The V4 hypervariable region was sequenced using the Miseq platform. Serum vitamin D was determined by chemiluminescence immunoassay. Serum concentrations of acyl-carnitines and amino acids were determined by electrospray tandem mass spectrometry. Diet was assessed by a food frequency questionnaire. The low-BMD group had fewer observed species, higher abundance of γ-Proteobacteria, lower consumption of lycopene, and lower concentrations of leucine, valine, and tyrosine compared with the normal-BMD group. These amino acids correlated positively with the abundance of Bacteroides. Lycopene consumption positively correlated with Oscillospira and negatively correlated with Pantoea genus abundance. Finally, the intestinal microbiota of women with vitamin D deficiency was related to Erysipelotrichaceae and Veillonellaceae abundance compared to the vitamin D non-deficient group. Associations mediated by the gut microbiota between diet and circulating metabolites with low-BMD were identified. The effect of microbiota composition and its health on bone tissue is a novel field for research. However, their associations with bone mineral density (BMD) have not been established in postmenopausal women. The present study investigates the relation of diet, the microbiota composition, and the serum metabolic profile in postmenopausal women with normal-BMD or with low-BMD. Ninety-two Mexican postmenopausal women were classified into normal-BMD ( Osteoporosis (OP) is a skeletal disease characterized by the loss of bone mineral density (BMD) that leads to bone fragility. OP predisposes people to fragility fractures, mainly at the hip, spine, and wrist. The prevalence of OP increases with age and is elevated among postmenopausal women due to estrogen deficiency . In MexiGenetic, hormonal, immune system, nutritional and lifestyle factors play a determining role in bone tissue mineralization. Several mechanisms accelerate bone loss through alteration of osteoblast and osteoclast activity. For instance, bone mass loss mediated by estrogen deficiency is associated with the differentiation of and activity of osteoclasts, due in part to the increase in the production of proinflammatory cytokines  and the activation of immune cells . Althoug+/Ca2+ exchange, regulating the absorption of minerals [The absorption of calcium is determined by several factors: the levels of vitamin D, the intake of Ca, the consumption of phosphates, phytic acid, oxalates, the consumption of excess sodium, and the Ca/phosphorus ratio, among others. Although most of the calcium is absorbed in the small intestine, the colon also has an absorptive capacity. One possible mechanism is the fermentation of dietary fiber by the intestinal microbiota, inducing short-chain fatty acid (SCFA) production, lowering the luminal pH. The low-pH environment increases calcium ions. However, the acidifying medium alone does not ensure calcium transport in the colon. Therefore, attributing the pH reduction entirely to solubilizing the minerals seems like a simplistic approach. Another possible explanation is that SCFAs could directly affect the signaling pathways in the Hminerals .Prevotella copri and Bacteroides vulgatus were associated with increased concentrations of branched-chain amino acids (BCAAs) and with the presence of IR [Due to its contribution to modulating several host processes, which include energy metabolism, intestinal permeability, intestinal hormone secretion, and the regulation of the immune response, the intestinal microbiota composition has been proposed as an important factor in the development of chronic non-communicable diseases and in several inflammatory alterations, such as Crohn’s disease, obesity, and rheumatoid arthritis . Thus, ice of IR . Furtherce of IR . OtherwiSome studies have recently shown a tight relationship between bone metabolism and intestinal microbiota, hence their potential effect on the risk of developing osteoporosis ,10,11. HAccordingly, the study of the microbiota composition and the health of the bone tissue is a novel field for research, representing a potential new biomarker for diagnosis and prognosis of metabolic diseases in the early stages, as well as contributing to improvements in the treatment . This stNinety-two unrelated postmenopausal women were selected from “The Health Workers Cohort Study (HWCS),” from the Mexican Social Security Institute (IMSS), located in Cuernavaca, Morelos . Data foAll women selected for the analysis accomplished the inclusion criteria , and available bone mineral density measurements). Women with metabolic diseases  and those under medication therapy  were excluded from the analysis . The pro2) were calculated using the software in a Lunar DPX NT dual-energy X-ray absorptiometry (DXA) instrument . All subjects in the analysis were classified into two groups according to the hip T-score measurement. Normal-BMD group included the participants with T-scores from −1.0 to +1, and the low-BMD group included participants with T-scores from −1.1 to −4. Serum vitamin D was determined by the chemiluminescence immunoassay (CLIA) in a LIAISON Analyzer . Based on the serum vitamin D levels, two groups were performed. The vitamin D deficiency (VDD) group was defined as serum 25 hydroxyvitamin D (25(OH)D) levels <20 ng/mL, and the vitamin D non-deficient (VDND) group as serum 25(OH)D ≥20 ng/mL, as previously reported [2), the following anthropometric measurements were obtained: weight and height following standardized procedures applied by trained personnel.All parameters measured were performed following standardized procedures. Femoral neck and lumbar spine BMD  derived from a study reported previously for the Mexican population ,19. The Each woman was instructed to collect a fecal sample in a sterile container and were then transported in ice-filled coolers. The samples were processed as previously described . BrieflyThe specific primers 515F and 806R were used for the sequencing of the V4 hypervariable region, as suggested by the Earth Microbiome Project  and as pProcessing of the Illumina fastq reads was performed using the QIIME 1.8.(16). The UCHIME algorithm was used for detection and removal of chimeric sequences. The Greengenes database was used as the reference 16S database. Taxonomy was assigned by the Ribosomal Database Project (RDP) classifier using a threshold of 80% . The taxg. Finally, 10 µL of each sample were injected into the flow at 4-min intervals. The Micromass Quattro equipment  was used coupled to an ESI source in positive mode. Nitrogen gas was used for desolvation and nebulization, and argon as the collision gas.Concentrations of serum acyl-carnitines, free carnitine, and amino acids were measured using the approach of targeted metabolomics by electrospray tandem mass spectrometry . Metabolite levels in serum were analyzed using the commercial kit , as previously described . Brieflyt-test was used to evaluate the parametric variables. For the non-parametric variables, the Kruskal–Wallis test was used to determine the significant variables among the groups. The results were expressed as mean and median. p-value < 0.05 were considered as statistically significant. Plots were generated using the ggplot2 package. Differential metabolite levels were evaluated by PLS-DA between groups. Student’s t-tests was performed for the differences between BMD high and BMD low. The Galaxy interface  was used to perform a linear discriminant analysis effect size (LEfSe) method for assessment of microbial communities’ differences (LDA < 2). The web-based tool Microbiome Analyst  was used to performed heat trees. The heat trees analysis uses the hierarchical structure of taxonomic classifications to quantitatively (using the median abundance) and statistically (using the non-parametric Wilcoxon Rank Sum test) Benjamin Hochberg (FD) corrected depict taxonomic differences between microbial communities.Student’s p < 0.05) were found. No significant differences were achieved between both groups in variables, such as BMI, nutritional status, waist circumference, body fat proportion, physical activity, smoking, glucose, diabetes status, vitamin D status, vitamin D intake, calcium intake, and dietary measurements. However, hemicellulose and dietary fiber showed a statistically significant difference. Additional characteristics of the postmenopausal women included in this study are shown in Significant differences among the groups in age, femoral neck (FN-) BMD, lumbar spine (LS-) BMD, and total cholesterol    A. Concerctively) B,C.Bacteroidetes, with a mean abundance of 53% across the dataset, followed by Firmicutes with 40%. Other phyla, such as Proteobacteria, Verrucomicrobia, Tenericutes, and Cyanobacteria, accounted for 6%, while Actinobacteria was less abundant (1%) (Firmicutes/Bacteroidetes ratio (F/B), although the low-BMD group showed a higher F/B ratio than the normal-BMD group, this was not statistically significant (p = 0.22) , Anaeroplasmataceae (family), and Yersinia (genus) was found in the low-BMD group . In contrast, an over-representation of Akkermansia (genus) and Actinobacteria (class) were found in the normal-BMD group   A. Regard = 0.22) B. The inMD group C. To anactively) D.Prevotellaceae and Prevotella were decreased in middle-age women with low-BMD compared to middle-age women with normal-BMD , middle-old (60–74 years), and old (>74 years) as well as normal-BMD and low-BMD. rmal-BMD A. The γ-rmal-BMD B. Similarmal-BMD C.Lachnospiraceae, Vagococcus, and Dialister positively correlated with fiber consumption. Vitamin D intake was negatively correlated with Lachnospiraceae and positively correlated with Oscillospira. Calcium consumption was negatively correlated with Butyricimonas (genus). Lycopene consumption was decreased in the low-BMD group, and positively correlated with Oscillospira and negatively correlated with Pantoea genus abundance , starch, and manganese consumption A. In conbundance B.Christensenellaceae and Mogibacteriaceae family and positively correlated with Bacteroides , tyrosine, were negatively correlated with the teroides B. Intereteroides A.Vitamin D deficiency is related with bone disorders, including low BMD, and increases fracture risk. Remarkably, postmenopausal women deficient in vitamin D have higher concentrations of citrulline and ornithine than deficient women with higher serum concentrations of BCAAs, glucogenic, and AAAs B.Firmicutes and Bacteroidetes, succeeded by Proteobacteria, Verrucomicrobia, Tenericutes, Cyanobacteria, Actinobacteria, and Euryarchaeota, which were limited  and Erwinia genus abundance   E.The gut microbiota is recognized as a significant contributor to many human chronic diseases . OtherwiOur results are consistent with other studies suggesting that low-BMD subjects have fewer observed species than the normal-BMD group ,29. The Klebsiella and Erwinia genus belonging to γ-Proteobacteria phylum were increased in the group with low-BMD compared with the normal group. This finding coincides with that recently reported by He et al. in postmenopausal women with osteopenia [Proteobacteria [Proteobacteria phyla have been associated with dysbiotic processes that lead to inflammation, which correlates with osteoclast activation, thus affecting bone health [In the present study, the abundance of teopenia . Interesbacteria . He et ae health . Lachnospiraceae in people with low-BMD, and a positive correlation between BMD and T-score [Clostridium cluster XlVa, Lactobacillus, Actinomyces, and Eggerthella in OP patients compared to healthy donors [Wang et al. reported the relationship between osteoporosis/osteopenia and the intestinal microbiota. The authors point out that composition and bacterial diversity is different in patients with OP and osteopenia than in healthy subjects . Li et a T-score . Recently donors , which iActinobacteria was significantly more abundant in the normal-BMD group. Interestingly, some members of the Actinobacteria phylum have been positively associated with bone density. The possible mechanism would be related to the decrease in intestinal inflammation and the improvement of the intestinal barrier [Otherwise,  barrier . AdditioBifidobacterium, the proportion of Bacteroides/Prevotella, Lactobacilli, and Faecalibacterium prausnitzii. Age also increases the abundance of Ruminococcus, Atopobium, and certain Proteobacteria, which contains several pathobionts [Lactobacillus plantarum PBS067, Lactobacillus acidophilus PBS066, and Lactobacillus reuteri PBS072 decreased serum markers associated with inflammation, such as C-reactive protein and tumor necrosis alpha-factor, as well as several risk factors associated with metabolic syndrome [The gut microbiota composition changes related to age include a decrease in saccharolytic bacteria like hobionts ,34. Aginhobionts . Howeversyndrome . InteresPrevotella copri and Bacteroides vulgatus have been associated with increased concentrations of BCAAs and with the presence of IR [Bacteroides. Recently, Ponsuksili et al. correlated Bacteroides abundance with several miRNAs and transcripts involved with bone metabolism and inflammatory response [Bacteroides fragilis evoke an anti-inflammatory response by inducing Treg cells. Therefore, microbiota composition may impact bone health by affecting T cell differentiation, eliciting an anti-inflammatory response by inducing Treg cells. Thus, it is possible that microbiota composition can impact bone health by influencing T cell differentiation [Diet is involved in modifying bone health and is an important driver in the intestinal microbiota composition. Hence, nutrients influence gut microbiota composition, and, in turn, microbiota produces metabolites via the degradation of nutrients that may affect several cellular functions and modulate the immune response . Some ofce of IR . Otherwice of IR , althougce of IR . In the ce of IR . Howeverce of IR ,40,41,42response . Bacterontiation .Oscillospira and negatively correlated with Pantoea genus abundance. Carotenoids are natural pigments that are considered key players in the regulation of bone metabolism [Oscillospira abundance is associated with microbial diversity, leanness, and high levels of HDL [Pantoea is a potentially pathogenic bacteria belonging to γ-Proteobacteria phylum and Enterobacteriaceae family associated with inflammatory responses [As mentioned above, diet is an important driver for the intestinal microbiota composition. In our study, lycopene consumption was decreased in the low-BMD group, and positively correlated with tabolism . Carotentabolism  and thattabolism ,47. Furts of HDL ; meanwhiesponses . The resErysipelotrichaceae and Veillonellaceae abundance compared to the vitamin D non-deficient group. Interestingly, both taxa are affected in patients with cystic fibrosis-associated vitamin D deficiency [Erysipelotrichaceae family possess opportunistic pathogenic bacteria and have been correlated to obesity, colorectal cancer, and inflammation. Collectively, we speculate that the observed increase in Erysipelotrichaceae and Veillonellaceae in vitamin D deficiency in postmenopausal women has the potential to contribute to an increased risk of inflammation and metabolic disorders. This speculation is worth further investigation with long-term follow-up of postmenopausal women with vitamin D deficiency. Finally, bone tissue mineralization and intestinal microbiota are determined by several factors, including vitamin D levels . Previouficiency . The EryOur study has some limitations but several key strengths. Even though we analyzed a modest sample size, this is the first study of diet, the microbiota, and the metabolome conducted in postmenopausal women with a low BMD. The information on the diet was collected from a self-reported questionnaire including 116 items. So, the error derived from the questionnaire may minimize other possible correlations in our measurements. Additionally, all the participants were recruited from the central region of Mexico and probably have similar dietary habits. The gut microbiota residing in the intestine is a complex community. Hence, the dynamics of the composition and function are influenced by non-modifiable factors, such as age, sex, and geographical location, and modifiable factors, such as diet, illness, and medications, especially antibiotics. Only postmenopausal women were selected, excluding women with exposure to antibiotics or hormone replacement therapy within six months before the collection of the fecal sample and excluding women who had non-communicable diseases. A limitation of our study is that the sampling was cross-sectional (third HWCS assessment). Longitudinal studies are required to determine whether changes in dietary patterns affect the metabolism mediated by the gut microbiota and whether these variations can influence bone health.By performing a multi-omic analysis in a subsample of a widely phenotyped cohort study, for the first time, microbiome-mediated relationships between circulating metabolites and diet with low-BMD were identified. The findings of our analysis provide helpful evidence to elucidate the underlying microbiota–diet–metabolites relevant mechanism in loss of bone mineral density.


We report the case of a successful management with combined aggressive surgery and negative pressure therapy, to treat a severely ill-septic patient, affected by multiple chronic enterocutaneous fistulas.A 26-year-old female patient presented with multiple pelvic and intra-abdominal abscesses, enterocutaneous fistulas and central venous catheter-related bacteraemia in extremely poor general conditions.The patient underwent both an abdominal CT which showed multiple digestive loops stuck and apparently fistulised and an abdominal-pelvic MRI, confirming the CT findings, and demonstrating a third fistula involving the Pouch and responsible for a pelvic and retroperitoneal chronic abscess.Given the patient's septic condition, despite several attempts of conservative therapies, an aggressive surgical approach was adopted.After temporary abdominal wall closure, the patient underwent Vacuum Assisted Closure therapy in order to close the abdominal wall and drain the residual abscess. The patient was discharged at the 35th post-operative day in good general conditions.This case is about a complex, long-lasting clinical scenario, progressively leading a young woman to death despite several attempts of conservative therapy, sometimes allowed to treat enterocutaneous fistulas. The use of negative pressure therapy to manage open abdomen is still controversial. Patients affected by enterocutaneous fistulas are in need of adequate nutritional support due to their hypercatabolic state, secondary both to the fluid loss and the concomitant inflammatory status.When conservative management fails and the patient shows septic complications, a multidisciplinary aggressive approach, including surgery, negative-pressure therapy and hyperbaric oxygen therapy is required to treat this life-threatening condition. •Using negative pressure therapy to deal with difficult abdominal wall closure.•Multidisciplinary team work is essential to optimize patients' surgical outcomes.•Highly-demolitive surgery can benefit from the use of negative pressure therapy. They can be classified according to several aspects such as their daily-output , their location , their aetiology (iatrogenic or spontaneous), and their numerosity .The global incidence of ECFs is still unknown but data from retrospective studies state that in the 25% of cases they are secondary to other clinical conditions  while in the 75% ECFs arise from a post-operative anastomotic leak [The use of negative pressure (VAC Therapy) is still controversial as there is not clear evidence from literature: some studies report an increase in the incidence of ECFs while some others state the opposite ,4.In the present case report, we will discuss about the successful management with combined aggressive surgery and negative pressure therapy (NPT), to treat a severely ill-septic patient, affected by multiple chronic enterocutaneous fistulas.2A 26-year-old female patient was admitted at the Infectious Diseases Department at Federico II University Hospital, because of multiple pelvic and intra-abdominal abscesses, enterocutaneous fistulas and central venous catheter-related bacteraemia. Three years before the admission, the patient underwent proctocolectomy with ileal pouch for Familial Adenomatous Polyposis type 1 (FAP1) .2.At the time of the admission, the patient was in extremely poor general conditions , her nutd-glucan, Rectal Buffer, urine culture, haemoculture. Furthermore, the patient underwent abdominal CT (with/without contrast) showing a diffuse inhomogeneity of the adipose peritoneal cellular layer in the pelvic cavity, with multiple digestive loops stuck and apparently fistulised. Then, an abdominal-pelvic MRI was performed, confirming the CT findings, and demonstrating a third fistula involving the Pouch and responsible for a pelvic  and retroperitoneal chronic abscess.  and peripheral catheter (PVC) in case of fever.During her post-operative course, the patient was administered with antibiotics, antifungin, albumin and anticoagulant drugs. Her phlogosis indexes (CRP and PCT) were daily tested whilst there was a weekly evaluation of B-The VAC therapy device was replaced every 72 hours and completely removed after re-granulation of the anterior abdominal wall on 25th POD. The patient underwent hyperbaric oxygen therapy (HBOT) for 15 days. She was finally discharged on 35th POD with trophic, active and well-functioning stoma with haematochemical values within the normal range, in definitely improved general conditions. As far as the nutritional status in concerned, we experienced a significant improvement. In addition to the BMI, the laboratory tests improved too: preoperative albumin value was 1.8 g/dL versus 4 g/dL postoperatively (n.v. 3.5–5.2 g/dL) while the preoperative serum total protein value of 4 g/dL increased to 6 g/dL (n.v. 6.4–8.3 g/dL) at 12th POD. Immediately after surgery, the patient received TPN which was accompanied by oral nutrition from the 7th POD. Exclusive oral nutritional therapy was administered since the 15th POD. According to both nutritionists’ and our opinion, the rapid BMI increase was due to both to the resolution of her chronic catabolic inflammatory status and the nutritional therapy provided with high calorie and protein intake.2) with a remarkable improvement of her performance status.The patient has been followed-up at our general surgery ambulatory for post-operative care and is currently in very good general conditions. She is monthly followed-up by our nutritionists team and is getting continuous improvement . Six mon3Entero-cutaneous fistulas (ECFs) are such a challenging surgical scenario and still represent an important cause of post-operative morbidity/mortality: this is the reason why they are a highly-debated topic in General Surgery.As previously stated, the use of NPT is still controversial as there is not clear evidence from literature: some studies report an increase in the incidence of ECFs while some others state the opposite ,4.Literature studies regarding the use of NPT to manage open abdomen present consistent limitations due to enrolled-patients’ heterogeneity ,8, to thThe National Institute for Health and Care Excellence (NICE) recommends  the dresPatients affected by ECFs are in need of adequate nutritional support due to their hypercatabolic state, secondary both to the fluid loss and the concomitant inflammatory status. Therefore, it is mandatory to correct electrolyte disorders, daily evaluation of the acid-base balance and the fluids input/output.Parenteral nutrition (PN) has been the standard therapy for many years even though, nowadays, several studies demonstrated that enteral nutrition (EN) can be approachable and effective in reducing infectious complications .Good prognostic factors of possible conservative fistula resolution include good nutritional status, serum albumin > 3g/dL, diverticular disease-related fistula, low fistula output, single fistula, intestinal continuity, whereas negative prognostic factors are generally poor nutritional status, low serum albumin/ferritin levels, intestinal abnormality >1cm, high fistula output, previous radiotherapy and surgical interventions .Spontaneous fistula healing is very tough to be obtained, even more when dealing with deep fistulas and patients with underlying diseases.In particular, once an ECF is diagnosed, it should be necessary to explore the abdominal cavity to evaluate the presence of misrecognised fistulas, underlying gastrointestinal diseases, foreign bodies, neoplasms and intestinal obstruction. If one of these conditions is found, the initial management should include its resolution, dealing with the fistula afterwards .When dealing with small, superficial, distal and low-output fistulas, a primary closure could be attempted with sutures and several types of glue (fibrin glue and cyanoacrylate glue) .In most cases, surgeons tend to perform a two-stage procedure: ECF removal at first and secondly the closing of the abdominal wall, once a proper physiologic homeostasis is obtained (8–12 months after surgery).However, in our case, given both local and general patient's clinical conditions, also including multiple abdominal abscesses and pouch leak, we decided to proceed with a resolution of the ECFs as first instance. We performed a challenging adhesiolysis of the small intestine, the removal of the pre-existing fistulising pouch, the 50cm-long ileal resection and the construction of a terminal high ileostomy.Then, the residual abdominal abscesses were drained thanks to a negative-pressure device  and the The risk for intestinal failure (IF) and short-bowel is another issue to take into account when dealing with ECFs.IF is characterised by the inability of the gut in absorbing macronutrients , micronutrients  and water with the mandatory parenteral integration of them . It can -Type 1: secondary to an acute condition and, usually, auto-limiting (ex. post-operative ileus)-Type 2: due to an abdominal surgical intervention in critic patients with multiple fistulas. It requires a multidisciplinary approach along with weekly or monthly-lasting parenteral nutrition.-Type 3: the chronic one, in which patients are in need of a long-lasting parenteral nutrition. It can be reversible . Also, iIt can be classified as :-Type 1:IF average survival is high when it is due to a benign condition, reaching the 80% in adults and 90% in children . Unfortu4ECFs fistulas are a challenging and high risk situation. When conservative management fails and the patient shows septic symptoms, a multidisciplinary aggressive approach, including surgery, patient optimization, negative-pressure therapy and HBOT is required to treat this life-threatening condition.Written informed consent was obtained from the patient for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal on request.Not commissioned, externally peer reviewed.None.This paper was not a research study, so ethical approval not required.Name of the registry:Unique Identifying number or registration ID:Hyperlink to your specific registration (must be publicly accessible and will be checked):Prof. Gaetano Luglio is the Guarantor of this case report.Gaetano Luglio: Conceptualization, Writing - original draft. Alfonso Amendola: Writing - original draft. Gianluca Pagano: Writing - original draft. Francesca Paola Tropeano: Writing - original draft. Chiara Errico: Data curation, Writing - original draft. Enrica Esposito: Data curation, Writing - original draft. Giuseppe Palomba: Writing - original draft. Paola Dinuzzi: Writing - original draft. Giuseppe De Simone: Writing - original draft. Giovanni Domenico De Palma: Conceptualization, Writing - original draft.All authors disclose any financial and personal conflict of interest.


Tumor necrosis factor (TNF)-alpha polymorphisms have been repeatedly indicated to play a pathogenetic role in various mental disorders, but none of these studies focused on the susceptibility to suicidal behavior in SCZ. We recruited 1087 chronic inpatients with SCZ and 576 controls to assess the psychopathological symptoms of SCZ using the Positive and Negative Syndrome Scale scales. We selected 2 polymorphisms (-308G>A and -1031C>T) in the TNF-alpha gene and analyzed their associations with SCZ and suicide. Our results showed that TNF-alpha -308G>A and -1031C>T were not related to SCZ and suicide. However, we found that suicide attempters with the C allele carriers exhibited suicidal behaviors significantly later than those with TT genotype in the SCZ patients. The haplotype containing the T allele of the -1031 was significantly associated with the age of suicide initiation. Further logistic regression analysis showed that -1031C>T interacted with psychopathological symptoms and drinking, age of smoking, and related to the initiation age of suicide attempts. Our study demonstrated that the TNF-alpha variants may affect the age at which suicide attempts started among SCZ suicide attempters.Patients with schizophrenia (SCZ) exhibit higher suicide rates than the general population. However, the molecular mechanism remains poorly understood.  Patients with schizophrenia (SCZ) have a high risk of attempting and completing suicide. Suicide is considered as one of the most common causes of premature death in SCZ patients . The aveRecent findings have recognized the possible role of the abnormal immune system in the primary pathophysiological mechanism of suicidal behavior –4. It isTNF-alpha polymorphism and suicide in SCZ. At this time, only the -308G>A polymorphism in the TNF-alpha gene has been studied in relation to suicide attempts in individuals with MDD [Several lines of studies of peripheral biomarkers have shown higher serum TNF-alpha levels in those persons with suicide attempts, suicidal ideation, or suicide –13, but with MDD .TNF-alpha gene has multiple frequent polymorphisms [TNF-alpha gene displayed better cognition functions [Inflammation is not only related to suicidal behavior, but also to mental disorders. Many studies by our team and others have suggested that the pathogenesis of schizophrenia involves an alteration of peripheral immune system that leads to altered blood levels of TNF-alpha –23. A reorphisms  and seveorphisms –27. Our unctions  and decrunctions . Taken tTNF-alpha gene and alteration of TNF-alpha serum levels in SCZ, it would be beneficial to investigate the association between the TNF-alpha gene and suicidality of SCZ patients. Based on previous literature, we selected -308G/A (rs1800629) and -1031T/C (rs1799964) single nucleotide polymorphisms (SNPs) to assay among the patients with SCZ in this study. -308G/A polymorphism has been confirmed by several studies to be a susceptibility factor for suicide in patients with MDD [Considering the high suicide rate in the patients with SCZ and the pathogenic role of with MDD , while -with MDD . Thus, wThe demographic characteristics and clinical data are shown in TNF-alpha -308G>A (rs1800629) and -1031C>T (rs1799964). Also, there was no genotypic or allelic correlation between the two SNPs and attempted suicide  and PANSS total scores  reached significantly different values in the subgroup analysis. However, the association between -308G>A and suicide still was not statistically significant after a logistic regression analysis (p>0.05). Given that most of the samples were male patients, we performed stratified analysis for each sex and still did not find significant differences in the allele and genotypic frequencies of the two SNPs between male suicide attempters and male non-attempters or female attempters and female non-attempters .No deviation from Hardy-Weinberg equilibrium was found in the patients and controls, or in the subgroup analysis based on suicide attempt. No significant differences in the genotype and allele frequencies between patients and controls were found for  suicide . In a stTNF-alpha polymorphism and clinical variables, the quantitative trait test was used to determine the relationship between TNF-alpha -308G>A or -1031C>T and individual phenotype. We found no association between -308G>A or -1031C>T and any clinical phenotypes, including gender, age, education, onset of illness, BMI, antipsychotic treatment , hospitalization, duration of illness as well as the symptoms of schizophrenia that were measured by the PANSS  and -1031C>T genotype  to be related to age at suicide attempt initiation.As shown in TNF-alpha gene and risk for attempted suicide in Han Chinese patients with SCZ, showing no relationship between TNF-alpha gene -308G>A and -1031C>T polymorphism and SCZ and suicide attempt. However, we found a link between the -1031C>T polymorphism and the age at which SCZ patients began to attempt suicide. Moreover, the age of suicide initiation was significantly positively correlated with age of symptoms onset in the patients.This is the first investigation to explore the relationship between TNF-alpha gene and SCZ, but with inconsistent results [TNF-alpha gene 1031T-863A-857C-308G, 1031C-863C-857C-308G, and 1031C-863C-857T-308G and increased risk of SCZ [TNF-alpha polymorphism and SCZ might be due to ethnic differences, as the frequency of -1301C>T and -308G>A genotypes varied significantly between different ethnicities.Several studies have investigated the association of polymorphisms in the  results , 30–33.  results –36, espe results . MoreoveTNF-alpha gene, our results indicate that there was no association between two tag SNPs in the TNF-alpha gene and suicide attempt. The patients with SCZ were associated with higher risk of suicide than the general population, and previous studies speculated that the pathophysiology of suicide might originate as a disturbance within the immune system [TNF-alpha gene in suicide. Results from these groups showed that -308G>A was related to suicidal behaviors in MDD [TNF-alpha gene polymorphism and suicide in SCZ [TNF-alpha gene polymorphism. Furthermore, neither study showed any association between suicide and TNF-alpha haplotype in patients. We attributed the failure to find a relationship between suicide and TNF-alpha polymorphism to low sample size of suicidal patients with SCZ in both studies, since there were only 120 suicide attempters in this study and 74 attempters in the above study for the genetics analysis. The sample size of suicide attempters was small, resulting in an even smaller number of patients found to have the CC genotype of -1031 polymorphism, which may produce false negative results. Therefore, additional studies with larger samples containing several ethnic populations are needed to confirm these findings. It is worthy of mentioning that in a stepwise logistic regression analysis for relationships between attempted suicide and TNF-alpha polymorphisms, with age, gender, education, BMI, smoking and clinical symptoms shown on PANSS scores as covariates, we found that -1031C>T genotype distributions  and PANSS total score  reached significance. This result suggests that under the influence of clinical variables, combined with clinical psychopathological symptoms, the TNF-alpha -1031C>T polymorphism may play a role in attempted suicide in SCZ patients. Why was there no significant difference in the allele or genotype distribution of TNF-alpha -1031C>T polymorphism between suicide attempters and non-attempters in SCZ patients, and then this difference became significant when combined with clinical variable, especially psychopathological symptoms? However, due to our cross-sectional design in this study, we are currently unable to provide a reasonable explanation for this phenomenon, which deserves further exploration in future research.Regarding the relationship of suicide attempt and the e system –39. Howes in MDD –21 and te in SCZ . HoweverAnother important finding was that the -1031C/T polymorphism may have an effect on the age at which suicidal behavior initiation occurred in SCZ. Patients attempting suicide with the -1031C allele started committing suicide later than those who carried the TT genotype. Further haplotype analyses found that the age at which suicidal behavior initiated in patients carrying 308(A)-1031(T) was earlier than those with haplotype counterparts, which further supports that the -1031TT genotype may be associated with earlier age of suicide initiation. Suicide is a complex behavior, and recent scientific literatures analyzed the age of suicide initiation of suicidal attempters as a potential candidate marker to characterize different subgroup attempters and reduce the heterogeneity of suicidal behavior –42. ThesTNF-alpha -1031 TT genotype and the earlier age at which suicide initiation occurred in the patients with SCZ. Many studies have indicated that the two tag SNPs (-1031C>T and -308G>A polymorphisms) have an effect on the protein production; although these results were contradictory [TNF-alpha mRNA was associated with impulsivity and hopelessness after controlling for demographics and substances used [It would be extremely meaningful to explore the possible mechanism underlying the associations between the adictory . For exaadictory . Anotheradictory . Moreoveadictory . Taken tadictory –51. Howeadictory . In partces used . In addices used . Taken tTNF-alpha gene knockout mice showed increased emotional responses when exposed to stress compared with wild-type mice [On the other hand, our previous results demonstrated that -1031C/T polymorphism was related to the onset age of SCZ in long-term hospitalization patients with SCZ . Interesype mice . Hence, ype mice –56. Animype mice , while pype mice , 58. Furype mice . Activatype mice . A recenTNF-alpha gene and suicidal tendencies in SCZ was preliminary, and further validation needs to be carried out in a larger, independent sample to increase statistical capacity before a definitive conclusion can be drawn. Second, there are several known polymorphisms in the promoter of TNF-alpha gene, but we had measured only two of those polymorphisms. Whether the other variants affected the results of this study is unclear. A complex analysis of the influences of polymorphisms in the gene should be examined in further researches. Third, the levels of TNF-alpha and other inflammatory cytokines in the cerebrospinal fluid and blood were not measured in this study. Therefore, we were unable to assess whether the T allele of the -1031T/C caused a decrease in TNF-alpha levels in the brain. Fourth, one of the major limitations of the current study is short of data on suicide method and associated lethality. Therefore, suicide attempts were not classified as serious suicide attempts. Therefore, we were able to only explore various factors related to suicide attempts, rather than the severity of the suicidal behaviors. In further investigations, an appropriate tool to evaluate the severity of suicide attempts would be warranted. Fifth, the hidden demographic stratification in our sample may be a confounding factor. We had a small number of female patients that may cause a discrepancy in results. Sixth, all SCZ patients enrolled in this current study were inpatients with long-term duration of illness. Hence, our findings were limited to chronic inpatients with longer duration of symptoms and more severe psychopathological symptoms compared to the typical psychiatric outpatients. Therefore, our findings in this study could not be generalized to other patients. Seventh, it is important to tease out how the genetic variation at the specific locus is getting translated into a phenotypic variation. However, we did not test the functional relevance of -1031C/T by using an in vitro cellular model, which should be remedied in the future study. Finally, although previous study showed that the C allele of TNF alpha-1031C/T was associated with higher expression of TNF-alpha , we did not measure TNF-alpha levels in this study. It would be better if the blood level of TNF-alpha could be provided in the same patient sample to increase the validity of this paper. Thus, we could not provide the mechanistic explanation linking the C allele to later age of suicide attempt in SCZ patients, which deserves further investigation.The study has several limitations that should be noted. First, although the sample was large, only 152 patients with suicidal behaviors were included. Therefore, the relationship between TNF-alpha gene did not appear to modify the propensity to develop schizophrenia and suicidal behaviors, but they did influence the age of suicide initiation in patients with SCZ. In addition, environment factors, such as drinking status, smoking, and psychotic symptoms, interacted with TNF-alpha gene and appeared to be related to the age at suicide initiation in SCZ. This suggests that the interaction between the genetics and environment might influence some pathological aspects of suicide in SCZ. However, our results may have appeared by chance, due to limited sample size of suicide subgroup. Therefore, the current association finding in this study should be confirmed in other independent samples.In conclusion, we found that the -1031C>T and -308G>A in the 1087 inpatient with schizophrenia were randomly recruited from Beijing HuiLongGuan hospital and HeBei Province Veteran Psychiatric Hospital. The current study was conducted from August 2015 to September 2017. All patients were of the chronic type, with duration of illness for at least 5 years. The inclusion criteria in the study were as our previous studies in detailed descriptions: age 20-75 years; and diagnosis of SCZ according to the Structured Clinical Interview for DSM-5 (SCID) by the trained psychiatrists. All subjects had been taking stable doses of oral antipsychotic medication for at least one year prior to recruitment. Antipsychotic medications consisted mainly of drug monotherapy, including clozapine (n = 488), risperidone (n = 239), chlorpromazine (n = 79), sulpiride (n = 55), perphenazine (n = 51), quetiapine (n = 47), haloperidol (n =38), aripirazole (n = 32), and others (n = 58). According to the method provided by Woods , the aveAccording to the definition of the World Health Organization (WHO 2014), the above four psychiatrists evaluated the history of suicide attempts based on chart review and interview data. Attempted suicide refers to any non-fatal suicide, which is intentional self-inflicted poisoning, injury, or self-harm, with or without fatal intentions or consequences (WHO 2014). In this study, there were 152 SCZ patients with a history of suicidal attempt and suicidal ideation and 935 SCZ patients without suicidal behaviors. The mean number of attempted suicide was 1.51 ± 0.94 times (ranging from 1 time to 5 times).In this study, 576 healthy controls were recruited. They were randomly recruited through advertisements in the local community, and they matched patients with fewer years of education. Patients and healthy controls had comparable socioeconomic backgrounds. Psychiatrists used the standardized SCID diagnostic assessment to rule out controls with Axis I disorders. Individuals taking psychoactive drugs  were also excluded.The current study was approved by institutional Review Board of Beijing HuiLongGuan hospital. All subjects provided signed informed consent form to participate in this study.On the same day of blood drawing, the symptoms of schizophrenia were assessed by PANSS. Four psychiatrists participated in a training session on the use of PANSS and conducted repeated assessment tests prior to the study. In this present study, a five-factor model of SCZ symptoms was analyzed, labeled as ‘positive factor’, ‘negative factor’, ‘cognitive factor’, ‘depression factor’, and ‘excitement factor’ .TNF-alpha gene based on previous studies, -308G>A (rs1800629) and -1031C>T (rs1799964). These two polymorphisms were genotyped as the protocol described in our previous study [Based on previous published studies, we selected 2 tag SNPs in the >A rs18009 and -10A technician who was blind to clinical conditions genotyped each DNA sample of all subjects twice for the accuracy of genotyping. For quality control, 5% of all samples were randomly selected for repeated genotyping. Quality control tests showed that the error rate of regenotyping data was less than 0.1%.http://analysis.bio-x.cn). Analysis of variance (ANOVA) for continuous variables and the Chi-Square test (χ2 test) for categorical variables were used to analyze the differences in patients and controls. ANOVA and 2χ test were used to find the associations between demographic and clinical variables and the suicidal behaviors. The differences in allele and genotypic frequencies of -308G>A and -1031C>T polymorphisms between patients and healthy controls and between suicide attempters and non-attempters within the patient group were tested by the 2χ test. SHEsis software was utilized to analyze pairwise LD statistics for two tag SNPs, haplotype frequency, haplotype block, and haplotype association with schizophrenia or suicidal behavior. Quantitative trait tests were conducted to analyze the relationship between gene polymorphisms and characteristics of suicidal behavior. Less than 1% of rare haplotypes were excluded in the association analysis. The effects of the TNF-alpha genotypes on the demographic characteristics and clinical symptoms in schizophrenia attempters were examined by ANOVA and effects of genotypes on the age of suicide initiation were examined by one-way analysis of covariance (ANCOVA) to adjust for clinical confounding factors using the SPSS 18.0 software. Bonferroni corrections were used to each test to adjust for multiple testing.Adherence to Hardy-Weinberg (HW) equilibrium was evaluated by SHEsis (TNF-alpha genotypic association with suicidal behavior within the patient group while considering recessive, dominant and codominant genetic models. Only the significant genetic models in the binary logistic regression and those variables with significant associations in the ANOVA analyses between schizophrenia attempters and non-attempters were included in the stepwise multiple logistic regression analyses. In the model, the genetic models and confounders were used as independent variables, and the diagnosis was used as a dependent variable.Furthermore, we used binary logistic regressions to calculate Statistical power of the sample was computed using Quanto Software with known risk allele frequencies and the suicide attempt prevalence in the patients with schizophrenia.


Collectively, this compendium of scRNA-seq data provides a valuable entry point for understanding the transcriptional and dynamic changes of non-cardiomyocytes in healthy and nonhealthy hearts by integrating multiple datasets.Advances in single-cell RNA sequencing (scRNA-seq) technology have recently shed light on the molecular mechanisms of the spatial and temporal changes of thousands of cells simultaneously under homeostatic and ischemic conditions. The aim of this study is to investigate whether it is possible to integrate multiple similar scRNA-seq datasets for a more comprehensive understanding of diseases. In this study, we integrated three representative scRNA-seq datasets of 27,349 non-cardiomyocytes isolated at 3 and 7 days after myocardial infarction or sham surgery. In total, seven lineages, including macrophages, fibroblasts, endothelia, and lymphocytes, were identified in this analysis with distinct dynamic and functional properties in healthy and nonhealthy hearts. Myofibroblasts and endothelia were recognized as the central hubs of cellular communication via ligand-receptor interactions. Additionally, we showed that macrophages from different origins exhibited divergent transcriptional signatures, pathways, developmental trajectories, and transcriptional regulons. It was found that myofibroblasts predominantly expand at 7 days after myocardial infarction with pro-reparative characteristics. We identified signature genes of myofibroblasts, such as Postn, Cthrc1, and Ddah1, among which Ddah1 was exclusively expressed on activated fibroblasts and exhibited concordant upregulation in bulk RNA sequencing data and  Cardiovascular diseases such as myocardial infarction (MI) are a leading cause of morbidity and mortality worldwide. Generally, the difficulties faced while treating ischemia involve not only the poor regeneration of cardiomyocytes after MI, but also the complexity of non-cardiomyocytes (non-CMs), including macrophages, fibroblasts (FBs), and T cells, which play a pivotal role in cardiac remodeling after MI . In the Recently, various studies have been dedicated to the exploration of the dynamic and functional changes of non-CMs after MI using a new method called single-cell RNA sequencing (scRNA-seq) , which pin vivo and in vitro experiments.In this study, we explored recently published articles on scRNA-seq investigating the functional changes of non-CMs in healthy or ischemic hearts and integrated three representative datasets containing cells collected at 0, 3, and 7 days after MI for a deeper analysis. A total of 27,349 cells, including major non-CM lineages, such as macrophages, FBs, endothelia, and smooth muscle cells (SMCs), were clustered into 20 subpopulations with distinct gene signatures. Analysis of the dynamics and intercellular communication of non-CMs revealed that FBs and endothelial cells (ECs) played a dramatic role in the cellular crosstalk via ligand-receptor interactions. Moreover, the endothelial-like macrophages (MAC-Endo) and FB-like macrophages (MAC-Fib) that exhibited a transcriptional profile of endothelia or FBs under a macrophage background were described in this study. By integrating these scRNA-seq data, we pointed to a novel marker of activated FBs, dimethylarginine dimethylaminohydrolase 1 (Ddah1), which was found to be sharply upregulated after MI in bulk RNA sequencing data and To our knowledge, this is the first study inquiring the possibility of integrating published scRNA-seq data to investigate the transcriptional and dynamic changes of non-CMs in healthy and nonhealthy hearts with unprecedented resolutions.in vivo as described previously  and maintained under a light/dark cycle. All animal experimental procedures were approved by the Animal Care Committee of Shanghai Jiao Tong University School of Medicine. The MI model was used to induced cardiac ischemia https://www.ncbi.nlm.nih.gov/geo/) or from ArrayExpress database (https://www.ebi.ac.uk/arrayexpress/) with full expression matrix and mapped Ensemble genes. Firstly, quality control of single cells was performed in every dataset by selecting single viable cells. Specifically, the percentage of mitochondria and the number of features in every cell were set as the limitations. Data on single cells were then filtered according to the source article criteria before the next analysis. After this quality-control procedure, each feature of the cells was normalized by dividing the total unique molecular identifiers and multiplying by 10,000 to obtain a value in transcripts per million, followed by logarithmic transformation using the R package Seurat  analysis or Gene Set Enrichment Analysis (GSEA)  in RStudp-value using the Benjamini-Hochberg (BH) method, significant connections between ligands and receptors were included for other analysis at a p-value < 0.01. A crosstalk network graph was generated using the R packages circlize (https://gephi.org).Cell-cell communication networks were assessed according to a previously published algorithm . Brieflycirclize  and GephImmunostaining was performed on the hearts of mice at 0, 3, and 7 days after MI surgery as reported previously . After iTotal protein was extracted from the heart tissues of mice after MI or sham surgery using a sodium dodecyl sulfate lysis buffer, and then protein concentrations were measured using a BCA assay kit. Western blot analysis was performed as described previously . Briefly−3.3, and their pseudotime trajectory was determined using the “learn_graph” function. The results were plotted in three-dimensional space. To analyze differentially expressed genes triggering a particular pseudotime distribution of cell populations, we used the “graph_test” function to investigate differential genes among cell clusters.Pseudotime analysis was specifically performed on macrophage and FB lineages from our integrated single-cell data using the R Package Monocle . The uniCore regulatory transcription factors of macrophages and T cell lineages were predicted using the R package SCENIC . The GENt-test. For experiments with more than two groups, after confirming normality and homogeneity of variance, one-way analysis of variance (ANOVA) followed by Tukey's post hoc test was used for comparison. For comparison between multiple experimental groups with a control group, the Dunnett's post hoc test was used in SPSS Statistics . For all analyses, statistical significance was set at p < 0.05.Data were presented as box-and-whisker plots or bar plots with all points, which were evaluated and organized using the commercial software GraphPad Prism . Statistically significant differences between every two groups were analyzed using Student's two-sided n = 1,866) or sham-operated hearts (n = 703) , and 7 days  after MI or sham  surgery   (Supplemn = 703) ; (2) iso surgery   col surgery  under a In this analysis, cells were clustered and designated depending on the concordant expression of well-characterized marker genes . SpecifiTaken together, these findings demonstrate that, by integrating different datasets, we were able to analyze non-CMs in healthy and injured hearts in a single-cell resolution.To delineate the composition of each cluster reflecting disease-related cell fluxes under ischemic conditions, separated UMAP plots including cells from sham, 3 or 7 days after MI  were mapped for a more directed visualization of cell composition during healing process . We obseIncreased cell-cell communication is a hallmark of cardiac repair and plays a pivotal role in cardiac remodeling . To inveAs macrophages constitute the largest number of immune cells in the heart and exhibit a dramatic cell flux in injured hearts , it was in vivo experiments  analysis  to invesIn our previous work, we observed that macrophages with varied origin exhibit divergent developmental trajectory and distinct transcriptional regulons. To explore the differences of gene signatures and pathways among TR and blood-derived macrophages, we compared the expression patterns of Ccr2, a well-known marker that distinguishes TR macrophages from blood-derived macrophages. Markedly downregulated expression of Ccr2 was observed in MAC-TR, MAC_Endo, and MAC_Fib clusters, compared to other cell populations . Next, tAs FBs constitute the largest number of non-CMs under homeostatic conditions and play an indispensable role in fibrotic scar formation and cardiac remodeling after MI, our next aim was to explore the functional changes of FBs in healthy and MI-operated hearts. In total, six FB clusters were identified , 5A in tWe then compared differentially expressed genes between Fibro_Myo and five other clusters and observed dramatic upregulation of Cthrc1, which has been confirmed to be a marker of myofibroblasts , and DdaNext, to investigate the trajectory development of FBs and myofibroblasts, we reclustered the FB populations in the Monocle package for pseudotime analysis . IntriguIt has been confirmed in a previous analysis that Ddah1 has a distinct function in the development and activation of FBs . To invein vivo and in vitro. Consistent with boosted Ddah1 protein level at 7-days after MI, we showed increased Ddah1+vimentin+ cells at 7-days after MI surgery compared with sham hearts  and border zones at 3-days after MI operations vs. sham group, and these cells were significantly expanded at 7-days post-surgery  secrete predominant ligands for cell communication, whereas ECs possess a large number of receptors. Among all ligands and receptor pairs, the integrin family  was identified as a pivotal interactive regulator during cardiac remodeling. Additionally, distinct functions of blood-derived and TR macrophages exhibiting altered gene profiles, regulons, and pathways were documented, allowing deeper understanding of the molecular mechanism of macrophages with different origins in response to ischemic stimulation. In addition, we showed that activated FBs (myofibroblasts) are characterized by a predominant level of Postn and Cthrc1. We also identified Ddah1, which is exclusively expressed in myofibroblasts in this study and was further confirmed using bulk RNA-seq, western blot, and immunofluorescence Notably, MI leads to sudden death of CMs, accompanied with the extravasation of leukocytes and lymphocytes from the blood vessels for clearance of necrotic debris, followed by replenishing of reparative cells, such as myofibroblasts, ECs, and anti-inflammatory macrophages (M2 macrophages) . It is vPrevious researches have confirmed that integrins contribute to myofibroblast differentiation by interacting with ECM or cytoskeleton proteins and activating the transcriptional expression of TGF-β in the heart, lungs, liver, and kidneys –42. ConsBesides FBs, recent studies have emphasized the important role of immune cells, especially macrophages, in cardiac functional modulation under hemostatic and perturbed conditions . In thisTo our knowledge, this is the first study raising the question of whether it is effective to have a comprehensive understanding of non-CM flux as well as cellular crosstalk and functions under homeostatic and ischemic conditions by integrating multiple single-cell datasets. We believe that single-cell sequencing analysis of multiple cell populations in a cell-type- and disease-dependent manner would provide new opportunities for discovering novel drugs and intervention targets for the repair processes of the heart.The original contributions presented in the study are included in the article/The animal study was reviewed and approved by the Animal Care Committee of Shanghai Jiao Tong University School of Medicine.LZ performed the study, analyzed the data, and wrote the manuscript. LL and RZ contributed to manuscript preparation and revision. KC and XY participated in the experimental design, data interpretation, manuscript preparation, and revision. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In the last years, the life expectancy of multiple myeloma (MM) patients has substantially improved thanks to the availability of many new drugs. Our ability to induce deep responses has improved as well, and the treatment goal in patients tolerating treatment moved from the delay of progression to the induction of the deepest possible response. As a result of these advances, a great scientific effort has been made to redefine response monitoring, resulting in the development and validation of high-sensitivity techniques to detect minimal residual disease (MRD). In 2016, the International Myeloma Working Group (IMWG) updated MM response categories defining MRD-negative responses both in the bone marrow (assessed by next-generation flow cytometry or next-generation sequencing) and outside the bone marrow. MRD is an important factor independently predicting prognosis during MM treatment. Moreover, using novel combination therapies, MRD-negative status can be achieved in a fairly high percentage of patients. However, many questions regarding the clinical use of MRD status remain unanswered. MRD monitoring can guide treatment intensity, although well-designed clinical trials are needed to demonstrate this potential. This mini-review will focus on currently available techniques and data on MRD testing and their potential future applications. The treatment course of multiple myeloma (MM) has been strongly improved during the last 20 years: the introduction of modern 3-drug regimen therapies combined with transplantation increased the achievement of deeper responses and the acquisition of minimal residual disease (MRD) negativity in up to 40/50% of patients enrolled in clinical trials . Consist5 normal cells  recently revised the response criteria and introduced the definition of MRD in CR patients as the persistence or re-emergence of very low levels of cancer cells, equal to about 1 tumor cell in at least 10al cells . These ral cells . HoweverThere are two techniques commonly used to detect MRD in the bone marrow (BM): multiparameter flow cytometry (MFC) and next-generation sequencing (NGS) molecular technology. Both techniques show positive and negative aspects .MFC can detect and quantify tumor vs. normal plasma cells using cell surface and cytoplasmic markers. For the identification of plasma cells, the combined use of CD38 and CD138 is recommended even if they are also expressed on other BM cells. In particular, the aberrant expression patterns of CD19, CD56, CD45, CD38, CD27, CD20, CD28, CD33, CD117 and surface membrane immunoglobulin can characterize the phenotype of monoclonal plasma cells (−4 and 10−5) is due to the simultaneous assessment of ≥8 markers in a single tube. In this way, if sufficient cell numbers are evaluated , it is possible to promptly identify aberrant PC phenotypes at MRD levels  panel in order to increase sensitivity and standardization of MFC , 7. This7 nucleated cells ; the LOD as 20 among 107 nucleated cells . This evaluation allows to discriminate between positive and negative samples. Interestingly, a baseline sample is not mandatory for MRD evaluation. After the multicenter evaluation of patients with very good partial response (VGPR) or CR, 110 follow-up bone marrows showed a higher sensitivity for NGF-MRD, as compared to conventional 8-color flow-MRD: MRD-positive rates were 47 vs. 34% (P = 0.003), respectively. Thus, 25% of patients who were categorized as MRD-negative by conventional 8-color flow were categorized as MRD-positive by NGF. This translated into a significantly longer PFS using NGF to discriminate between MRD-negative and MRD-positive CR patients (P = 0.02). Importantly, NGF can also provide a qualitative assessment of the patient sample by allowing the complete analysis of the normal B-cell compartment and the detection of a significantly decreased number of non-PC BM cells  revealing potentially hemodiluted BM samples. Finally, treatment with CD38 antibodies such as daratumumab and isatuximab can alter the antigen expression in MM cells. This sets a limit for the use of CD38 as a marker for the detection of plasma cells during MRD assessments at follow-up. The use of multi-epitope CD38 antibody in an advanced flow cytometry panel can solve this problem, since this conjugate can bind to a specific site (not covered by daratumumab) of the CD38 antigen. Nonetheless, in case of CD38 surface downregulation, the solution is the analysis of intracellular CD38 through the same protocol used for intracellular k- and λ-chain staining  and the limit of detection (LOD) of the NGF-MRD method. The LOQ is calculated as 50 among 10Allele-specific oligonucleotide polymerase chain reaction (ASO-PCR) was first explored to evaluate molecular MRD in MM, but even if its prognostic role was confirmed, different issues limited its use in favor of the NGS technique. First, its applicability ranged from 40 to 60% due to the low rate of diagnostic marker identification, since this technique does not take into account the somatic hypermutation rate of immunoglobulin loci and this translates into sequencing problems. Moreover, patient-specific reagents raised the complexity of this technique –11.NGS was developed to overcome all these disadvantages. ClonoSEQ® Assay  is the most frequently adopted commercial platform in the United States. In this test, DNA is extracted from patient's BM, a multiplex PCR amplifies VDJ, IgK, and IgL gene sequences and a common PCR prepares DNA for sequencing and creates a sequencing library. At the end of the process, a bioinformatic tool is essential to extrapolate and analyze all NGS data.Using this assay, we can define as “clonotypes” two identical sequencing reads. A clonotype with frequency >5% at diagnosis is considered a clonality , thus becoming a target for the detection of MRD in follow-up samples , 13. In −6 allowed to predict the best outcomes in MRD-positive vs. -negative patients .In the IFM2009 clinical trial, a comparison between NGS and 7-color MFC has been made, showing that the higher sensitivity with NGS at 10−5) regardless of response in patients achieving ≥CR, indicating that both techniques performed similarly in evaluating MRD  was observed using the same sensitivity  is a sensitive, non-invasive imaging technique available to detect the bone involvement in the spine and to provide details regarding the soft tissue disease and the pattern of marrow infiltration .Fluorodeoxyglucose positron emission tomography/computed tomography (FDG PET/CT) can be used to analyze the vitality of the focal lesions and is therefore the current standard of care to evaluate the post-therapeutic residual infiltration –19.Different studies showed the prognostic and predictive role of FDG PET/CT –22. InteFinally, different researchers confirmed the complementarity of PET/CT and BM techniques. Rasche et al. showed how patients who were both Flow-MRD- and PET/CT-negative had the best PFS outcome when compared with those who were Flow-MRD-negative but PET/CT-positive . Paiva ePET/CT has some limitations, some of which are linked to the tracer used (FDG). Indeed, a low expression of the enzymes responsible for the glycolysis process  in MM cells could lead to false-negative cases with FDG PET/CT . AlternaIn a head-to-head prospective comparison in a heterogeneous MM patient population, 11C-Methionine PET/CT was more sensitive than FDG PET/CT in the detection of focal lesions, both within and outside the bone. More data are needed in a homogenous patient population to understand whether this tracer could be an alternative to FDG in the detection of residual disease after treatment. Currently, other tracers targeting lipid membrane  and CXCR4 are also under study .In this section we focus on clinically relevant questions regarding MRD, reviewing available data on newly diagnosed MM (NDMM) patients. Single studies are summarized in −4-10−5, Lahuerta et al. nicely demonstrated that MRD-negative patients with a conventionally defined CR had better PFS  and OS  than MRD-positive CR patients  (post-induction and at different post-ASCT timepoints (3 to 24 months), showing that patients with persistent MRD negativity post-induction until 24 months after ASCT (n = 33) had better time to progression  and OS (not reached vs. 35.2 months), as compared to patients that were MRD-negative post-induction but MRD-positive within 24 months post-ASCT (n = 5)  . These d (n = 5) . The low−4, Rawstron et al. demonstrated that each log depletion in MRD levels predicted a 1-year median OS advantage , suggesting that MRD level is a continuous rather than a discrete variable  were indeed MRD-positive using NGS with a sensibility of 10−6 . This is especially important in clinical trials designed to explore treatment interruption based on MRD levels because a low sensibility of the technique can lead to unacceptable risk of patients' undertreatment.Another question is whether the sensibility of the technique impacts the reliability of MRD. Using MFC with a sensibility of 10variable . Recentlable  receiving thalidomide maintenance remained in a MRD-negative state more often than patients not receiving maintenance treatment . Regarding MM patients who are at high risk according to baseline prognostic factors , MRD-negative patients at a low level of sensitivity (10−4) still showed inferior clinical outcomes than standard-risk patients , the timing of evaluation and the need for a sustained MRD negativity. Moreover, safety should be closely addressed, as it was demonstrated by higher MRD (13.4 vs. 1%) but worse OS rates  in the experimental arm of the BELLINI trial (M14-031) comparing venetoclax-Vd vs. Vd , PFS is inappropriately becoming a late endpoint. MRD is considered the best candidate as a PFS/OS surrogate marker for provisional drug approval by regulatory agencies. Indeed, ClonoSEQ® Assay is now authorized by FDA  and MRD d vs. Vd , 68. MorMM is a spatially heterogeneous disease and simultaneous MRD negativization inside and outside the bone marrow showed synergistic predictive values .Moreover, MRD analysis within the bone marrow is done on bone marrow aspirates coming from a single random site and, in some patients, MM cells show a patchy infiltration . To overAs an example, applying the ClonoSEQ® assay to peripheral blood ctDNA and paired BM samples, Mazzotti et al. showed that residual disease in the peripheral blood was undetectable in 69% of patients with concurrent MRD-positive bone marrow samples . This waMRD has not yet entered the clinical practice, but it represents an attractive tool to potentially guide treatment choices. To address this hypothesis, many MRD-driven trials are beginning to explore treatment intensification in MRD-positive patients after standard treatment  or treatment deintensification in sustained MRD-negative patients . Ongoing and future MRD-driven trials will contribute to solve the unanswered question: is it recommended to evaluate other induction cycles until the achievement of MRD negativity in patients who are MRD-positive after 4 induction cycles? Can we perform post-transplant consolidation on the basis of MRD status? Can we stop maintenance after 1 year of sustained MRD negativity?Ongoing and future clinical trials will evaluate the definition and the role of sustained MRD-negativity in treatment decision-making. On the one hand, the achievement of a MRD-negative status does not necessarily mean that treatment should be stopped. Indeed, it should be noted that what we define as “MRD-negative” is a MRD undetectable with the current techniques, each one of them having a sensitivity limit. This means that we are not sure that the disease is eradicated even in MRD-negative cases. On the other hand, the achievement of a MRD-positive status after treatment brings the question of whether it is necessary to change treatment, improving the depth of response. However, before developing response-adjusted treatment strategies based on MRD status—either intensifying/changing treatment for MRD-positive patients or de-escalating treatment for MRD-negative patients—we need to understand if sustained MRD negativity should be the treatment goal and to define the most appropriate timepoint for its evaluation (after 1 year or after more years).SO, MD'A, MB, and AL: substantial contributions to the conception or design, acquisition, analysis, or interpretation of data, critical revision for important intellectual content, final approval of the version to be published, and agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. SO, MD'A, and AL: first draft. MB and AL: supervision.SO has received honoraria from Amgen, Celgene, and Janssen; has served on the advisory boards for Adaptive Biotechnologies, Janssen, Amgen, and Takeda. MD'A has served on the advisory board for GSK. MB has received honoraria from Sanofi, Celgene, Amgen, Janssen, Novartis, AbbVie, and Bristol-Myers Squibb; has received research funding from Celgene, Janssen, Amgen, Bristol-Myers Squibb, Mundipharma, Novartis, and Sanofi. AL has received honoraria from Amgen, Bristol-Myers Squibb, Celgene, and Janssen; has served on the advisory boards for Bristol-Myers Squibb, Celgene, Janssen, and Takeda.


Cancer immunotherapy using immune-checkpoint inhibitors (ICI) has revolutionized the therapeutic landscape of various malignancies like non-small-cell lung cancer or melanoma. Pre-therapy response prediction and assessment during ICI treatment is challenging due to the lack of reliable biomarkers and the possibility of atypical radiological response patterns. Positron emission tomography/computed tomography (PET/CT) enables the visualization and quantification of metabolic lesion activity additional to conventional CT imaging. Various biomarkers derived from PET/CT have been reported as predictors for response to ICI and may aid to overcome the challenges clinicians currently face in the management of ICI-treated patients. In this narrative review, experts in nuclear medicine, thoracic oncology, dermatooncology, hemato- and internal oncology, urological and head/neck tumors performed literature reviews in their respective field and a joint discussion on the use of PET/CT in the context of ICI treatment. The aims were to give a clinical overview on present standards and evidence, to identify current challenges and fields of research and to enable an outlook to future developments and their possible implications. Multiple promising studies concerning ICI response assessment or prediction using biomarkers derived from PET/CT alone or as composite biomarkers have been identified for various malignancies and disease stages. Of interest, additional major incentives in the field may evolve from novel tracers specifically targeting immune-checkpoint molecules which could allow not only response assessment and prognosis, but also visualization of histological tumor cell properties like programmed death-ligand (PD-L1) expression in vivo. Despite the broad range of existing literature on PET/CT-derived biomarkers in ICI therapy, implications for daily clinical practice remain elusive. High-quality prospective data are urgently warranted to determine whether patients benefit from the application of PET/CT in terms of prognosis. At the moment, the lack of such evidence as well as the absence of standardized imaging methods and biomarkers still precludes PET/CT imaging to be included in the relevant clinical practice guidelines.  Positron emission tomography/computed tomography (PET/CT) constitutes a major progress in oncology imaging, as it augments CT with the additional dimension of metabolic activity. Primarily used in staging and to some extent in response assessment of various malignancies, research for additional applications of PET/CT is currently evolving towards prognosis estimation and prediction of response to certain therapies, especially in the field of immunotherapy . Cancer immunotherapy through immune-checkpoint inhibitors (ICI) has revolutionized the world of medical oncology, achieving major and long-term treatment responses also in metastatic disease that would have been unthinkable only a few years ago. Fields of application of ICI therapies have rapidly expanded to different tumor entities and a multitude of ICI substances in various treatment regimens has subsequently become available . Still, 18F-FDG are discussed in this article, the term “PET/CT” is used synonymously for 18F-FDG PET/CT, unless otherwise specified.As the term “immunotherapy” is rather heterogenous and applies to a multitude of different antineoplastic therapies, this review aimed to give a focused clinical perspective on the current application and research advances of PET/CT, especially in the context of ICI therapy. Furthermore, although other newly developed tracer substances beyond Checkpoint inhibitor therapy itself, as well as the value of PET/CT in different malignancies and disease stages, are highly heterogenous. There is a general lack of high-quality large-scale trials and prospective randomized data for PET/CT-based response evaluation in ICI-treated patients. Although countless studies in that field have been reported, evidence is mostly derived from retrospective study settings with low patient numbers. Therefore, up to now, there is no standard of care (SOC) regarding ICI therapy assessment using PET/CT.(a)Give an overview of the current applications of PET/CT in the context of ICI therapy in current clinical practice within the respective field.(b)Identify up-to-date studies on PET/CT and ICI therapy that might be relevant to the future clinical approaches in the field.(c)Identify current challenges, perspectives, and possible research targets concerning PET/CT and assessment of ICI therapy response within the respective clinical field.Thus, we chose a structured clinical approach to this review: Experts in the fields of nuclear medicine, thoracic oncology, internal- and hemato-oncology, dermatological, urological and head and neck tumors conducted individual literature reviews based on their respective relevant current clinical practice guidelines as well as on literature research based on MEDLINE using PubMed search of articles within the last 5 years. Each expert was then asked to:Because of the lack of larger prospective trials, the inclusion of relevant literature into this narrative review, coordinated by M.G. and D.L., was based on the individual decision of each expert with regard to their specific perspective .D.L. accomplished the compilation of the manuscript, which was then critically revised and finally approved by all other authors.As mentioned above, this review article mainly refers to the application of PET/CT imaging in patients undergoing immunotherapy with programmed cell death protein 1 (PD-1)/PD-L1 or cytotoxic T-lymphocyte-associated protein 4 (CTLA-4)-directed immune checkpoint inhibitors (ICI), while other targeted anticancer therapies, sometimes also referred to as “immunotherapy”, e.g., antibodies against other tumor-specific antigens or tyrosine kinase inhibitors, are not discussed. However, our approach also includes a brief section on PET/CT and bi-specific T-Cell Engagers (BiTEs) as well as Chimeric Antigen Receptor (CAR-T) cell therapy in the hemato-oncology section of the article. The advent of ICI in the treatment of various malignant tumors constitutes a milestone in clinical oncology. However, the complex immunological response to these novel agents involving the tumor microenvironment has only been incompletely understood and poses a major challenge in molecular imaging strategies.18F-FDG uptake in malignant lesions as quantified by the standardized uptake value (SUV) can be used as a biomarker for therapy response. This metabolic biomarker dimension has been included in many oncological treatment concepts. A summary of quantitative biomarkers derived from PET/CT imaging that can be easily (semi)-automatically determined is shown in Accurate assessment of response to antineoplastic therapy is essential to recognize treatment failure at an early stage and to be able to adapt therapy timely. Classically, this would be indicated by an increase in number and/or size of tumor lesions . Using p18F-FDG uptake may be diminished and sometimes misleading. Enhanced 18F-FDG uptake can also be triggered by the activation of the tumor microenvironment with an increased influx and activity of immune cells like T-lymphocytes induced by ICI therapy itself [In the context of ICI therapy, however, the efficacy of quantitative measurement of y itself ,14. In cy itself . At the y itself ,17. ThisBasic evaluation of therapy response using PET/CT can be accomplished using qualitative parameters, like the decrease or increase in metabolic of active tumor lesions. Such binary “good or poor” categorization is rather robust and can be used, e.g., for end-of-treatment assessment. Particularly complete metabolic response in PET represents an important individual decision-making criterion and generally indicates a favorable long-term outcome . An examHowever, an such approach does not allow a fine-tuned therapy assessment and is not suitable for clinical trials, where reproducible and standardized response data are warranted. In conventional CT, this led to the introduction of standardized response evaluation criteria in solid tumors (RECIST) using the classifications complete/partial remission (CR/PR), stable disease (SD) and progressive disease (PD) . ConcernOne major point that needs to be considered in that context is that quantitative indices  are dependent on the quality of the PET images. Results may vary distinctly between up-to-date imaging technology and equipment ten or twenty years old. Also, comparability is usually impaired when different reconstruction methodologies are used. Both can pose difficulties especially when comparing quantitative measures between institutions ,24.In ICI therapy, atypical response patterns pose additional challenges to classical radiological as well as PET-based response evaluations. One such feature is pseudoprogression , which mDissociated response is a similar atypical response pattern, with a mixture of lesions responding and progressing simultaneously ,27. Its Durable responses to ICI therapy are not comprehensively defined but can occur both after primary partial or complete remission or out of stable disease . As show18F-FDG PET-CT, using quantitative parameters, e.g., total lesion glycolysis (TLG) [An unfavorable feature of atypical response is hyperprogression, which denotes patients presenting with an accelerated tumor growth rate early after ICI initiation, occurring in up to 7% ,28. It ris (TLG) .To account for these imaging challenges associated with atypical response patterns, RECIST criteria have been expanded to iRECIST, which is now commonly used for CT re-staging in ICI patients . Analogo18F-FDG tracer uptake in various involved organs. These therapy-related findings represent possible pitfalls in PET/CT interpretation, especially as such side effects may not necessarily be clinically evident [Immune-related adverse events (IRAE) such as thyreoiditis, pneumonitis or hypophysitis may lead to unusual patterns of  evident ,36,37. H evident ,39,40.A novel approach for baseline PET/CT imaging and prognosis estimation apart from traditional staging and response criteria refers to textural features within the tumor lesions. Texture analysis means a systematic, computer-aided evaluation of image data with special regards to features like heterogeneity within one or between individual tumor lesions. Such biomarkers have been correlated with clinical outcome parameters and may pose a major step towards a truly personalized therapy approach. Promising first results have been reported in patients with malignant melanoma treated with vemurafenib and ipilimumab . For lun18F-FDG as a tracer for cancer treated with ICI is that therapy itself leads to an influx of inflammatory cells leading to enhanced PET activity [89Zr-atezolizumab for PET imaging showed very promising results assessing clinical response to PD-L1 blockade in 25 patients with locally advanced or metastatic bladder cancer, non-small-cell lung cancer (NSCLC) or triple-negative breast cancer. Interestingly, responses to atezolizumab therapy in these patients had a better correlation with the pretreatment 89Zr-atezolizumab PET signal than with immunohistochemistry- or RNA-sequencing-based predictive markers [As already discussed, a general problem concerning activity . Thus, m markers . Further89Zr-IAB22M2C for PET-CT imaging is ongoing among patients with Melanoma, Non-Small-Cell Lung Cancer, Renal Cell Carcinoma, and Squamous Cell Carcinoma of the Head and Neck, aiming to predict response to ICI (NCT03802123).A very interesting novel approach is based on the use of Zr-89-labeled minibodies that target CD8-positive T cells . Since tAnother novel promising imaging approach was reported by Chatterjee et al., who developed highly specific radiolabeled peptides for PET imaging of PD-L1 tumor expression ,47,48. PAccording to current clinical practice guidelines, PET/CT is recommended in the staging process of both small-cell lung cancer (SCLC) and non-small-cell lung cancer (NSCLC), as it can improve diagnostic accuracy as compared to conventional staging using CT ,51,52,53General limitations of PET/CT in thoracic tumors are its low sensitivity in small lesions, movement artifacts due to breathing and the possibility of false-positive results due to inflammatory processes like in infectious or inflammatory diseases, after surgery, radiotherapy or pleurodesis. False negative findings may also occur in tumors with low cell density and low PET avidity, e.g., in bronchoalveolar carcinoma ,52,54.Despite the fact that current guidelines do not routinely recommend PET/CT for follow-up of any of the mentioned thoracic tumor entities ,50,51, eThis concept was recently refined by Jreige et al., who established the ratio of metabolic-to-morphological lesion volumes (MMVR), which delineates the fraction of necrosis within a tumor lesion. MMVR was found to be negatively correlated to PD-L1 status, and low MMVR was associated with better survival rate. In line with the imaging aspect derived from PET/CT, a retrospective evaluation of histological samples revealed that tumors with necrosis showed a greater frequency of CD8+ lymphocytes . This coA major field of scientific advance is the combination of PET/CT-derived biomarkers with other patient- or tumor-related biomarkers: Recently published data demonstrated that a composite biomarker combining the PET/CT-derived variable total (T) MTV together with the derived neutrophil-to-lymphocyte ratio (DNLR) was highly predictive of OS in NSCLC patients treated with ICI . On the Another recently reported approach is the assessment of PET/CT early after ICI initiation: Kaira et al. showed that uptake dynamics after 1 month of nivolumab therapy in NSCLC patients were a better predictor of the further course of disease than corresponding CT measures . SimilarIn another noteworthy study, Humbert et al. investigated NSCLC patients under therapy with pembrolizumab or nivolumab, who had PD according to PERCIST at an interim PET/CT at week 7 after treatment initiation, but without clinical worsening. In those patients, therapy was continued until another PET/CT evaluation after 3 months. Of 19 patients with initial PD but ICI treatment being continued, 42% had a homogeneous disease progression and did not reach a durable clinical benefit (DCB), defined as ICI continuation of six months. In addition, 26% had immune-dissociated response and 32% had pseudoprogression, both categories leading to a DCB . This su18F-FDG uptake in the thyroid gland under therapy with ICI in lung cancer patients can predict thyroiditis even before laboratory testing [Of interest, PET/CT may also have implications for predicting the development of IRAE: Hashimoto et al. showed that the frequency of IRAE was higher in patients with low tumoral FDG uptake . Data fr testing . Our ownimmune-related response criteria (irRC), PERCIMT or iRECIST have been established [For staging and therapy monitoring of patients with advanced melanoma undergoing immunotherapy, PET/CT is increasingly used in clinical practice. Due to the possibility of atypical response patterns previously mentioned, classical imaging response criteria such as RECIST have been shown to frequently over- or underestimate the therapeutic effect of ICI . Hodi etablished .18F-FDG-PET/CT in melanoma patients, potential new benefits of this imaging technique arose recently: multiple authors suggest PET/CT as a prognostic tool, as pre-treatment- or therapy-monitoring-examinations may provide information regarding future therapy response [18F-FDG PET/CT biomarkers as a prognostic tool in patients with cutaneous and mucosal melanoma. Their data suggested a negative correlation between high baseline metabolic tumor burden or bone marrow metabolism and ICI response for cutaneous melanoma. On the other hand, for mucosal melanoma, high baseline SUVmax was found to be the only prognostic baseline imaging biomarker [Despite the challenges that come with evaluating ICI therapy response with response . Seban eiomarker . Partly based on these data, Hindié et al. recently suggested that patients with high tumor burden as defined by total metabolic tumor volume (TMTV) in baseline PET/CT were unlikely to respond to ICI monotherapy. For these patients, alternative therapies such as BRAF/MEK inhibitor therapy or combined ICI therapy might be a better primary therapy option. Furthermore, CR in 18F-FDG PET was discussed as a strong indicator for safe discontinuation of ICI therapy .Nakamoto et al. investigated the prognostic value of wbMTV three months following initiation of ICI and concluded that it was a strong indicator of OS in advanced melanoma patients. They further stated that the possibility of pseudoprogression must be considered during early restaging images but noted that it had only occurred in one of their eighty-five patients evaluated .18F-FDG PET/CT scans was suggested by Wong et al., who discovered that a pre-treatment spleen-to-liver ratio of >1.1 was associated with poor outcome after ipilimumab treatment in advanced melanoma. This could not be shown for patients treated with PD-1 antibodies, but the authors criticized the small sample size of the anti-PD-1 cohort. Further investigation of this 18F-FDG-PET/CT signature with larger cohorts of patients treated with PD-1 antibodies or combined ICI was recommended [Another promising feature of ommended .Very recently, a first systematic review and metanalysis concerning prediction and monitoring of immunotherapy response in patients with metastatic melanoma was performed by Ayati et al. The authors concluded that baseline metabolic tumor burden parameters such as SUVpeak, MTV and TLG were promising biomarkers for response prediction and that image evaluation using modified response assessment criteria such as PERCIMT was generally regarded as superior compared to conventional criteria in most applications. Finally the authors stated that the occurrence of particularly severe IRAE was correlated with response, but was not essential for clinical benefit .18F-FDG-PET/CT, possibly even ahead of clinical diagnosis [18F-FDG-PET/CT. Interestingly, detection rates varied distinctly for various organs. Further investigation regarding this topic is advisable because of low patient numbers in this study [Besides its role as a follow-up—and potentially prognostic—tool, recent data suggest that relevant IRAE can be detected by iagnosis , whereasis study . In our There are limited data regarding the significance of PET/CT in ICI therapy of urothelial cancer. A case report of a patient with lynch syndrome who underwent sequential immunotherapy with atezolizumab, pembrolizumab and ipilimumab/nivolumab for urothelial cancer of the bladder and ureters, which occurred alongside a recurrence of colorectal carcinoma, showed a metabolic response on 18F-FDG PET/CT . In a muDue to the high activity of FDG in 18F-FDG, PET/CT has shown disappointing results in the differentiation of renal masses, with a pooled sensitivity of 50–60% . Its pooWhile there seems to be no added benefit of 18F-FDG PET/CT in comparison with conventional CT in primary diagnosis and initial staging of renal malignancies, beneficial results have been shown in the evaluation of treatment response. High SUVmax correlated with high-grade, poor prognosis and prediction of metastatic disease ,82,83. L68Ga- and 18F-bound tracer substance provides excellent contrast-to-noise ratio and only suffers from few false positives like other non-prostatic malignancies, sarcoidosis or benign bone diseases. In addition to computed tomography, a recent systematic review has shown superior diagnostic accuracy for PSMA-PET/MRI when compared to the current gold standard, multiparametric magnetic resonance imaging (mpMRI) [68Ga-PSMA PET/CT a pooled sensitivity and specificity for lymph node involvement before treatment of 75% and 99%, respectively [In prostate cancer, no ICI substance has yet shown efficacy sufficient for approval, but trials are ongoing,  and prel (mpMRI) . There i (mpMRI) . Recentlectively . In the Staging with PET/CT is critically important for patients with aggressive hemato-oncological diseases such as lymphomas. Precise staging before therapy initiation reduces the risk of overtreatment and consecutive toxicity due to extended-field radiation or prolonged chemotherapy .Despite the well-established role of PET-imaging at baseline and under therapy, surveillance imaging is discussed controversially in literature, as relapse is mostly detected by clinical examination or the patients’ awareness.With novel immunotherapy options like ICI, Bi-specific T-Cell Engagers (BiTEs) and Chimeric Antigen Receptor (CAR-) T cells having emerged especially in the therapy of aggressive lymphomas, PET/CT for staging, and response evaluation is gaining increasing impact based on a growing body of evidence.Concerning ICI, the value of early PET/CT two months after nivolumab initiation as compared to regular CT imaging in relapsed or refractory Hodgkin Lymphoma has been recently analyzed retrospectively by Mokrane et al. Both PET/CT and CT response criteria were correlated with OS, but PET/CT could identify more patients with complete responses .BiTEs are novel bispecific antibodies or antibody fragments connecting T cells and tumor cells directly, binding CD3 on the T cell and CD19 on the lymphoma cell, enabling the malignant cell to be attacked. In a 2016 phase 2 study on blinatumomab by Viardot et al., PET/CT and the “2007 revised response criteria” were used for the first time in such a therapeutic setting. Three patients who had a CT-defined PR that was confirmed by PET at week 10 later achieved CR in the follow-up period (3–12 months), indicating a prolonged immune response or a lag time in the demonstration of CR . These oCAR-T cell therapy is based on patient T cells being genetically reprogrammed. These cells synthesize a protein on the T-cell surface binding to the surface proteins of the tumor cell , which then acts as a reprogrammed killer T cell after binding to the tumor cell. Usage of PET/CT in patients treated with CAR-T cells has recently been examined by Shah et al. in seven patients with diffuse large B-cell lymphoma (DLBCL). In PET/CT one-month post-treatment, three patients concurrently showed no sign of MTV activity, two had an increase in MTV and two had residual MTV. Two years post-treatment, only those three patients without residual MTV remained in CR. Development of cytokine release syndrome (CRS) did not confound PET/CT findings .PET/CT has become an important tool in the evaluation of head and neck cancer (HNC) in the last years. Especially in advanced stages, clinicians benefit from higher sensitivity regarding stage migration . Relapse89Zr-durvalumab in 58 participants with relapsed or metastatic HNSCC [Summarized data analyzing PET/CT in the evaluation of ICI therapy in HNC is not yet available. However, one promising clinical PD1/-L1 imaging study is upcoming with the PINCH trial , measuring the uptake of ic HNSCC . Data on18F-FDG PET/CT currently plays an important role in staging and follow-up of FDG-avid tumor entities, like NSCLC, melanoma or aggressive lymphomas. However, PET/CT follow-up of patients under antineoplastic therapy is not routinely recommended in most situations. With ICI therapy rapidly evolving in numerous tumor entities, and due to the challenges associated with assessing radiological response to such therapies, there is an urgent clinical need for additional imaging-derived biomarkers. The large number of studies on PET/CT biomarkers in ICI therapy response evaluation featured in our review reflects this quest and testifies that there already is a growing body of evidence for such metabolic imaging strategies. Biomarkers derived from PET/CT are promising for the development of personalized treatment strategies, especially concerning the choice of therapy and for its longitudinal management. Their potential prognostic appliance is especially appealing considering the current lack of established highly predictive blood- or tumor-related biomarkers.In synopsis, 18F-FDG-PET/CT have been previously discussed and include false-positive findings in inflammatory lesions, tumor entities showing no or little FDG uptake or physiological uptake like in the urinary tract. It is likely that these challenges associated with the low specificity of FDG uptake can be overcome with novel, more specific, tracer substances that are currently under development. Using tracers linked to the respective ICI substance, actual “theranostic” approaches may become possible, including more specific pre-therapy response prognosis and follow-up assessment. Also, the idea of non-invasive estimation of immunohistochemical features like PD-L1 expression using special tracers appears highly attractive.Inherent technical limitations of Another limiting factor to be considered in the application of PET/CT in ICI-treated patients is the obvious shortage of PET/CT resources in some regions . Also, iReviewing the large number of studies published concerning PET/CT for response assessment to ICI therapy, we note that, to date, there still is a striking discrepancy with the relevant clinical practice guidelines which, across the board, do not suggest using PET/CT in this indication. On the one hand, this is due to the extensive lack of prospective data allowing to draw conclusions as to whether there is a clinically significant benefit of using PET/CT versus conventional imaging in such therapy settings. On the other hand, PET/CT imaging and various derived biomarkers frequently lack standardization, which precludes their broader implementation in clinical practice. Interim PET/CT examinations could aid an early response assessment and help to identify atypical response patterns, especially hyperprogression or pseudoprogression. Standardized response criteria like PERCIST may be helpful in that field; however, we assume that also in future they will rather be applied in clinical trials than in daily clinical practice as they do not have immediate implications on the individual patient’s level. Also, we believe it is likely that, rather than singular PET-derived biomarkers, the combination of a broader panel also including blood and histological parameters will be necessary for a significant clinical impact.Due to these challenges, it is still unclear how PET/CT can be embedded in clinical decision-making apart from staging and re-staging. We believe that there is large future potential, especially for pre-ICI therapy PET/CT biomarkers to stratify patients for maximizing the therapeutic effect of ICI therapy. However, unless high-quality clinical trials demonstrate a significant survival or response benefit and thereby also define the methods to be used in future, PET/CT for ICI therapy response assessment is not yet ready for broader application. Right now, it is time to focus the numerous existing pieces of evidence, to standardize technique, sequencing and biomarkers and to translate that knowledge into high-quality clinical trials. Only by doing so, PET/CT assessment in ICI therapy can finally be implemented in daily clinical practice based on a solid body of evidence.