The growth of malignant tumors is influenced by their microenvironment. Glioblastoma, an aggressive primary brain tumor, may have cysts containing fluid that represents the tumor microenvironment. The aim of this study was to investigate whether the cyst fluid of cystic glioblastomas contains growth-stimulating factors. Identification of such growth factors may pave the way for the development of targeted anti-glioblastoma therapies.We performed hormone analysis of cyst fluid from 25 cystic glioblastomas and proteomics analysis of cyst fluid from another 12 cystic glioblastomas.Glioblastoma cyst fluid contained hormones within wide concentration ranges: Insulin-like growth factor 1 (0–13.7 nmol/L), insulin (1.4–133 pmol/L), erythropoietin (4.7–402 IU/L), growth hormone (0–0.93 µg/L), testosterone (0.2–10.1 nmol/L), estradiol (0–1.0 nmol/L), triiodothyronine (1.0–11.5). Tumor volume correlated with cyst fluid concentrations of growth hormone and testosterone. Survival correlated inversely with cyst fluid concentration of erythropoietin. Several hormones were present at concentrations that have been shown to stimulate glioblastoma growth in vitro. Concentrations of erythropoietin and estradiol (in men) were higher in cyst fluid than in serum, suggesting formation by tumor or brain tissue. Quantitatively, glioblastoma cyst fluid was dominated by serum proteins, illustrating blood–brain barrier leakage. Proteomics identified several proteins that stimulate tumor cell proliferation and invasiveness, others that inhibit apoptosis or mediate adaption to hypoxia and some that induce neovascularization or blood–brain barrier leakage.The microenvironment of glioblastomas is rich in growth-stimulating factors that may originate from the circulation, the tumor, or the brain. The wide variation in cyst fluid hormone concentrations may differentially influence tumor growth.The online version contains supplementary material available at 10.1186/s12987-022-00333-z. Glioblastoma, an especially aggressive form of primary brain tumor, develops due to gene mutations . Tumor gThe purpose of this study was to see what growth factors glioblastoma cells might be exposed to in their microenvironment. Identification of tumor growth factors may pave the way for the development of new diagnostic methods and targeted anti-glioblastoma therapies. Therefore, we performed hormone and proteomics analyses of cyst fluid from patients with cystic glioblastomas.The study was approved by the Regional Committees for Medical and Health Research Ethics of Norway  and was conducted in accordance with the Declaration of Helsinki. All participants gave informed, written consent.g for 10 min at 4 °C, and the supernatants (1 mL) were stored in aliquots at − 70 °C in Axigen 1.8 mL microtubes  until analysis. In 15 patients, parallel blood samples were drawn during tumor surgery for serum analysis.Patients with cystic glioblastomas were recruited at the Department of Neurosurgery, Oslo University Hospital, 2012–2020. Patients underwent magnetic resonance imaging (MRI) investigation at 1.5 Tesla. The following MRI modalities were available for all patients: T1-weighted images obtained before and after the administration of intravenous gadolinium-based contrast agent, T2-weighted images, and T2-weighted fluid attenuated inversion recovery (FLAIR) images. During tumor surgery, which took place between 10 a.m. and 4 p.m., the cyst fluid volume, which corresponded closely to the volume measured by MRI (see below), was harvested in Vacuette blood collection tubes without additives . Samples were rapidly centrifuged at 3000The following parameters were recorded from the MRIs: tumor volume, cyst volume, peritumoral edema, tumor location, cyst location , non-contrast enhancing tumor, and whether the tumor was multifocal or not. Tumor size, cyst volumes and peri-tumoral edema were measured in the MR images by two of the authors (JR and DD), who were blind to the protein or hormone profiles of the patients. Segmentation of MR images was done semi-automatically with Smartbrush  in the group of patients that underwent hormonal analysis or manually with Iplan Cranial Planning Software (Brainlab) in the group of patients that underwent proteomics analysis. Peritumoral edema was classified as none (0), mild (1), or moderate/severe (2) as described . In addiCyst fluid from 25 cystic glioblastomas was analyzed with respect to growth hormone, insulin, triiodothyronine, estradiol, progesterone, serum albumin, apolipoprotein A-I, electrolytes and glucose. These analyses were performed as per hospital routine on a Cobas platform  according to the manufacturer’s instructions. Insulin-like growth factor 1 (IGF-1), erythropoietin, and sex hormone-binding globulin (SHBG) were analyzed by an immunoluminometric assay . Testosterone was analyzed by liquid chromatography-tandem mass spectrometry . Serum samples (n = 15) were analyzed with respect to hormones, serum proteins, and electrolytes with the above methods. All cyst fluid and serum concentrations of hormones and serum proteins were within the methods’ ranges and coefficients of variation were within accredited ranges.g for 10 min. Before LC–MS injection, the sample peptides were purified on C18 microcolumns .Cyst fluid from a separate cohort of 12 patients with glioblastoma underwent proteomics analysis. Cyst fluid supernatants (see above) were treated as described . BrieflyProtein separation and identification were done on an Easy nLC1000 nano-LC system connected to a quadrupole–Orbitrap (QExactive) MS  equipped with a nanoelectrospray ion source . All MS raw files were submitted to MaxQuant software version 1.5.2.8 for protein identification . Paramett-test, paired or unpaired, as appropriate. Correlations are given as Pearson’s correlation coefficients, except for correlations between degree of edema  and cys3 . Glioblastoma cyst volume was 2–85 cm3 . Cyst volume correlated with tumor size . Tumors were localized in any of the cerebral lobes: 11 patients had a tumor that primarily resided in a frontal lobe, 8 of them on the right side; 19 patients had a tumor primarily in a temporal lobe, of which 11 were on the right side; 5 patients had a tumor in a parietal lobe, of which 2 were on the right side; 2 patients had a tumor in an occipital lobe, of which one was on the right side. Nine patients had multifocal tumors. The cyst was located centrally in the tumor in 12 patients; in the rest of the patients, the cyst was eccentric/exophytic relative to the main tumor mass, but lined with tumor tissue, as could be verified at surgery. There was no significant difference between patients with central or eccentric cysts with respect to tumor volume (p = 0.71), cyst volume (p = 0.17), extent of peri-tumoral edema (p = 0.45), or survival (p = 0.69).The 37 glioblastoma patients were 15 women and 22 men, who were 42–79 years old at surgery (median age 59 years). Glioblastoma tumor size was 3–47 cmFive tumors harbored IDH1 mutations; these were not different from wild type tumors with respect to tumor size (p = 0.55) or cyst volume (p = 0.29). All tumors were contrast-enhancing lesions on MRI, and all but one had a peri-tumoral edema , several hormones were present: IGF-1, insulin, erythropoietin, growth hormone, testosterone, estradiol, and free triiodothyronine  or similar to  the serum concentration , whereas the concentrations of IGF-1, insulin, erythropoietin, growth hormone, estradiol, and triiodothyronine in cyst fluid and serum were not significantly correlated .In the cysts that lay centrally in the tumor, the concentration of SHBG tended to be higher than in those that lay eccentrically: 25 ± 14 vs. 16 ± 9 nmol/L . For the hormones and the other plasma proteins there were no significant differences between central and eccentric cysts .To examine whether a dysfunctional BBB (or blood-tumor barrier) might contribute to the influx of hormones into the glioblastoma cyst, we looked for correlations between cyst fluid levels of serum proteins and hormones Table . The serCyst fluid concentration of insulin correlated with duration of methylprednisolone treatment, whereas the concentration of growth hormone correlated inversely with treatment duration Table . In contCyst fluid concentrations of growth hormone, testosterone, SHBG, and albumin correlated with tumor volume Table . The conSurvival of the glioblastoma patients after surgery correlated inversely with cyst fluid level of erythropoietin . The cyst fluid concentration of the other hormones and serum proteins did not correlate with survival .Glioblastoma cyst fluid from a separate cohort of 12 patients underwent proteomics analysis. In each sample, 314–901 different proteins were identified, illustrating the complexity and the variability of the tumor microenvironment; 146 proteins were detected in all 12 glioblastomas , α-2-HS-glycoprotein, leucine-rich α-2-glycoprotein (LRG), secreted GRP78, macrophage migration inhibitory factor (MIF), VEGFA, and hepatoma-derived growth factor. Kininogen-1 promotes neovascularization through its product bradykinin.Identified proteins that are known to participate in inflammatory processes and cause capillary leakage included kininogen-1/bradykinin, kallikrein, HRG, α-2-HS-glycoprotein, LRG, MIF, VEGFA, TREM2, and soluble cadherin-5. However, proteins with presumed anti-inflammatory properties were also detected: apolipoproteins A-I, A-II, and A-IV , 14, 44,Here we show that glioblastoma cyst fluid, which represents the microenvironment of glioblastoma cells, contains a host of tumor growth factors, hormonal as well as non-hormonal. Growth factor concentrations varied widely between patients: in some samples, growth factors were present at high concentrations, in others, certain growth factors could not be detected.Some of the hormones in glioblastoma cyst fluid were present at concentrations that have been shown to promote glioblastoma cell growth in culture. In one study, IGF-1 and insulin stimulated the growth of glioblastoma cells at 0.7 nmol/L and 80 pmol/L, respectively , which iAvailable evidence suggests roles for growth hormone , erythroThe proteomics analysis did not yield absolute concentrations of the various proteins in the glioblastoma cyst fluid, and so we cannot establish whether they were present at levels that would be necessary to support tumor growth. However, previous studies suggest that several of the identified proteins are potent stimulators of tumor cell proliferation or invasiveness, including SPARCL1 , IGF-BP2All of the tumor growth-promoting factors identified in glioblastoma cyst fluid are known to be present in blood, including hormones, kininogen-1, osteopontin, GRP78, SPARCL1, HRG, LRG, α-2-HS-glycoprotein, MIF, VEGFA, kallikrein, IGFBP2, CD166, clusterin, CD5L, and hepatocyte growth factor activator . This coMethylprednisolone treatment prior to surgery may have increased the cyst fluid level of insulin, as suggested by the correlation between length of treatment and cyst fluid insulin concentration. This would agree with previous studies showing that corticosteroids given over several days stimulate insulin secretion . SimilarWe asked if the microenvironment of glioblastomas, as reflected in the cyst fluid of cystic glioblastomas, contain growth factors that may contribute to the growth of these aggressive tumors. We found that the glioblastoma cyst fluid contained a host of growth factors, both hormonal and non-hormonal. Many of these growth factors have been shown to stimulate growth of cultured tumor cells. In the present study, cyst fluid concentrations of testosterone and growth hormone correlated with tumor size, whereas the concentration of erythropoietin correlated inversely with patient survival. The concentration of growth factors varied greatly among patients, which could differentially influence tumor growth.Additional file 1: Table 1. Proteins identified in cyst fluid from cystic glioblastomas. Cyst fluid was harvested from 12 cystic glioblastomas. Data are number of samples in which the protein was detected, log-10 values of raw data (area under the curve) from the mass spectrometry analysis; mean ± SD as calculated from the samples with positive identification of the protein in question. On the first page, proteins are listed according to abundance and number of positive samples in glioblastoma cyst fluid. On the second page, proteins are listed alphabetically. In the rightmost columns, data on peptide counts are given together with gene names and FASTA headers.


Images were analysed for SUVmax, SUVmean, metabolic tumour volume (MTV), and total lesion glycolysis (TLG). Major and partial pathological responses  to immunotherapy were identified based on the residual viable tumour in the resected primary tumour specimen . Pathological response in lymph node metastases was assessed separately. Response for the 2 [18F]FDG-PET-analysable patients who did not undergo surgery was determined clinically and per MR-RECIST v.1.1. A patient with a primary tumour MPR, PPR, or primary tumour MR-RECIST-based response upon immunotherapy was called a responder.In the IMCISION trial (NCT03003637), 32 patients with stage II‒IVb HNSCC were treated with neoadjuvant nivolumab with . A ΔMTV or ΔTLG of at least − 12.5% detected a primary tumour response with 95% accuracy, compared to 86% for the EORTC criteria. None of the patients with a ΔTLG of − 12.5% or more at the primary tumour site developed a relapse (median FU 23.0 months since surgery). Lymph node metastases with a PPR or MPR (5 metastases in 3 patients) showed a significant decrease in SUVmax . However, a SUVmax increase (median + 2.1) was observed in 27 lymph nodes (in 11 patients), while only 13 lymph nodes (48%) contained metastases in the corresponding neck dissection specimen.Median ΔSUV18F]FDG-PET-based ΔMTV and ΔTLG accurately identifies pathological responses early upon neoadjuvant ICB in HNSCC, outperforming the EORTC criteria, although pseudoprogression is seen in neck lymph nodes. [18F]FDG-PET could, upon validation, select HNSCC patients for response-driven treatment adaptation in future trials.Primary tumour response assessment using [https://www.clinicaltrials.gov/, NCT03003637, December 28, 2016.The online version contains supplementary material available at 10.1007/s00259-021-05610-x. Immune checkpoint blockade (ICB) of programmed cell death protein 1 (PD-1) leads to objective responses in 13–17% of patients with recurrent or metastatic head and neck squamous cell carcinoma (HNSCC) and significantly improves their overall survival compared to chemotherapy . Recent 18F]fluorodeoxyglucose(FDG)-PET-based metabolic response evaluation [18F]FDG-PET in a 4-week timeframe [18F]FDG-PET as an imaging biomarker for early pathological response to neoadjuvant ICB in HNSCC remains unclear, as does its susceptibility to false-positivity (i.e. pseudoprogression) by ICB-induced immune activity in the primary tumour or involved or reactive lymph nodes [18F]FDG-PET scans acquired in the context of the IMCISION trial, wherein patients with locoregionally advanced HNSCC were treated with two cycles of nivolumab (anti-PD-1) monotherapy or nivolumab plus ipilimumab (anti-CTLA-4) before definitive surgery [18F]FDG-PET scanning’s ability to predict pathological response early upon immunotherapy in patients with resectable HNSCC.Such a response-driven treatment adaptation requires a reliable biomarker to identify individual patients with a pathological response in the neoadjuvant time frame. With its widespread availability and established position in the clinic, imaging-based response evaluation is an attractive option. However, evaluation of CT and MR images according to the response evaluation criteria in solid tumours (RECIST ) has shoaluation , on the aluation . In addiimeframe . Still, ph nodes . Here, w surgery . We aim IMCISION (NCT03003637) was an investigator-initiated, non-randomized, open-label phase Ib/IIa trial carried out at the Netherlands Cancer Institute (NKI), of which the methods and main results have been reported previously [18F]FDG-PET. Staging was performed according to the 8th edition of the American Joint Committee on Cancer (AJCC) staging manual. Enrolled patients received 2 cycles of neoadjuvant ICB. Figure 18F]FDG-PET were obtained at the end of week 4. Standard-of-care surgery was performed in week 5, or ultimately in week 6. Adjuvant (chemo)radiotherapy was performed if indicated according to institutional and national guidelines.Patients underwent staging investigations at baseline (week 0), including tumour biopsy, laboratory investigations, MR imaging of the head and neck, ultrasound of the neck with fine-needle aspiration cytology, and total body 8FFDG-PETThe pathological response of the primary tumour was determined by a head and neck pathologist (LS) on H&E-stained sections of the resected specimen. The proportion of viable tumour cells within the histologically identifiable tumour bed was quantified as a percentage and compared to the percentage of viable tumour cells within the baseline biopsy to compensate for a low baseline viable tumour cell count. The degree of pathological tumour regression was determined by calculating the percentage change in the primary tumour viable tumour cell percentage from baseline biopsy to the on-treatment resected specimen. Patients with ≤ 10% residual viable tumour cells and 90‒100% tumour regression at the primary tumour site at time of surgery had a major pathological response (MPR). Patients with ≤ 50% residual viable tumour cells and 50‒89% regression had a partial pathological response (PPR), and patients with any percentage of residual viable tumour cells but < 50% regression had no pathological response (NPR) . Two pat18F]FDG-PET-identifiable lymph nodes after ICB, the head and neck surgeon marked the different cervical nodal levels during surgery using beads of different colours, according to our institutional neck dissection protocol. Lymph nodes were evaluated by LS. If a metastasis was present, ICB response was determined using the same cut-offs for pathological response at the primary tumour site .To facilitate pathological correlation of FDG (according to BMI) intravenously. Sixty minutes later, 3D PET images were obtained with 3 min per bed position for the head-neck area and 2 min per bed position for the neck-thighs. For anatomical correlation, low dose CT was acquired with parameters including 120 kV, 40 mAs with dose optimization, and slice interval and thickness 2 mm. All image sets from all scanners used were acquired and reconstructed according to EARL specifications to allow standardized quantification.PET-scans were obtained at baseline and, if the patient consented, in the week prior to surgery using a Gemini TF, TF Big bore, or Vereos PET/CT scanner . One patient underwent baseline scanning in the referring hospital on a Biograph M20 . Patients were instructed to fast for at least 6 h before the scan. If the blood glucose level did not exceed 12 mmol/L, the patient received 190‒280 MBq [18F]FDG-PET images were evaluated jointly by two researchers (JV and WV), one of whom is a head and neck nuclear physician (WV), using Osirix software v11.0.1 . A spherical volume of interest containing the whole area of [18F]FDG-activity was manually grown around the primary tumour, in which SUVmax was determined. Next, SUVmean (the mean SUV of voxels within the volume of interest) was calculated in the subvolume with an intensity ≥ 50% of SUVmax, as is the clinical standard in our institute. This volume also defined the metabolic tumour volume (MTV). Total lesion glycolysis (TLG) was calculated by multiplying MTV with SUVmean. For the on-treatment scan, 50% SUVmax of the baseline scan was used to calculate MTV and TLG. SUVmean, MTV, and TLG could not be reliably calculated if the primary tumour could not be clearly visualized or accurately distinguished from background [18F]FDG-uptake. As determined at the immunotherapy symposium of the European Association of Nuclear Medicine 2017 annual meeting FDG-avid lesions was not classified as PMD. All patients not meeting the criteria for CMR, PMR, or PMD were classified as having stable metabolic disease (SMD).The FDG-PET prior to treatment were clinically diagnosed by ultrasound and, if needed, fine needle aspiration cytology. If an [18F]FDG-PET-identifiable lymph node resided in a level where at least one node was pathologically tumour-positive, that avid lymph node was assumed to be tumour-positive. A lymph node detected by [18F]FDG-PET was considered tumour-negative only if all dissected nodes in that particular level were pathologically tumour-negative. We defined a lymph node as pseudoprogressive if there was an increase in SUVmax from baseline to on-treatment in the absence of HNSCC metastasis upon pathological examination.In case a lymph node, with or without metastasis, showed notable metabolic activity on the baseline or on-treatment scan (or both), SUVmax, SUVmean, MTV, and TLG values at baseline and on-treatment and the (percent) change between the two time points are reported as medians with their interquartile range (IQR). Median values are compared between responders and non-responders using a Wilcoxon rank-sum test. Within the same patient, baseline and on-treatment values were compared using a Wilcoxon signed-rank test. Time to progression (TTP) was defined as the time from surgery to the first local, regional, or distant HNSCC relapse. The 2 patients that did not undergo surgery were thus excluded from TTP analysis. Overall survival (OS) was defined as the time between the first ICB dose and death from any cause (i.e. including the 2 patients that did not undergo surgery). Survival estimates were made using the Kaplan–Meier method; responders and non-responders are compared using a log-rank test. Median follow-up time was calculated using the inverse Kaplan–Meier method. The performance of a PET parameter as a diagnostic test for detecting response was assessed per receiver operating characteristic (ROC) and the area under the ROC curve. All tests were two-sided, and a P-value < 0.05 was considered statistically significant. All statistical analyses were performed in SPSS Statistics version 25.0  and GraphPad Prism version 9.0.0 .All statistics were descriptive. SUV18F]FDG-PET scans were obtained at baseline and a median of 24 (IQR 3) days after the start of ICB in 21 of 32 IMCISION patients. Thirteen patients underwent imaging on the same scanner at both time points. Different scanners from the same manufacturer were used in 7 patients, while 1 patient was scanned on an EARL-calibrated machine from another manufacturer in the referring hospital. Definitive surgery was performed in 19 of these 21 patients, a median of 3 days (IQR 0) after the on-treatment scan: 2 patients were ineligible for surgery due to progressive disease or synchronous incurable oesophageal carcinoma.FDG-PET . Three other lymph node metastases had a decrease in SUVmax in the absence of a pathological response ; the metastasis with a − 5.2 SUVmax decrease showed evidence of an ICB response, though not sufficient for a PPR. Thirteen lymph node metastases had an increase in SUVmax , all without a pathological ICB response.Twenty-one of 33 pathologically confirmed lymph node metastases could be reliably identified on [PET Fig. a. The 5 18F]FDG-PET turned out to be pathologically tumour-negative  and were considered pseudoprogressive  for all 27 lymph nodes with a SUVmax increase after neoadjuvant ICB (13 tumour-positive and 14 tumour-negative) are shown in Table max of the 13 tumour-positive nodes  was significantly higher than of the 14 tumour-negative nodes . Still, distinguishing between true- and pseudoprogression in the cervical lymph nodes on [18F]FDG-PET is problematic, not least because these phenomena may be present simultaneously within the same patient and irrespective of the ICB responses in other lymph node metastases and the primary tumour, as illustrated in Fig. Median baseline and on-treatment SUVDue to insufficiently avid and bulky disease at the metastatic sites, MTV and TLG could only be determined at baseline and on-treatment in 2 lymph node metastases with a response  in IMCISION. In a trial investigating neoadjuvant sintilimab (anti-PD-1) in patients with resectable non-small cell lung cancer, Tao et al. noted that one patient with a PPR after neoadjuvant sintilimab (anti-PD-1) was classified as having PMD per PERCIST, while MTV and TLG did decrease with 60 and 50%, respectively FDG-avidity in the surrounding tissue. While this is in general not a problem in the locally advanced HNSCC setting, one patient in the present trial had a barely avid T2 tumour of the cheek mucosa, of which MTV and TLG could not be calculated at baseline. Similarly, metastatic HNSCC in cervical lymph nodes is often not sufficiently bulky and avid. Second, while MTV and TLG are more accurate than SUVmax, they too are most likely not free from false-negativity through immune-induced pseudostable or -progressive disease, as has been shown in non-small cell lung cancer FDG-PET scan at a relatively early time point: median 14 days after ICB initiation, compared to 24 in our study [18F]fluorothymidine  may help distinguish between true- and pseudoprogression and has previously been proven an early indicator of a favourable outcome after (chemo)radiotherapy in HNSCC FDG-PET-based, primary tumour volumetric metabolic response assessment may be an early and accurate surrogate biomarker to identify individual HNSCC patients with a clinically relevant pathological response to neoadjuvant nivolumab or nivolumab + ipilimumab. In addition, an MTV or TLG decrease seems a promising tool to identify individual patients who are very unlikely to develop a tumour relapse, irrespective of mixed responses or pseudoprogression in the cervical lymph nodes, and may therefore serve as an on-treatment surrogate biomarker to guide response-driven treatment adaptation in future trials.In conclusion, our data suggest that [Visualization of the patient with metabolic evidence of response, but without sufficient tumour regression to be a pathological responder. a, A patient with cT3N1 HNSCC of the retromolar trigone and soft palate had no primary tumour pathological response, yet still demonstrated 22% tumour regression (not shown) and a decrease in SUVmax (-22%), SUVmean (-7%), MTV (-47%), and TLG (-51%). b, A level 2 lymph node metastasis of the same patient shows a decrease in SUVmax (-36%), SUVmean (-24%), MTV (-99%) and TLG (-99%). Correlative pathology (not shown) revealed a major pathological response in the lymph node metastasis (PDF 1582 KB)Supplementary file1 Below is the link to the electronic supplementary material.


Our understanding of hyponatremic conditions has undergone major alterations. There is a tendency to treat all patients with hyponatremia because of common subtle symptoms that include unsteady gait that lead to increased falls and bone fractures and can progress to mental confusion, irritability, seizures, coma and even death. We describe a new approach that is superior to the ineffectual volume approach. Determination of fractional excretion (FE) of urate has simplified the diagnosis of a reset osmostat, Addison’s disease, edematous causes such as congestive heart failure, cirrhosis and nephrosis, volume depletion from extrarenal salt losses with normal renal tubular function and the difficult task of differentiating the syndrome of inappropriate secretion of antidiuretic hormone (SIADH) from cerebral/renal salt wasting (C/RSW). SIADH and C/RSW have identical clinical and laboratory parameters but have diametrically opposite therapeutic goals of water-restricting water-loaded patients with SIADH or administering salt water to dehydrated patients with C/RSW. In a study of nonedematous patients with hyponatremia, we utilized FEurate and response to isotonic saline infusions to differentiate SIADH from C/RSW. Twenty-four (38%) of 62 hyponatremic patients had C/RSW with 21 having no clinical evidence of cerebral disease to support our important proposal to change cerebral to renal salt wasting (RSW). Seventeen (27%) had SIADH and 19 (31%) had a reset osmostat. One each from hydrochlorothiazide and Addison’s disease. We demonstrated natriuretic activity in the plasma of patients with neurosurgical and Alzheimer diseases (AD) in rat clearance studies and have now identified the natriuretic protein to be haptoglobin related protein without signal peptide (HPRWSP). We introduce a new syndrome of RSW in AD that needs further confirmation. Future studies intend to develop HPRWSP as a biomarker to simplify the diagnosis of RSW in hyponatremic and normonatremic patients and explore other clinical applications that can improve clinical outcomes. Hyponatremia, defined as a serum sodium of <135 mmol/L, is the most common electrolyte problem encountered in clinical practices worldwide and is an independent risk factor for higher morbidity and mortality rates . SymptomThe diagnostic approach to hyponatremia has traditionally started with an assessment of the state of extracellular volume, designated as being euvolemic, hypervolemic or hypovolemic, despite a general acceptance that we cannot accurately assess the volume status of patients by usual clinical criteria .Foremost among the limitations is to resolve the conundrum of differentiating “euvolemic” patients with the syndrome of inappropriate secretion of antidiuretic hormone (SIADH) from hypovolemic patients with cerebral/renal salt wasting (C/RSW) by this ineffectual and technically inaccurate volume approach, as all credible volume determinations in SIADH have shown them to be hypervolemic and not euvolemic ,9,10,11.Beck proposed that the coexistence of hyponatremia and hypouricemia, defined as serum urate < 4 mg/dL, differentiated SIADH from most other causes of hyponatremia . As noteInterestingly numerous studies have shown that the increased FEurate >11% in SIADH occurred only when the patient was hyponatremic and normalized to <11% after correction of the hyponatremia, A reset osmostat (RO), which occurs in about 30% of hyponatremic patients, can be readily identified by having a normal FEurate of 4–11% ,28,29. RSince urate is transported exclusively in the proximal tubule, the excretion rates of urate and other solutes will be low as seen in prerenal azotemic conditions where there is under perfusion of the kidneys and an increase in solute reabsorption in the proximal tubule . In brieThe early diagnosis of Addison’s disease is not only important but very difficult to achieve because of nondescript signs and symptoms such as fatigue, weight loss, nausea, vomiting, postural dizziness and hyperpigmentation. As much as 50% are diagnosed when they present with life threatening adrenal crisis . Of the The plasma levels of ADH are inappropriately increased in SIADH because ADH does not respond to the usual volume or osmolar stimuli. ADH in C/RSW, however, responds appropriately to both stimuli. This important physiologic difference can be appreciated by the dissimilar responses to isotonic saline infusions in SIADH as compared to C/RSW. As Bartter stated in 1967, “a striking and consistent finding in patients with SIADH is the persistence of hyponatremia even when large quantities of sodium are administered” . IsotoniBecause of the difficulty in differentiating SIADH from C/RS, we decided to test the response to isotonic saline in unequivocally diagnosed cases of SIADH and C/RSW. We performed blood volume studies by radioisotope dilution methods utilizing 51 chromium labeled red blood cells and radio-iodinated serum albumin. Two patients with SIADH had increased blood volume, low plasma renin and aldosterone levels as compared to decreased blood volume and high plasma renin and aldosterone levels in C/RSW, which along with clinical and laboratory findings collectively strengthened the diagnoses of SIADH and C/RSW, respectively. Isotonic saline infusions in the 2 patients with SIADH failed to induce excretion of dilute urines or correct the hyponatremia as compared to diluting the urine after 13 h of isotonic saline infusions and correcting the hyponatremia within 48 h in the patient with C/RSW, We decided to determine whether hyponatremic patients with increased FEurate >11% diluted their urine and corrected their hyponatremia with infusions of isotonic saline and determined FEurate before and after correction of their hyponatremia in 62 hyponatremic patients recruited from the general medical wards of the hospital . AccordiIt should be pointed out that it is extremely difficult to differentiate SIADH from RSW by the methods described above. FEurates must be determined after correction of the hyponatremia or isotonic saline infusions initiated with frequent urine osmolality determinations to see if isotonic saline infusions diluted or failed to dilute the urine when there are length of stay concerns. Successful execution of this difficult protocol was possible by a funded study, which provided the necessary personnel to perform these studies on a timely basis, especially the unexpected high prevalence of RSW in the general medical wards of the hospital. Challenges continue to confront us as we attempt to simplify methods to identify the different causes of hyponatremia, especially to differentiate RSW from SIADH.RSW of the type described in this manuscript does not include other causes of salt wasting such as chronic kidney disease, Addison’s disease, Bartter’s or Gitelman’s syndromes. It occurs in patients with normal kidney function with creatinine not exceeding 1.3 mg/dL and normal thyroid function. It goes through two important physiologic phases that start with the upregulation of a natriuretic protein such as the recently identified HPRWSP by a multitude of cerebral and many non-cerebral conditions with or without hyponatremia .The natriuretic factor, HPRWSP, has its major effect on proximal tubule sodium transport to increase sodium and water excretion, which exceeds sodium and water intake to decrease extracellular volume. The extent of the volume depletion depends on the balance between the potency of the natriuretic factor in increasing salt and water excretion and salt and water intake. The deficits in sodium and water can be significant enough to cause hemodynamic instability with lower blood pressure and even postural hypotension with reflex tachycardia. This non equilibrated state is difficult to document in the majority of cases because of its short duration. This phase was documented in a 74 year old female hypertensive who sustained a subarachnoid hemorrhage. Her admission systolic blood pressure of 166 mmHg decreased to 98 and 93 mmHg when saline infusions lagged behind an increasing urine output between days 2 and 5. She received four 250 mL IV boluses of isotonic saline to maintain hemodynamic stability untilthe saline infusion matched the urine output of 4–5 L/day . She wasThe patients eventually escape the effects of the salt wasting natriuretic protein by undergoing humoral, hemodynamic and neuronal compensations and enter an equilibrated state where sodium and water intake matches sodium and water output but at lower total body salt and water volumes. In this volume-depleted state sodium excretion and even serum sodium concentration will depend on the intake of sodium and water. As noted in our 62 hyponatremic patients, 10 of the 24 patients with RSW had UNa <20 mmol/L at a time when they were in the equilibrated phase when sodium input equals output and in a state where their comorbid conditions were severe enough to reduce appetite and sodium intake . An unapWe previously injected the plasma of 21 patients with neurosurgical diseases and 18 AD patients into rats and demonstrated significant increases in FENa, FElithium and urine flow rates without changing blood pressure or glomerular filtration rates as compared to normal and gender-matched controls in both studies and vascular dementia in the AD study ,44. BecaUtilization of the sophisticated protein analysis revealed levels of haptoglobin related protein to be highest as compared to control sera. Haptoglobin related protein with signal peptide did not have natriuretic activity but HPRWSP was the only protein with natriuretic activity in SAH and AD sera. As noted in It is our hope that these data will be an impetus for others to study the effect of this relatively unknown protein in many organs in the body, including brain.Blood volume studies in patients with subarachnoid hemorrhage revealed 8 of 9 hyponatremic and 8 of 12 normonatremic patients to be volume depleted, suggesting that RSW occurs commonly in hyponatremic and normonatremic patients with SAH . Water rHyponatremia in AD is also an unusual occurrence because these patients limit their water intake by the natural reduction in thirst with aging in addition to the dementia that reduces water intake for multiple reasons. As stated above, RSW is not only present but appears to worsen as the MMSE levels decrease below 12 and future studies must determine the prevalence of RSW at all stages of dementia and whether HPRWSP can serve as a biomarker of RSW in AD. It should be noted that the identification of HPRWSP as the natriuretic factor in RSW was accomplished in normonatremic SAH and AD patients. The essential development of HPRWSP as a biomarker for RSW will give us the opportunity to identify RSW in hyponatremic and a potentially large group of normonatremic patients.HPRWSP is the first legitimate inhibitor of proximal tubular sodium transport and can significantly mobilize fluid overload in patients with congestive heart failure when combined with a distal diuretic such as furosemide. Congestive heart failure induces pre renal azotemia by increasing the reabsorption of sodium and other solutes in the proximal tubule, which reduces the effectiveness of distal diuretics. As noted, the significant increases in FElithium quantitates the profound effect on proximal tubular sodium transport so its combination with the traditional distal diuretics will significantly increase sodium and water excretion with removal of the excess fluid in heart failure. The benefit of combining acetazolamide, a weak proximal diuretic, with furosemide in patients with acute heart failure improved outcomes and decreased length of stay . CombiniThe development of a physiologically derived approach to hyponatremia has brought clarity in identifying many causes of hyponatremia with consequential improvement in clinical outcomes as we apply the appropriate management for each group of patients. The physiology of these conditions have many subtle nuances that must be addressed by well-founded physiologic principles that lead to credible outcomes. As previously discussed, the dictum that conclusions are based on data would be better expressed as conclusions being based on credible data . We feelRSW is common in the general medical wards of the hospitalChange cerebral salt wasting to RSW. Twenty one of the 24 patients with RSW did not have clinical evidence of cerebral disease. RSW would not be considered without the presence of cerebral disease. We must incorporate this important change in nomenclature.RSW can occur in hyponatremic and in a potentially large number of normonatremic patients.We have introduced and support the conclusion that RSW is not only a new syndrome in AD but is probably common.Determining urine sodium concentrations in work up of hyponatremia is not as informative as professed to be. It should be viewed as being less informative.a.Serve as a biomarker of RSW to simplify the diagnosis of RSW in a wide variety of comorbidities, including a new syndrome of RSW in AD, on first encounter with the patients and to deliver the appropriate management and improve clinical outcomes.b.Because increasing salt and water intake in patients with RSW will increase excretion of large urine volumes that includes a distressing nocturia, there is a need to develop an inhibitor to HPRWSP to improve outcomes.c.HPRWSP satisfies our long search for a potent proximal diuretic, which can be combined with a distal diuretic to effectively eliminate the fluid overload of congestive heart failure and improve clinical outcomes.The identification of HPRWSP as the natriuretic protein that probably causes RSW can have the following clinical applications:


Kirsten rat sarcoma (KRAS) protein is an essential contributor to the development of pancreatic ductal adenocarcinoma (PDAC). KRAS G12D and G12V mutant tumours are significant challenges in cancer therapy due to high resistance to the treatment.  To determine how effective is the ATO/D-VC combination in suppression of PDAC the mouse transgenic model. This study investigated the antitumour effect of a novel combination of arsenic trioxide (ATO) and D-ascorbic acid isomer (D-VC). Such a combination can be used to treat KRAS mutant cancer by inducing catastrophic oxidative stress.  In this study, we examined the effectiveness of ATO and D-VC on xenograft models—AK192 cells transplanted into mice. Previously, it has been shown that a high concentration of Vitamin C (VC) selectively can kill the cells expressing KRAS.  The results of this study demonstrated that the combination of VC with a low dose of the oxidizing drug ATO led to the enhancement of the therapeutic effect. These findings suggest that the combined treatment using ATO and D-VC is a promising approach to overcome the limitation of drug selectivity and efficacy. KRAS, a member of the RAS family of protooncogenes, has been associated with a range of high-grade malignancies. KRAS has been identified as the most frequently mutated gene in human cancers that is responsible for 30% of all human carcinomas –4. Studiβ to cause cell death. The classical pathway of cell signalling involves epidermal growth factor receptor (EGFR), hepatocyte growth factor (HGF), and insulin-like growth factor (IGF-I) binding to their respective ligands [The KRAS gene, a protooncogene, encodes a 21-kD guanosine triphosphate (GTP)/guanosine diphosphate (GDP) binding protein. It is involved in the transcriptional regulation of gene products involved in the response to many extracellular stimuli. It is located on the short arm of chromosome 12 and represents the RAS family of oncogenes . KRAS is ligands .Previous studies have shown that cancer cells with KRAS mutations exhibit rewired glucose metabolism and higher levels of GLUT1 expression. KRAS mutant tumors tend to be more glycolytic than wild-type tumors and require glycolysis for survival and proliferation . Further2O2 can impair cancer cells development as a result of an increase of endogenous ROS levels or decrease in the cellular glutathione levels.Redox homeostasis is an important process that provides both normal and cancer cells with the energy they need to grow and spread. However, malignant cells often show abnormal redox reactions. Therefore, cancer cells are not able to regulate their intracellular levels of reactive oxygen species (ROS) in response to oxidative stress and signals that control growth and differentiation . In thisAscorbic acid functions as an enzymatic cofactor and is the primary antioxidant in vivo, protecting cells from damage by reactive oxygen species such as free radicals. The prooxidant effect of vitamin C with subsequent release of oxygen free radicals at pharmacological/milli-molar concentrations is responsible for the anticancer effect . In humaBoth VC isomers have the same mechanism of action in provoking oxidative stress. However, xenograft studies' findings suggest that an unnatural form of Vitamin C (D isomer of Vitamin C) produces better anticancer effects than its natural L-Ascorbic acid analog . The ratPreviously, we confirmed the antitumour effect of oxidative drug combination on the HCT116 xenograft mouse model . Here, wIn this paper, we provide evidence that ATO/D-VC combination therapy is effective against KRAS mutant tumour growth.μg/ml).The mouse AK192 cell line was provided from the original sources by Drs. Haoqiang Ying. AK192 cells were cultured in DMEM/F-12 (Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12)  supplemented with 10% foetal calf serum (FCS), 2 mMl-glutamine, and penicillin (100 units/ml)-streptomycin  or combination of drugs (5 uM ATO and 1 mM D-VC). In some experiments cells were pretreated for 30 min with 5 mM NAC before adding ATO and D-VC treatment.For 6 cells/ml and stained with propidium iodide (PI) (1ul for 5 min in the dark) to assess % of dead cells and MitoSOX (2.5 nM for 30 min and 37°C) for mitochondrial superoxide production (both from ThermoFisher).Adherent AK 192 cells were harvested by gentle trypsinisation at 24 and 48 h (depending on the experiment), counted using MultiSizer (Beckman). The cells were then resuspended at 1 × 10μl of 1 × 106 cells/ml were stained with 3ul of AnnexinV-FITC and 1 ul of PI for 15 min at RT in the dark. Afterwards 400 ul of buffer was added and cells were kept on ice until analysis.To assess induction of apoptosis by combination of oxidative drugs cells were trypsinsed and combined with collected DMEM to account also for floating apoptotic cells. Suspension was washed once with PBS and then with 1X annexin binding buffer (ABB) and 100Attune NxT Flow Cytometer (ThermoFisher) for all 3 florescent staining according to the manufacturer's instructions. For PI and apoptosis assays 30.000 events were acquired and 10.000 for MitoSOX.Samples were acquired using The Ascorbic Acid , d-(-) isoascorbic acid , and Arsenic Trioxide  used in our study was obtained from Sigma. To prepare 340 mM stock solution of VC, 30 g of D-VC was dissolved in sterile 400 ml PBS solution following the addition of 39 g of sodium bicarbonate . The solution was constantly stirred for another 5 minutes. Sodium bicarbonate was used as a buffer to bring the acidic (2.3) pH of VC up to physiological levels (pH 7.35) for injection into laboratory animals. The final D-VC solution was adjusted to 500 ml by adding PBS. The D-VC solution was filtered, aliquoted, and kept in a -20°C refrigerator to ensure that the drug was as pure and effective as possible. The stock solution of 330 mM ATO was prepared by mixing and dissolving 13 g of ATO in 1 N sodium hydroxide (NaOH). Then, the total volume of ATO was adjusted to 200 ml. The final solution was filtered and stored in a -20°C refrigerator.The animal studies were performed according to the guidelines provided by National Laboratory Astana. Xenograft studies were conducted to investigate the growth of human Pancreatic ductal adenocarcinoma (PDAC) xenografts in Nu and NOD Scid strains of a mouse obtained from the Institute of Cytology and Genetics, Novosibirsk. A mixture of 2×106 AK 192 cells and Matrigel  were injected subcutaneously under isoflurane anaesthesia. Tumour growth was monitored every 2 days with a flexible ruler. Body weight was measured once a week during the experimental period. The drug injections started after 10 days when the tumours reached a size of >0.6 cm in diameter. The animals were treated with a combination of ATO/D-VC at 6 mg/kg and 1.5 g/kg for 2 weeks, respectively. Each group that included xenografts also contained animals that received PBS instead of drugs as a negative control. Mice were sacrificed at 2 weeks' postinjection. The xenograft tissues were examined histologically and quantitatively (weight) by standard protocol. Formalin-fixed, paraffin-embedded cancer tissues were stained with hematoxylin and eosin. Histological analysis was done by microscope Olympus CX41 with the morphometric program.Statistical significance was determined using GraphPad Prism 9 . Data were considered to be significant when p values are <0.05.μM. Slowed growth was observed at ATO 10μM and significant morphological changes at 20 and 30μM ATO , treatment with ATO/D-VC induced 7.3 and 7 fold increase in mtROS at 24 and 48 h, respectively Figures . PreincuKRAS mutated pancreatic cancer is characterized by high KRAS expression and the presence of a chirality-dependent tumour microenvironment. The action of VC is sensitive to chirality and thus the activity of ATO can be selectively potentiated when combined with VC at low doses. Considering that the ATO/VC combination is effective in cell culture, we tested it in a xenograft mouse cancer model. AK 192 cancer cells were transplanted into NOD Scid and Nu mice subcutaneously and the drugs were injected after 10 days when the tumours reached a size of >0.6 cm in diameter . The comTo avoid fluctuations in the blood glucose level, mice were fasted two hours before receiving the injections of ATO or VC and were fed 2 hours after the administration of D-VC.The dose of VC was 1.5 g/kg. Within the second week of injections, we observed tumour shrinkage in mice that were treated with an ATO/D-VC combination . During We employed two strains of mice: NOD Scid and Nu. After the 15th (last) injection of the drug, analysis of the tumour by weight showed that the combination of ATO/D-VC caused potent tumour shrinkage in NOD Scid mice; tumour shrinkage was slightly less in Nu mice .p ≤ 0.0006, p ≤ 0.004, p ≤ 0.0404, and p ≤ 0.04, respectively  compared to the negative PBS control, ectively .ATO/D-VC has been shown an effective in suppressing tumour growth of the KRAS G12D mutant PDAC tumours.In NOD Scid mice treated with ATO/D-VC, the tumour node within the subcutaneous soft tissues was examined histologically and confirmed to be in a state of total necrosis meaning cells had been destroyed by inflammation . The tumA productive inflammation on the periphery of a necrotic tumour node and adipose tissue inflammation was observed. Tumour tissue is represented by slit-like cavities and short strands of tumour cells .Pseudobulbar and glandular structures remained intact in subtotal necrosis. Tumour tissue was with desmoplastic changes .Histological analysis of nontreated NOD Scid xenograft mice tumour, revealed the nodular formation that is surrounded in places by a thin fibrous capsule with extensive areas of necrosis and focal haemorrhages . Under tThe tumour tissue was preserved on the periphery of the node and represented by glandular, pseudo-tubular and nested structures, consisting of epithelial cells with oxyphilic cytoplasm and polymorphic nuclei with an uneven distribution of chromatin. In places, the pseudo tubular structures were unevenly expanded. The presence of papillae and cribriform structures was observed. Mitoses take place. In tumour epithelial cells, areas of cytolysis and polymorphism were observed.In the zone of tumour tissue necrosis, fibroblastic changes were noted in the form of sclerosis of the papillary stroma and atrophy of tubular structures. Stromal oedema, stasis of erythrocytes in micro vessels, and scattered lymphocytic reaction are present on the periphery of the tumour. We observed small diapedetic haemorrhages. In addition, we found stasis of erythrocytes with an admixture of leukocytes in the lumen of the vessels. Results demonstrated the leukocyte infiltration around the necrosis area. There is an accumulation of secretion with an admixture of leukocytes (inflammatory infiltration) in the lumen of some glands. Stromal oedema, muscle cells in a state of myolysis, and atrophy in the thickness of the tumour tissue were also detected.Histological analysis showed the signs of pronounced fibrosis in the stroma . The nodIn Nu ATO/D-VC, the tumour capsule invasion was detected. The glands were separated by proliferating connective tissue. In tumour necrosis with sclerosis, in places, the zone of necrosis reaches the capsule. The analysis showed tumour necrosis, discrete tumour cells under the capsule, inflammation, extensive subcapsular fibrosis, capsule invasion, sclerosis, and lipomatosis of the tumour.The signs of necrosis and shrinkage of tumour indicated that ATO/D-VC is effective in reducing tumour of both NOD Scid and Nu mice. Thus, we demonstrated that the unnatural enantiomer of VC (D-VC) in combination with ATO is effective in suppressing the KRAS mutant tumour.The results showed that ATO/D-VC is capable of inhibiting tumour growth of the KRAS G12D mutant PDAC cancers. ATO/D-VC was effective in reducing tumour weight of both NOD Scid and Nu mice.Histological analysis of mouse organs  did not detect significant abnormalities or signs of necrosis Figures . The difWe noticed the effect of xenograft tumour on the studied organs of mice. These processes are a normal immune response to the presence of a tumour.Our findings suggest that the combination of ATO and VC can lead to oxidative stress-mediated KRAS mutant cancer cell death. A tumour analysis revealed that the average weight of tumour of ATO/D-VC treated NOD Scid and Nu mice were lower by 72% (3.57 times) and 38% (1.62 times) compared to the control group, respectively .At the beginning of the injection, the implanted tumor under the skin of the animals was a uniform round structure with a diameter of 0.6 cm. On the 9th day, in the experimental group of animals treated with a combination of oxidizing drugs ATO/D-VC, macroscopic examination showed primary signs of a decrease in tumour volume compared to animals without treatment. In the following days, these signs intensified and the tumor took on a flattened shape. At the end of the experiment, depletion of the tumour was observed, followed by the scabbing process.The exact mechanism of how ROS production triggers apoptosis is still unclear but there is an evidence of suicidal ROS production by mitochondria (SRPM) under oxidative stress . The metChronic oxidative stress has been considered as a factor of cancer-specific vulnerability, as well as an adaptive strategy for cancer cells under hypoxic conditions. As a concept, it is based on the redox biology paradigm. The idea is that increased reactive oxygen species generation plays a major role in tumourigenesis. Therefore, dysregulation of the redox system represents a promising target to prevent tumour growth and progression. Despite the therapeutic potential, many current conventional therapies lack a selectivity. Up to date, researchers have developed an inhibitor that selectively induces oxidative stress in tumours while leaving healthy cells unharmed. Here, we tested a novel combination of FDA-approved drugs that have already been considered safe for clinical use.Arsenic trioxide is a standard chemotherapy agent for treating solid cancers and leukaemia. It kills cancer cells via chemically-induced apoptosis—excess generation of reactive oxygen species (ROS)—which in turn cause intracellular glutathione levels to go down. Cancer cells that show a high level of glutathione (GSH) are more resistant to arsenic trioxide. Researchers have found that cancer cells can be killed by combining arsenic trioxide with agents that deplete intracellular GSH levels such as Vitamin C .ATO and D-VC in combination induce catastrophic oxidative stress and death of cells expressing mutant KRAS in vivo. Mice xenografted with the KRAS G12D oncogene responded well to a drug combination consisting of ATO and D-VC. Moreover, this study found that the ATO/D-VC combination treatment has better efficacy in inhibiting tumour growth in the AK192 xenograft model than the HCT116 cell line transplanted xenograft  .A synergistic cytotoxic impact induced by the ATO and D-VC combination indicates that surpassing an oxidative threshold in KRAS mutant cancer cells affects the redox-sensitive cellular systems including the most vulnerable mitochondrial oxidative phosphorylation system (OXPHOS) complexes. Taking it into account, a synergy of the ATO and VC combination in killing KRAS mutant cancer cells might take place by the two step actions. In the first step, a depletion of GSH is mediated by a high dose of VC that disarms a cellular antioxidative shield. In the second step, ATO acts in a full capacity by attacking thiol-reactive groups on cellular proteins with a preference of attacking mitochondrial OXPHOS complexes. It is likely that a direct oxidation of thiol-reactive proteins within OXPHOS complexes leads to uncontrolled ROS production or suicidal mitochondrial ROS production.To summarize, the proposed combination of drugs could effectively inhibit KRAS-mutant cell growth by generating high concentrations of reactive oxygen species that induce apoptosis and suppress angiogenesis.A novel anticancer drug combination has been found to be effective in promoting tumour cell death in vivo. These findings indicate the therapeutic potential for the development of new type of anticancer therapy. More studies are needed to prove clinical relevance, efficacy, and safety of the proposed method.


Immune checkpoint blockade (ICB)8-109 distinct TCRs,,The TCR, originally described by Tak Wah Mak,TCR sequence diversity is afforded by differences in the complementarity determining regions 1, 2, and 3 , made up of short protein loops forming a binding surface for the target antigen. αβTCRs recognise short peptides produced by endogenous or exogenous antigen processing and bound within the binding groove of the major histocompatibility molecules (MHC) . The most variable CDR3 regions of the TCR interact directly with the peptide, and hence are key in determining antigen specificity of individual T-cell clones.Specific antigen recognition is fundamental to T-cell activation. The process of T-cell activation is reviewed in detail elsewhere.TCR sequencing enables accurate and reproducible estimation of the relative frequency of different TCRs in blood or tissue, defined as the ‘TCR repertoire’. Therefore, analysis of the TCR repertoire provides a window into the nature of the adaptive immune response for an individual.There are several methods to profile pooled T-cell populations referred to as ‘bulk’ TCR sequencing. Bulk TCR sequencing enables the study of TCR diversity and clonality, and allows comparison of repertoires between samples based on the frequency of single TCR chains.Currently available bulk TCR sequencing protocols can use either gDNA or RNA as starting material  with pros and cons to each approach.Bulk TCR library preparation is achieved through one of three approaches: (i) multiplex PCR, (ii) targeted in-solution enrichment, and (iii) 5′ rapid amplification of complementary DNA ends (5′RACE) PCR .Figure 1Multiplex PCR methods amplify the TCR CDR3 region, by combining primers for all known TCR variable (V) and joining  or constant  regions. A key limitation is amplification bias leading to misrepresentation of the relative abundance of TCR genes, ultimately impacting TCR clonality read-out. There are several approaches to reduce amplification bias including adjustment of primer concentration,,Targeted enrichment involves fragmentation of gDNA or purification of mRNA followed by end-repair, A-tailing, and adapter ligation. Target sequences are then enriched with bespoke RNA baits complementary to V, J, and C gene segments followed by target DNA hybridisation.5′RACE PCR requires reverse transcription of RNA to cDNA. Then, there is a two-step amplification reaction of cDNA using two different primer pairs. In this method,,The result from bulk TCR sequencing methods is a set of unpaired TCRα and TCRβ chains from different T cells. The unpaired nature of the data generated limits the resolution of TCR clonality and diversity estimations because it hinders the distinction between T-cell clones with the same TCRα chain paired to distinct TCRβ chains (or vice versa—with the same TCRβ chain but paired to different TCRα chains).,Despite these novel developments, the current optimal way to pair TCRα and TCRβ chain is through single-cell TCR sequencing (scTCR-Seq). scTCR-Seq approaches generally rely on the adaptation of bulk 5′RACE approaches (described earlier), and different reviews cover in greater depth the details of this methodology.de novo assembly of TCR reads,,,An alternative to dedicated targeted single-cell TCR sequencing is the computational reconstruction of TCR sequence from full-length single-cell RNA sequencing data. Computational methods to extract the TCR sequence are generally based on a combination of alignment and Collectively, single-cell sequencing platforms are currently limited by cost, number of cells processed, and requirements for high-quality (i.e. fresh) input material. In contrast, bulk TCR sequencing is considerably cheaper, thus scalable for the number of T cells profiled, and suitable for profiling of formalin-fixed paraffin-embedded (FFPE) tissue.,The tumour microenvironment (TME) is frequently infiltrated by a variety of T cells [tumour-infiltrating lymphocytes (TILs)], most of which are not tumour-reactive .TCR specificity can also be determined using functional assays. Peptide–MHC multimers can be used to identify tumour T cells that exhibit high affinity for the multimer. Commonly used MHC multimers are tetramers conjugated with fluorochromes to enable the identification and isolation of antigen-reactive T cells using flow cytometry. Such workflows depend on reliable MHC-binding predictions and the generation of specific MHC alleles. Traditionally, this was limited to only human leukocyte antigen (HLA)-A∗0201, but it is now possible to study T-cell specificity against antigens presented by a wide range of HLA class I alleles.A common application of peptide–MHC multimers is the identification of the TCRs that recognise tumour neoantigens in a given patient. To do so, putative tumour-derived neoantigens are first inferred by applying bespoken algorithms to sequencing data.,,,A simpler, though indirect compared to functional assays, way to study antitumour T-cell responses is the quantification of TCR repertoire diversity, clonality, and clustering, given how all of these metrics are related to T-cell activation and proliferation . Highly By analysing these TCR sequencing metrics derived from tumour samples, we have recently reported the presence of clusters and networks of related TCR sequences within populations of TILsWhether the key mechanism underpinning ICB response is through driving novel T cells into tumours  or reinvigoration of pre-existing TILs  is under debate .52 MultiYost et al.in vitro assay to directly test reactivity of expanded intratumoural and peripheral TCR clones against tumour neoantigens, and identified the neoantigen-specific T cells and their corresponding TCR sequences. Intriguingly, neoantigen-specific TCRs were detected in the peripheral blood in most patients but only in a third of tumour samples. However, detection of these TCRs did not correlate with ICB response in this study.Further evidence in NSCLC highlights clonal replacement as a key ICB mode of action. Zhang et al. analysed longitudinal blood, tumour, and normal lung samples from 21 patients with stage I-III NSCLC treated with neoadjuvant anti-PD-1.Collectively, clonal replacement postulates that ICB triggers tumour infiltration of novel CD8+ T cells, replacing pre-existing CD8+ T cells that are incapable of cancer killing, presumably due to chronic activation and exhaustion. However, these studies describing dynamic TCR repertoire changes did not consistently show correlation with clinical responses to ICB. Additionally, while the source of novel TCR clones may be tumour-extrinsic , tumour-intrinsic sources (i.e. expansion of rare pre-existing clones) cannot be excluded.P value = 0.08).Clonal revival proposes an alternate view in that ICB clinical response derives from pre-existing (i.e. tumour antigen primed) intratumoural T cells capable of cytotoxic reinvigoration despite displaying features of exhaustion. We have recently investigated this using paired longitudinal tumour and blood samples from patients with metastatic clear-cell renal cell carcinoma in a phase II study of first-line anti-PD-1 (nivolumab).n = 106) treated with anti-PD-1 or sequential anti-PD-1/anti-cytotoxic T-lymphocyte–associated antigen 4 (CTLA-4).In other tumour types, Valpione et al. analysed pre-treatment biopsies from 20 patients with metastatic melanoma undergoing anti-PD-1 treatment.One potential way to reconcile apparently contradicting reports supporting clonal replacement with those supporting clonal revival is that the mechanism underpinning ICB response is context-dependent and hence can change, for example, in distinct tumour types. However, contradicting data exist even within the same tumour type. Contrary to the aforementioned reports supporting clonal replacement in NSCLC,,,,Another explanation to these divergent read-outs is the existence of confounding factors unequally controlled in different studies: differences related to (i) tumour sample characteristics  due to recruitment of novel T cells, particularly from blood, or (ii) due to functional boosting of pre-existing intratumoural T cells, is relevant to predictive biomarker and cellular therapy development. In the first scenario, longitudinal analysis of the blood TCR repertoire combined with on-treatment tumoural TCR repertoire profiling may be required to predict ICB responders. In the second scenario, analysis of pre-treatment tumoural TCR clonality may be adequate to serve as a baseline predictor of ICB response. Further, if reinvigoration of pre-existing TILs is the dominant mode of action by ICB, harvesting tumour material before treatment followed n = 8; absence of colitis, n = 6).TCR sequencing has also helped to elucidate the mechanism underpinning immune-related adverse events (irAEs) associated with ICB. Luoma et al. carried out scRNA-Seq of colonic tissue from eight healthy individuals and patients with melanoma previously treated with ICB  and higher rates of irAEs.Another mechanism that could explain the link between ICB response and irAEs is that a more varied peripheral TCR repertoire can be observed following ICB,in vitro T-cell reactivity assays remain indispensable to identify tumour-reactive T cells. We are hopeful that the current intense focus in developing new TCR sequencing technologies and new computational approaches to extract biological information from TCR sequences, including the prediction of the cognate antigen of a given TCR sequence, will help to overcome many of these technical challenges, ultimately for patient benefit.TCR sequencing has revolutionised immuno-oncology research; however, several limitations hamper its clinical application. First, current single-cell and bulk TCR sequencing techniques are not standardised or clinically validated and require bespoke computational analyses. Outside of dedicated research settings, the vast majority of the clinically available tumour samples are FFPE samples. These are not suitable for high-quality single-cell TCR sequencing. Finally, a key limitation of TCR sequencing is the inability to determine the antigen specificity of a given TCR sequence; therefore, bespoke


Public catering has become increasingly important in recent years. With increasing annual customers, the sector’s impact on the environment is also growing continuously. At the same time, public catering offers a lever to promote sustainable nutrition that has rarely been used so far. Small changes in kitchen practices and food offers can thus be multiplied into a significant positive impact on environmental challenges, such as climate change or loss of biodiversity due to the large number of servings. In contrast to private households, management decisions in public catering can influence the food-related environmental impact of thousands of customers. This article deals with the nationwide level of greenhouse gas (GHG) emissions and resource use in the German public catering segment “business” and its saving potentials by different scenarios of unsupported and supported recipe revision. In this paper, we define "unsupported" as the intuitive optimization of recipes by employees of public catering businesses. In contrast, “supported” approaches had to meet specific target goals, for example of the Deutsche Gesellschaft für Ernährung; engl. German Nutrition Society or the sustainable level. Specifically, we will test how (A) an unsupported recipe revision, (B) a recipe revision based on dietary recommendations and (C) a recipe revision using scientific guidance affect the environmental impact of a dish. As a methodological framework, an online survey of public catering companies was conducted as well as a scenario analysis at menu level and at nationwide level. The results are based on empirical data on the one hand, and on extrapolations on the other. The results show that the nationwide implementation of recipe revision according to scientific guidance—such as concrete target goals for the GHG emissions per serving—can save up to 44% of resource use in the German business catering sector (which corresponds to 3.4 million tons of resources per year) and as much as 40% of GHG emissions (0.6 million tons GHG emissions per year). Even in the scenario of unsupported recipe revision, GHG and resource savings of up to 20% can be realized. The results show that public catering can reduce its material and carbon footprint by 20% overnight. Moreover, the findings show indications for the sustainable transformation of public catering. Nevertheless, it must be noted that these are some first steps of the transformation, which will require further changes with even greater impacts and political activities.The online version contains supplementary material available at 10.1007/s11625-022-01183-2. Thus, the following research question arises:For the revision of recipes and menus, there are different tools or guidance for use in practice to support a more sustainable offering. One approach is to implement qualitative and quantitative dietary recommendations or guidelines for the composition of dishes and menus, for example from the Planetary Health Diet (PHD)  were given access to the survey. The survey was conducted in the period between July and September 2020. During this period, the survey was completed by eight companies. Collectively, this sample sells more than 100,000 midday meals per day and thus has a wide outreach. The questionnaire contained both open and closed questions on basic data about the company, the application of the NAHGAST online tool and a survey section for original recipes and variants that had been optimized using the NAHGAST online tool. With regard to the recipe collection, all ingredients (excluding ingredients in insignificant amounts like herbs or spices) and their quantities were captured, as well as the preparation method and duration. A total of 20 recipes  were completely transmitted and could be taken into account in the final results. Five of the original recipes were vegetarian recipes, and the remaining five contained meat.For this purpose, a controlled online survey was conducted using the online survey tool SurveyMonkey. All public catering companies of the NAHGAST II project  were transferred to the Life Cycle Assessment software openLCA to model the recipes’ GHG emissions and use of natural resources per serving. In total, we analyzed 70 ingredients. The methodology followed the standardized approach of the life cycle assessment according to DIN EN ISO 14040:2021-02 . It compBased on the individual results of the recipes, an average value was also calculated. The difference between the result of the original recipe and the revised recipes from scenario A, B and C forms the absolute savings per portion, from which the relative saving potential can also be derived:Moreover, the average resource use and average GHG emissions for the original and optimized dishes are determined using the following formula, shown here as an example for the average material footprint of the original recipes:In addition to the assessment of the environmental data, an analysis of nutritional values was conducted. For this purpose, the considered nutrients were analyzed for all recipes to be considered as original recipes as well as optimized recipes. The nutritional values were calculated using the data set of the German federal food catalog (Bundeslebensmittelschlüssel) version 3.02 published by the Ministry of Food and Agriculture BMEL . This coIn the same way as described in Sect. Supplementary to the saving potential at recipe level, the results were used to model the environmental effects of the recipe revisions on a nationwide level. For this purpose, a scenario analysis was conducted to examine how many GHG emissions and resources could be saved by a nationwide recipe revision using the presented scenarios A, B and C as an example. Approximately 1.6 million meals are served in business catering per year DEHOGA . For eacWith the conservative assumption, the average material footprint and carbon footprint of the original recipes were taken for 50% of the menus in business catering . For the remaining 50%, the average material footprint or carbon footprint of the revised recipes according to scenario A, B or C was taken . This results in the absolute resource use and GHG emissions of the business catering assuming a conservative nationwide dissemination:For the ideal dissemination, the average material footprint and carbon footprint of the optimized recipes according to scenarios A, B and C were assumed for 100% of the offered meals in business catering :As a reference for both scenarios, the status quo resource use and GHG emissions were used. For this purpose, the average material and carbon footprints of the original recipes were estimated for 100% of the menus in business catering :2-eq per menu.First, the survey results were used to determine the average impacts per original recipe—more precisely the status quo of GHG emissions and the use of natural resources per menu. Based on the sample, the average resource consumption is 4.00 kg of resources and 0.91 kg of CORegarding the use of natural resources, the status quo could be improved in all three scenarios   were revised by the survey participants, in scenarios B and C only the recipes that did not meet the recommendations or target values were optimized. In scenario B, five out of 10 recipes were revised, in scenario C there were 6. In scenario A, even recipes that already have a very low impact were revised, such as a Mediterranean vegetable pan or a lentil sugo with spaghetti. Further on, there are differences in the selection of ingredients that are commonly substituted or reduced. While in scenario B and C mainly ingredients with a leverage effect were replaced, e.g., high-fat dairy products or meat products, in scenario A also ingredients with a low leverage were partially replaced, for example frozen vegetables. In addition, in scenario A, it appeared that the impact was reduced by smaller portion sizes. However, this measure is only useful if there is a lot of plate waste in the company. In the other scenarios, the portion size was kept constant. In scenario A and C, it can also be observed that the meat amounts were significantly reduced. In two of the meat-containing recipes, meat was completely removed. Although this leads to high savings in the carbon footprint and the material footprint, it can be associated with a loss of acceptance among customers of popular and traditional dishes. However, the character of each recipe was preserved as best as possible during the recipe revision. Thus, in the revision of the recipe spaghetti bolognese, the share of fresh vegetables was increased, while the amount of minced meat was partly reduced. In the rice pudding recipe, cow's milk was partly substituted by soy drink until the corresponding target values of the scenarios were met.In summary, recipe revision offers the greatest saving potentials according to quantitative environmental target values. In terms of the material footprint and the carbon footprint, almost twice as high savings can be realized as in scenarios A and B. At the same time, the revision by environmental target values is also more complex in application. For example, the replacement of a meat component with a vegetable alternative enables great saving potentials, but can also lead to significant losses in customer acceptance if the revision is too substantial.The analysis of nutritional values has been primarily intended to show whether and, if so, in which way the considered nutrients of a recipe revision may differ.Looking at Table In summary, there was no deterioration in the considered energy supplying nutrients because of the recipe optimization. On the contrary, the fiber content was increased and the fat content significantly reduced.Based on the average impact per menu  and the assumption of about 1.6 billion midday meals served in German business catering per year DEHOGA , this re2-eq. The nationwide savings in GHG emissions show a similar trend as the use of natural resources. While around 0.15 million tons of CO2-eq can be saved in scenario A and B assuming a conservative dissemination, scenario C offers savings of 0.29 million tons of CO2-eq. Again, the best result can be realized in scenario C with an ideal dissemination (100%) with savings of up to 0.57 million tons of CO2-eq.In terms of GHG emissions, the annual carbon footprint for business catering in Germany is 1.45 million tons COIn summary, it can be said that an unsupported recipe revision by the practitioners and/or the implementation of dietary recommendations can already make a significant impact on the reduction of GHG emissions and the use of natural resources in the field of business catering. However, twice as much can be saved if the target values of environmental indicators—such as material footprint and carbon footprint in this case—and their compliance is focused more stringently, as it is the case with nutritional indicators .The paper aims to understand the status quo of the material footprint and carbon footprint of business catering in Germany and to identify saving potentials as well as differences in various recipe revision scenarios. Further, a nationwide perspective complements this research. Based on the findings, it was possible to illustrate the leverage effect of the business catering sector and to show various possibilities to use this leverage and to reduce the use of natural resources and GHG emissions. The greatest saving potentials could be realized by recipe revision based on clearly defined environmental target values and their achievement. Compared to the status quo, a saving potential of up to 44% with regard to the material footprint and 40% with regard to the carbon footprint can be realized. Almost half of the savings can be realized if the revision is done by the kitchen staff without any guidance or if the dish includes less than 30% animal-based products complying with the recommendation.n = 10 recipes), the results are likely applicable to other samples and settings.Against this background, the saving potentials from scenarios A and B correspond to the results of other studies, which estimate a saving potential of about 25% in the public catering sector by recipe revision  were optimized and ingredients with minor or no positive effects were substituted. Thus, although this type of optimization is similarly effective as recipe revision based on dietary recommendations, it is less time efficient.Furthermore, the saving potential determined in scenario C is significantly higher, but also associated with more significant revisions in the recipe composition. Considering this, it can be assumed that the dishes will not receive the same acceptance as the other revised recipes. The compliance with the target values only allows very small amounts of meat and dairy products, so that in some cases only 20 g of meat per serving remained after the recipe revision. Theoretically, this would still be feasible for minced meat, but it is hardly feasible for meals such as schnitzel. Either way, for the customers, there is a large discrepancy between the expected dish and the received one, which can lead to a loss of acceptance . As a second point, it can be stated that an optimization of existing recipes is probably limited by the customers' expectations of the dish. Therefore, in addition to the optimization of established dishes, the focus should also be on the development of new dishes that are not associated with traditional expectations and that also meet the requirements of a sustainable diet. From a nutritional perspective, all recipe optimization scenarios do not represent a deterioration in the considered energy supplying nutrients, but it remains to be seen how the recipe revisions will affect other nutritional indicators, such as the biological quality of proteins.Nevertheless, an unguided optimization or a recipe revision according to dietary recommendations such as the DGE quality standards or the PHD DGE  can alsoAll in all, it can be said that the steps implemented within the framework show (1) good indications for the transformation of the sector, (2) that real-world lab approaches can be effective in practical implementation Below is the link to the electronic supplementary material.


In the Financial Disclosure, the number for the grant awarded by EPIDNA is incorrect. The correct EPIDNA grant number is 894489.