their current weight and height, which we used to calculate body mass index (BMI) as kg/m2. Based on their BMI, participants were placed in weight categories .Eating Disorders in Youth-Questionnaire (EDY-Q): The 14-item EDY-Q . Internal consistency of the EDY-Q total mean score  amounted to Cronbach's α = 0.67 and McDonald's ω = 0.51 : The EDE-Q8  referring to the past 28 days. The mean global score showed excellent internal consistency (Cronbach's α = 0.93). Two additional EDE-Q items were used to assess the number of objective binge-eating episodes and compensatory behaviours .Questionnaire on the Perception of One's Own Body-9 (FBeK-9): The FBeK-9  and measure attractiveness/self-confidence, accentuation of body/sensitivity and uncertainty/impaired sensation, with three items each. In the present study, only the subscale attractiveness/self-confidence was used to approximate body image. This subscale has shown acceptable reliability across representative and clinical samples (ω = 0.53–0.83).Patient Health Questionnaire-4 (PHQ-4): The four-item PHQ-4 . Two items each are summed up to form scale scores representing depressive and anxiety symptoms, with higher values indicating greater psychopathology. Both scales show acceptable to good internal consistency (Cronbach's α = 0.78 or 0.82).Descriptive statistics  are used to describe demographic and anthropometric characteristics of participants as well as prevalences of PB and RB. Differences by gender, weight status and education in the groups of individuals with recurrent PB and RB were computed using binary logistic regression models. In the case of a significant prediction of recurrent PB or RB by weight status, another model, controlling for age, gender and socio-economic status was computed. Differences in age, BMI, symptoms of ARFID, eating disorder psychopathology, body image, depression and anxiety between groups with and without recurrent PB and RB, respectively, were analysed using linear regression analyses with recurrent PB or RB, respectively, as the predictor variable. Again, in the case of a significant prediction of the level of BMI by either recurrent behaviour, regression analysis was repeated controlling for age, gender and socio-economic status. Prediction of the occurrence of any binge eating episodes and compensatory behaviours, respectively, by the presence of recurrent PB or RB was tested using binary logistic regression models.n = 1288 (53.60%) participants identified as women. The overall sample had a mean age of 49.49 ± 17.44 years (range from 18 to 94 years), and 1857 (77.29%) participants reported a duration of education of less than 12 years. Most participants were German . The mean BMI of the sample was 25.89 ± 4.68 kg/m2 (range from 11.47 to 67.76 kg/m2), with n = 24 (1.00%) individuals being assigned to the underweight group, n = 1108 (46.11%) to the normal-weight group, n = 892 (37.12%) to the overweight group and n = 339 (14.11%) to the obese group.A total of N = 2403) are shown in M = 0.13, s.d. = 0.12; RB: M = 0.13, s.d. = 0.65). A total of 128 participants  indicated having experienced PB and 132  RB at least once (scores ⩾1 on EDY-Q items). Recurrent PB was reported by 26 participants  and recurrent RB by 17 participants  (scores ⩾4 on EDY-Q items).Distributions of PB and RB in the total sample .Recurrent PB and RB co-occurred in six participants . This corresponds to six of 26 participants with recurrent PB  and six of 17 participants with recurrent RB . The frequencies of the two behaviours showed a large correlation .Regression models did not indicate differences in prevalences of recurrent PB and RB by gender, weight status or education see . Of noteN = 2403) by age groups in 10-year brackets are depicted in Individuals with recurrent PB or RB, respectively, did not differ from those without recurrent behaviours regarding age  and 4. RFurthermore, individuals with recurrent PB did not differ from those without recurrent PB in BMI. However, those with recurrent PB reported greater eating disorder psychopathology, more symptoms of ARFID, depression and anxiety, and less positive body image see . Group db(s.e.) = 1.62 (0.43), p < 0.001, OR = 5.08, 95% CI 2.17–11.85; RB: b(s.e.) = 1.67 (0.52), p < 0.01, OR  =  5.29, 95% CI 1.91–14.69). Compensatory behaviours were only more often reported by individuals with recurrent RB but not by those with recurrent PB compared to their counterparts without recurrent behaviours (PB: b(s.e.) = 0.96 (1.04), p = 0.35, OR = 2.61, 95% CI 0.34–19.92; RB: b(s.e.) = 3.08 (0.61), p < 0.001, OR = 21.73, 95% CI 6.61–71.48).In terms of specific eating disorder symptoms, compared to those without, individuals with recurrent PB or RB, respectively, more often reported the occurrence of binge eating episodes , more symptoms of ARFID, anxiety and depression, and less positive body image.We found that 5.33 and 5.49% of adults, respectively, reported having consumed pica substances or performed RB at least once. As expected, recurrent PB and RB were much less frequent (1.08 and 0.71%). In the presence of one recurrent behaviour, the prevalence of the respective other behaviour was significantly higher (35.29% recurrent PB in RB and 23.08% vice versa), and the two were strongly correlated. In contrast, the prevalence of recurrent PB or RB did not differ by gender, weight status or education. Lastly, while individuals with et al., et al., et al., et al., et al., et al., et al., In line with our hypothesis, both recurrent PB and RB were reported less frequently in adults than in youth  conducted by the Robert Koch Institute between 2008 and 2011 . This may partially be due to the fact that our participants self-reported their height and weight. Additionally, our sample comprised slightly more female participants compared to the data of the Federal Statistical Office from 2018 (53.6 v. 50.7%). Finally, compared to data from the Federal Statistical Office for 2020, we had slightly more participants from younger age groups (<60 years: 69.4 v. 63.0%). Thus, the generalisability of our findings across the German adult population is slightly limited. A further strength lies in the assessment of other general and eating disorder psychopathology using validated instruments. Limitations include the one-item assessment of PB and RB, which was used due to the lack of diagnostic instruments and does not allow for a precise diagnostic categorisation. Moreover, the EDY-Q measure only has anchors for both ends of the seven-point Likert scale . Furthermore, while the items are worded in the present tense, they do not clearly indicate the time point for which participants are asked to report behaviours. Thus, the kind of prevalence reported (lifetime v. period v. point) cannot be pinpointed. When interpreting the findings, particularly the group differences, it needs to be acknowledged that despite the large number of participants, the power of the study was limited due to the low prevalences. Additionally, the survey did not include tests measuring intellectual disabilities, which have proven to be a frequent comorbid presentation in individuals of various age groups . In view of the increased comorbid presentation of the two behaviours, the same holds true when patients report one of the behaviours. With regard to research implications, further population-based studies are needed to replicate the present findings, and German-language diagnostic instruments comparable to the English Pica, ARFID and Rumination Disorder Interview , they are comparable to those of anorexia nervosa and should not be overlooked. The clinical significance of the behaviours is further highlighted by their potential detrimental health consequences, and associations with greater symptoms of eating disorders, including ARFID, as well as depression and anxiety.Data pertaining to this study are available upon reasonable request from the last author (AH).