They provide a valuable body of knowledge on how tension influences relevant genes, proteins, and metabolites. However, no systematic review summarizing these findings has been conducted so far. Aim of this systematic review was to identify all related in vitro studies reporting tension application on hPDLCs and summarize their findings regarding force parameters, including magnitude, frequency and duration. Expression data of genes, proteins, and metabolites was extracted and summarized. Studies’ risk of bias was assessed using tailored risk of bias tools. Signaling pathways were identified by protein-protein interaction (PPI) networks using STRING and GeneAnalytics. According to our results, Flexcell Strain Unit® and other silicone-plate or elastic membrane-based apparatuses were mainly adopted. Frequencies of 0.1 and 0.5 Hz were predominantly applied for dynamic equibiaxial and uniaxial tension, respectively. Magnitudes of 10 and 12% were mostly employed for dynamic tension and 2.5% for static tension. The 10 most commonly investigated genes, proteins and metabolites identified, were mainly involved in osteogenesis, osteoclastogenesis or inflammation. Gene-set enrichment analysis and PPI networks gave deeper insight into the involved signaling pathways. This review represents a brief summary of the massive body of knowledge in this field, and will also provide suggestions for future researches on this topic.Orthodontic tooth movement is based on the remodeling of tooth-surrounding tissues in response to mechanical stimuli. During this process, human periodontal ligament cells (hPDLCs) play a central role in mechanosensing and mechanotransduction. Various  Cells described as “PDL fibroblast” are commonly isolated from the middle third of the tooth root. For cell isolation either the “explant” or the “digestion” technique is employed (for further details: see discussion). Yet, both techniques will result in a heterogeneous mixture of different cell types  or intermitted (“dynamic”) tension force along one principal axis  or along all axes in all directions  of a cell  as well as the effect of tension on the expression of genes and proteins. Specifically, this systematic review aimed 1) to identify all relevant studies applying tension on hPDLCs e.g. to simulate orthodontic force or other clinically relevant forces; 2) to make an assessment of the methodological and reporting quality of the included studies; 3) to summarize the biological and force parameters, and the commonly adopted methods for detecting biological regulation; and 4) to identify the most frequently investigated genes/proteins and their regulation, as well as the biological processes and pathways that might be affected by tension in hPDLCs.Therefore, this systematic review aimed to summarize the data on different in vitro studies were included.This systematic review was conducted following the “Preferred Reporting Items for Systematic Reviews and Meta-Analyses” (PRISMA) guidelines . The pro• P(atient): human periodontal ligament cells (hPDLCs) or human periodontal ligament derived stem cells (hPDLSCs);in vitro static and dynamic tension ;• I(ntervention): • C(ontrol): human periodontal ligament cells (hPDLCs) or human periodontal ligament derived stem cells (hPDLSCs) not subjected to mechanical force;• O(utcome): force parameters  and regulation of gene, protein and/or metabolite expression in response to tension.Inclusion criteria were defined in accordance with the “P.I.C.O.” framework :• P.The search strategy considered keywords concerning the specific objectives of the studies, the force applied and the cells exposed to experimental force and were summarized in First, unrelated studies were excluded after reading titles and abstracts according to the eligibility criteria defined. Then, full texts of the remaining studies were acquired. After full text reading, articles not fulfilling the eligibility criteria were excluded , and thoin vitro studies was assessed using the methods described by Risk of bias of the included All included articles were scored by two authors (C.S. and M.J.R.). Any disagreement was discussed internally until consensus was achieved. The results of the risk of bias assessment were recorded and summarized in predefined tables .After final selection of relevant studies, the following information on experimental design and outcome were extracted and summarized: reference ; cells used ; force applied ; genes analyzed  with reference to force application and the methods applied to measure their expression. “Gene or analyte investigated”, “Cells used” and the details on the “Force apparatus” were extracted using the original phrases from the studies and recorded in https://www.ncbi.nlm.nih.gov/tools/primer-blast/). All genes were reported using their official gene symbol according to the HUGO Gene Nomenclature Committee . For protein data, antibody or ELISA specificity was verified using information provided within the studies and the information given by the suppliers. If possible, official gene symbols according to HGNC were applied. If antibody specificity was not sufficient, e.g. no discrimination between isoforms or gene variants, antibody targets were recorded according to the manufacturer given in that publication.Specificity of primers used in PCR reactions was verified with Primer-BLAST (https://markummitchell.github.io/engauge-digitizer). To allow comparisons between gene or protein expression data from different sources, ratios  or fold changes were calculated if not given by the authors. Gene expression was reported as “fold change” (FC), if its calculation was done either according to −ΔΔCt” or “ΔΔCt”. Gene expression was designated as “ratio”, if the control was “defined as 1”, otherwise it was described as “relative change” (“rel”). Results reported from more than one donor were listed separately.The expression patterns of genes and/or proteins were described using specific terms  and nume® patent search (URL: https://patents.google.com) if applicable. Equibiaxial and uniaxial tension . Afterwards, publications using the same type of apparatus were combined into the same category for further analysis. A summary statistic on the different force parameters was compiled from a unified list of publications based on tension type and tension frequency. From each publication reporting dynamic tension application, maximum duration of force exposure and mainly adopted magnitude were summarized for the same frequency. Studies utilizing static tension were classified first by magnitude followed by maximum force duration. Replicates derived from the same study were removed and all analytes were ranked according to abundance.Based on the complete lists of examined genes, differential expressed gene (DEG) lists were compiled according to the following criteria: the gene was identified unequivocally and changes in gene expression due to force application were reported using the terms defined in https://string-db.org)    using thon 1.5.1  with Cyton 3.8.0 . A minimon 3.8.0  as impleon 1.3.1  with defsion 0.1  using deStringApp was also applied for gene list enrichment using the “GeneOntology/Biological Process” database (https://ga.genecards.org) (stringApp.database . The “Surds.org) . To incrThe whole process of study selection was summarized in the PRISMA flow diagram  Moher e. The appin vitro studies. The results of the other risk of bias criteria showed a large variability. “Sample size determination” and “Statistical analysis” were mostly assessed as “high risk of bias” (“HoB”). “Accounting for confounding variables”, “Optimal time window used” and “Test organism/system” were found to have a high level of “incomplete or unclear risk of bias”. With reference to the remaining criteria, most of the studies were assessed as “low risk of bias” (“LoB”)  .The reporting quality of the publications was higher in comparison to the methodological quality, since more studies were classified as “LoB” . Only thIn 30 out of 137 qualified studies (∼22%) static tension was applied, whereas 103 out of 137 included studies (∼75%) focused on the effect of dynamic tension . Direct Regarding the type of apparatus, all included studies were identified as either equibiaxial or uniaxial . VariousDynamic equibiaxial tension: Fifty three studies applied dynamic equibiaxial tension using the Flexcell Strain Unit  and its revisions   by application of a vacuum below the flexible membrane. In nine studies, Bioflex® plates were used together with individually constructed devices, including the “CESTRA” (®-plate based silicone or elastic membrane-based apparatuses including the “Cell Strain Unit (CSU)” (FX-5000) . This sy“CESTRA”  and the “CESTRA”  (Table 2“CESTRA” . Ten stut (CSU)” , which wDynamic uniaxial tension: Application of dynamic uniaxial tension using the “STB-140” system  was described in ten publications. The Flexcell Strain Unit® in combination with Uniflex® culture plates  was adopted in eight studies. Non Bioflex®-plate based silicone and other elastic membrane-based apparatus were adopted in ten studies. Four-point bending systems were employed in six studies  were adopted in eleven studies. Apparatuses based on the Petriperm™ dish were used in ten studies, in which the dish was deformed by the weight placed onto a spheroidal template. Bioflex®-based devices were used in seven studies, Lumox® culture dishes were adopted in two and the “tension incubator” in one study , alkaline phosphatase , bone gamma-carboxy glutamic acid-containing protein , interleukin 1β (IL1B), prostaglandin-endoperoxide synthase 2 , tumor necrosis factor-alpha receptor superfamily member 11B , TNF superfamily member 11 , collagen Iα1 (COL1A1), prostaglandin E2 (PGE2) and Osterix  (The most commonly investigated 10 genes or metabolites in these studies were (in descending order): runt-related transcription factor 2 (ith OSX) . Their eGene list analysis was done as described . The genSTRING-DB as described, and network statistics for both networks were calculated  whereas a few genetic loci were reported to be either up- or down regulated . In contrast, gene expression of the majority of genes included in the “static” PPI network was either up- or downregulated depending on the particular study  by different studies. Interestingly, 12 genes from the “dynamic” DEG list  networks were generated using both gene lists with lculated . For eaclculated  an upregar study . Up- andDEG list  and 4 geDEG list  were notDEG list  includin10(FDR) for GO or the SuperPathway’s score, respectively. Only GO terms containing at least 5% of the genes from the particular DEG list (ratio ≥0.05) were considered  and GeneAnalytics’ SuperPathway database. The top 10 enriched terms from the “dynamic” and “static” DEG lists were ranked according to lognsidered .Dynamic DEG list. The most significant GO terms describing biological processes were related to “responses to endogenous stimuli” (ratio: 0.05/log10(FDR) = 36.16; 0.05/30.14), the action of growth factors , ossification (0.15/29.10) or differentiation (0.06/27.36) and motility/movement   and the “Hippo signaling pathway” (0.14/66.29). Nevertheless, “Lung Fibrosis” SuperPathway (0.27/70.05) was also part of the “top 10 list” of enriched terms.6/27.54) . SuperPa6/27.54) . Lower sStatic DEG list. The most significant GO terms describing biological processes driven by genes from the “static” DEG list were ossification (0.06/10.08), responses to mechanical stimuli , regulation by glucocorticoids , positive regulation of small molecule metabolic processes (0.07/8.21), and apoptotic signaling pathways  (19/7.54) . SuperPa19/7.54) .To identify highly connected gene clusters within each network, MCODE clustering was performed. Seven different clusters were identified in the dynamic PPI network ; Table 4adenylate cyclase-inhibiting G protein-coupled glutamate receptor signaling pathway”-related (0.56/8.97). Cluster #3 from the same network was described significantly by the “ionotropic glutamate receptor signaling pathway” (0.24/14.10), but also by “excitatory chemical synaptic transmission” (0.38/6.97), whereas cluster #4 was dominated by terms related to bone mineralization and remodeling, e.g. “osteoblast differentiation” (0.10/12.50) and development (0.22/6.80), or “regulation of bone resorption” (0.08/4.09). In general, an increase in specificity (i.e. higher ratio) was also observed for SuperPathways enrichment  in both networks were identified with VEGFA, JUN, TNF, IL6, HMOX1) included into the “dynamic” PPI network was upregulated by dynamic tension forces. For the remaining six hub genes both up- and downregulation of gene expression was reported. For eight out of ten hub genes  upregulated expression was observed after exposure to static tension forces, and for the remaining two genetic loci (IL6 and IGF1) both up- and downregulated expression was reported.The expression of five of eleven hub genes  and to delineate the impact of different force parameters on the expression of relevant genes. Risk of bias assessment was conducted by application of published criteria using clear definitions. The commonly investigated genes, proteins, and metabolites were summarized and analyzed by gene enrichment and pathway analysis.in vitro three different major groups of apparatuses were identified here: 1) commercially available systems. i.e. the Flexcell® Tension System or the STREX® Cell Stretching System, 2) self-designed apparatuses using commercially available components  as a central part of a stretching device, and 3) solely self-constructed apparatuses. Irrespective of the particular design, cells were grown on a flexible surface undergoing cyclic (or static) equibiaxial or uniaxial stretching in all devices.To apply tension type of force on hPDLCs ® plates. As such, we propose, that the heterogeneous strain distribution might also apply to these setups. In contrast, in Flexcell Tension systems using Bioflex® plates with a “biaxial loading post” the constant biaxial strain region was located in the membrane area on the post, whereas off-post large radial strain was produced  was introduced to the market to be used in combination with the BioFlex cell culture plates. Limiting cell growth within a defined area providing uniform strain distribution increases the reproducibility of cell seeding and exposure against mechanical cues. However, none of the included studies reported the application of this device.According to this review, the Flexcell® Tension System was the most widely adopted apparatus for equibiaxial tension using Bioflex®, and uniaxial tension using Uniflex® cell culture plates. In various reports the mechanical characteristics of these elastic membranes used to apply tension to adherent cells have been studied and limitations of this method have been reported. Specifically, several studies reported a heterogeneous strain distribution within the surface used for cell cultivation and a considerable interference of other types of force (e.g. compression and shear stress) . In the produced . Applicaproduced . Irregulproduced . As a coproduced  and thusThe PDL comprises different types of collagen, including type I about 80%), type III (15%), type V, type VI, and type XII collagen . Other sout 80%, in vivo microenvironment. Providing structural support and signal transduction to the cells, ECM is involved in various biological processes, including cell migration, proliferation, differentiation and intercellular communication  cell culture studies have reached increasing significance since they allow for a better simulation of the cells’ extracellular matrix (ECM) and nication . For simnication . Though igration  and mechigration .Interestingly, only two studies reported on the effects of tension force applied to cells within 3D scaffolds . The firin vivo evidence from animal models simulating OTM or measurement of bite force in human subjects  was mostly based on the purpose of the specific study, being either the simulation of a clinical situation , or to investigate the force-related expression of specific genes, group of genes or pathways. Due to the general objectives of the studies analyzed herein, the selection of cell type and type of force seems plausible. The remaining parameters were selected according to: 1) cts e.g. ; 2) finicts e.g. ; 3) in vays e.g. .Force duration: Continuous exposure to a stretching force is considered as an appropriate surrogate for in vivo forces applied by fixed appliances during OTM  expression of a specific gene in response to tension force application allowing to define the dynamic range along with the range of optimum forces  ces e.g. . In mostces e.g. . This foic force , or studic force . Severalic force  took resic force .Force frequency: To apply dynamic tension different frequencies were adopted. Selection of appropriate force frequencies mostly relied on two different rationales: 1) experience from previous in vitro studies either using similar setups or defining appropriate frequency ranges to study the expression of specific genes  ic genes  or an avic genes . In thisic genes , a mean ic genes , or 1.5–ic genes .According to our review, the most commonly used frequency was 0.1 Hz for dynamic equibiaxial tension and 0.5 Hz for dynamic uniaxial tension. Although the real frequency of tension applied on the PDL cells during OTM yet remains unknown , the cliRUNX2, ALPP, BGLAP, IL1B, PTG2, TNFRSF11B, TNFSF11, COL1A1, PGE2 and SP7 , osteoclastogenesis , and inflammation .To identify the most relevant mechanical responses during OTM, we focused on the top 10 most frequently investigated genes, proteins and metabolites:  and SP7 . AccordiRUNX2 regulation is an integral and central part in the development and remodeling not only of osseous tissue but also of the periodontal ligament (RUNX2 upregulation was reported in 19/26 (73%) studies using either RT-qPCR or both RT-qPCR and western blotting. Decrease, temporary changes or other types of regulation were found in the remaining studies . Although several studies revealed partially contradictory results, it was well-supported that RUNX2 expression increased within the first 12 h of tension application.ligament . PDL celligament . RUNX2 uSP7 in response to tension was reported in 9/10 (90%) of the relevant studies, whereas only one study reported downregulation of gene expression and temporary upregulation of the corresponding protein , which belongs to the zinc finger-containing transcription factor SP family . Signifi protein . The aut protein .ALPP gene expression was determined using either semiquantitative or quantitative PCR. ALPP protein was quantified in cell lysates using western blots, ELISA or enzyme activity assays. Significant upregulation in response to tension <12% was reported in 17/24 (71%) of the studies, mostly during the first 12 h of force application.Alkaline phosphatase (ALPP) also plays a crucial role in the initiation of osteogenic differentiation and bone remodeling . In all BGLAP gene expression and it was assumed that an enhanced cell proliferation of young osteoblasts might be responsible  is a late marker of osteoblast differentiation and mineralization . Signifiponsible . Anotherponsible .COL1A1 gene expression (COL1A1 in response to tension was confirmed in the majority of the studies considered here.The extracellular matrix (ECM) of the periodontal ligament mainly consists of fibrillar collagens, among which type I collagen accounts for ∼75% . The latpression . AlthougBone remodeling is primarily regulated by a closely interrelated system of receptors and mediators including TNFSF11 , its cellular receptor, receptor activator of NF-kappaB (RANK), and TNFRSF11B  ultimately maintaining the balance between osteogenesis and osteoclastogenesis . As a deUpregulation of OPG gene expression and/or protein synthesis was reported in 11/14 (79%) of the included studies. Due to the heterogeneity of force parameters, the correlation between gene expression, force duration and/or force magnitude can only partly be defined. The regulation of RANKL expression showed large variability in comparison to OPG: only 57% (8/14) of the relevant studies reported an upregulation of both gene and protein expression after force application, whereas the remaining studies showed inconsistent expression patterns.It is commonly accepted, that an increased RANKL/OPG ratio favours osteoclastogenesis, meaning an upregulation of RANKL in parallel with a downregulation/lower induction of OPG . The RANThe response of the PDL to mechanical stress has been characterized as an aseptic transitory inflammatory process, which is regulated by various mediators, including cytokines and chemokines . Interle2 (PGE2) synthesis  found an increasing transcription of the PTGS2 gene and/or PGE2 concentration after mechanical stimulation correlating with force duration and force magnitude. The data of the remaining studies revealed mixed expression patterns of PTGS2 and PGE2 activity after force exposure, which might be attributable to anti-inflammatory effects of lower tension forces  is known as a key regulator enzyme of the eicosanoid biosynthesis pathway and is thus also involved in prostaglandin Eynthesis . Amongsting IL1B . PGE2 medontitis . Herein,n forces . Taken tConsidering the list of the most commonly considered genes, inconsistencies between gene and protein expression were found in several reports. The observed non-proportional relationship between gene expression and protein activity can be attributed to the time lag between transcription and translation. This time lag might be prolonged by post-transcriptional processing and degradation of the transcripts, as well as post-translational modifications like phosphorylations e.g.  or protein vitro life span clearly depends on the age of the donor . These cells were commonly isolated from the middle third of the roots from teeth removed due to mostly orthodontic reasons using two different cell isolation techniques, i.e. the “explant”  or the “n hPDLCs , that wen hPDLCs , osteogeposition , cell typosition , and immposition .Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE)” guidelines were established, with reference gene selection being one of the most crucial steps in RT-qPCR establishment  network construction were applied. Moreover, the most influential genes and sub-networks were identified in both PPI networks. The gene lists were created from the studies identified in this systematic review. To increase specificity, only those genes showing clear force-dependent expression were included.Gene-set enrichment analysis: In general, gene-set enrichment analysis is applied to expression data of individual genes obtained by techniques like microarrays, next-generation sequencing or proteomics  data was publicly not available. Nevertheless, data from these studies was included if qualified reanalysis using (s)qPCR or protein expression was reported additionally.Search Tool for the Retrieval of Interacting Genes/Proteins” database (STRING-DB), which incorporates regularly updated data from different biological pathway and scientific literature databases , being one of the most frequently analyzed genes/proteins identified herein. With the application of a confidence score cut-off of ≥0.400, thus including medium confidence interactions, ALPP was integrated into dynamic network due to co-mentioning in PubMed abstracts with bone sialoprotein 2 , osteopontin , bone morphogenetic protein 2 , RUNX2 (0.495), and osteocalcin . GOSR1, CDC42EP2, UNC50, ACY1, and AMDHD2 were still not integrated. Application of the same threshold to the static network would integrate all nodes including ALPP .Interestingly, cluster #7 of the dynamic network consisted of nodes, that were contributed to the gene list based on one study . This fiInitially, a meta-analysis of the ten most frequently analyzed genes or metabolites was intended to supplement the findings. Unfortunately, due to heterogeneity of the experimental conditions, including force parameters, cell culture, reference gene selection in RT-qPCR experiments and incomplete reporting especially concerning the statistical unit, this was not further considered.COL1A1 (RUNX2 (Several identified studies showed that not only gene and protein expression is regulated by tension application, but also post-translational modifications like proteolytic cleavage, activation by GTP-binding, phosphorylations and protein translocation between nucleus and cytoplasm, or cytoplasm and extracellular space. Regulation of second messengers like cAMP  and metaCOL1A1  and RUNX1 (RUNX2  are know1 (RUNX2 .in vitro and assessed potential reporting and methodology-associated risk of bias related to this issue. Due to the enormous variety of apparatus in both, dynamic and static tension experiments, it is not possible to universally define optimum force parameters including force magnitude, duration and frequency. However, clinically relevant parameters were identified, that can be used as a reference for in vitro studies. Taken together, quantitative and qualitative information on mechanical stimulated gene and protein regulation and a comprehensive network analysis have provided more clear insights into the mechanisms involved in the OTM.In this systematic review we summarized relevant information about tension application on hPDLCs in vitro model for the simulation of orthodontic force, and to provide more reliable evidence for clinical treatment.Future studies should focus on the comparison of dynamic and static tension. There is also a need to elucidate the differences between the application of equibiaxial and uniaxial tension in more detail, to develop an optimal


Lymphoid neoplasms are a heterogeneous group of lymphoid neoplastic diseases with multiple presentations, and varying prognoses. They are especially frequent in older patients (OPs) and the atypism of this frail elderly population can make the diagnostic process even more difficult. Blood lymphocyte immunophenotyping (BLI) is essential in rapid noninvasive diagnosis orientation and guides complementary investigations. To our knowledge, BLI prescription has never been evaluated in OPs. We hypothesized that, when there is a suspicion of lymphoid neoplasm in the geriatric population, a BLI is performed in view of various clinical or biological abnormalities. This study aimed to: (1) describe the characteristics of hospitalized OPs having undergone BLI for suspected lymphoid neoplasm, (2) identify the causes leading to BLI prescription, and (3) identify the most profitable criteria for BLI prescription. This was a descriptive retrospective study on 151 OPs aged ≥75 years who underwent BLI over a 2-year period. Regarding BLI prescriptions, eight had lymphocytosis, constituting the “lymphocytosis group” (LG+), while the 143 others had BLI prescribed for reasons other than lymphocytosis (LG−), mainly general weakness and anemia. In the LG−, we compared OPs with positive and negative BLI results. The criteria found to be profitable for BLI prescription were lymphadenopathy, splenomegaly, lymphocytosis, and thrombocytopenia. BLI identified circulating lymphoid neoplasms (positive BLI) in 21/151 OPs, mainly marginal zone lymphoma and chronic lymphocytic leukemia. In polymorbid OPs, as per our study population, the diagnostic and therapeutic complexity explained in part the sole use of indirect and minimally invasive diagnostic techniques such as BLI. Lymphoid neoplasms are a heterogeneous group of lymphoid diseases, which are clonal tumors originating from lymphocytes, B-cells (B-cell lymphoid neoplasms) or T-cells (T-cell lymphoid neoplasms) . LymphoiIn current practice, it is possible to perform blood lymphocyte immunophenotyping (BLI) by multiparameter flow cytometry for detection of small-cell circulating clonal lymphoid proliferation and subset identification in the event of suspected lymphoid neoplasm . BLI is We hypothesized that in case of lymphoid neoplasm suspicion in the elderly population, a BLI test is carried out in view of various clinical or biological abnormalities, including lymphadenopathy(ies), splenomegaly, hepatomegaly, general weakness, unexplained fever, formed elements of blood disorders , monoclonal gammapathy and unexplained inflammatory syndrome. To our knowledge, BLI prescription has never been evaluated in OPs.The main objective of this study was to assess the indications of BLI prescription in a hospitalized geriatric population with suspected lymphoid neoplasm. The second objective was to distinguish possible profitable clinical and/or biological characteristics justifying BLI prescription. This retrospective observational descriptive study was carried out in the two geriatric departments (an acute geriatric care unit and a geriatric rehabilitation unit) of the Dijon-Bourgogne University Hospital.Data were collected for OPs hospitalized between 1 January 2018 and 31 December 2019.This study was conducted in accordance with the Declaration of Helsinki and French national standards.We included all hospitalized OPs (aged ≥ 75 years) who had a BLI prescribed for the first time during their hospital stay. OPs were excluded when BLI was added by the hematology laboratory following morphological abnormalities on the blood smear.The Ethics Committee of our institution was consulted and approved this retrospective study, which had no effect on patient management.BLI was performed by the flow cytometry platform of the biological hematology laboratory of the Dijon-Bourgogne University. Total EDTA blood samples were processed in a “lysis-no wash” protocol within 24 h after blood collection. A first screening panel was used with the nine-color monoclonal antibody combination: Kappa-FITC/Lambda-PE/CD4-PECF594/CD5-PC5.5/CD8-PC7/CD3-APC/CD19-AA750/CD16 BV421/CD45-KO [Sample acquisition was performed using a Navios®  on at least 5000 lymphocytes. Data were then analyzed using Kaluza® software, version 2.1 .BLI was considered positive when it detected and identified a cluster of clonal lymphoid proliferation consistent with lymphoid neoplasm with circulating blood phase. BLI was considered negative when no circulating clone was identified .The collected epidemiological data included age, sex, and history of lymphoid neoplasm. BLI prescription was justified by the prescribing physician.The collected clinical data included tumoral syndrome (lymphadenopathy(ies), hepatomegaly, splenomegaly), and general symptoms .Collected blood data included leukocyte, lymphocyte, eosinophil, monocyte, hemoglobin, and platelet counts, albuminemia, and C-reactive protein, as well as serum protein electrophoresis and immunofixation .The collected geriatric data were: (1) age-adjusted Charlson comorbidity index (ACCI) , (2) numData for complementary investigations were also collected, such as histology confirmation (solid biopsy) and disease staging by imaging  and/or myelogram/bone marrow biopsy. Finally, data were also collected for follow-up care including hematological and/or oncogeriatric evaluation, and in confirmed lymphoid neoplasm, initiation or not of a specific treatment.Patients were divided in two groups depending on BLI prescription in the presence or absence of lymphocytosis. The “lymphocytosis group” (LG+) consisted of OPs with BLI prescribed for lymphocytosis ≥ 5 G/L. In the “absence of lymphocytosis group” (LG−), BLI was prescribed for other clinical or biological reasons.The LG− was divided into two subgroups depending on BLI results: BLI positive, identifying circulating lymphoid neoplasm, or BLI negative, defined by the absence of circulating lymphoid neoplasm.t-test for quantitative variables. Qualitative data were expressed as numbers and percentages. Quantitative data were expressed as means with standard deviations and extreme values. The two subgroups of LG− were compared using univariate analysis. The Chi-square test or Fischer test was used for qualitative variables and the Student’s BiostaTGV© , a free website, was used for all statistical analyses on Excel®, version 16.16.21 .p ≤ 0.05.Statistical significance was set at A total of 151 OPs had had a BLI prescribed during the study period. Their average age was 86.7 ± 5.7 years (range 75–97) and 51% were men; 8 (5.3%) patients had lymphocytosis (LG+), while 143 (94.7%) did not (LG−) .The mean ACCI was 6.4 ± 1.7 (range 3–15) and 102 OPs (67%) had an ACCI ≥ 6. The mean ADL score was 3.9 ± 1.6 (range 0–6); 57 OPs (38%) had an ADL score ≥ 5. In addition, 137 of the 151 OPs received a cognitive assessment by MMSE with an average score of 18.7 ± 6.2 (range 3–30).Finally, 28% of OPs had a level of medical intervention giving them access to intensive care in the event of deterioration.The LG+ included eight patients aged 85.0 ± 5.3 years, and 87.5% women. Five (62.5%) had positive BLI identifying three (60%) CLL and two (40%) circulating phase of marginal zone lymphoma (MZL) . Only onThree OPs (37.5%) had negative BLI with neither signs of a tumor nor cytopenia, and lymphocytosis was reactive.While BLI was prescribed to investigate lymphocytosis in the LG+, the most common reasons for prescribing BLI in the absence of lymphocytosis was general weakness (19.6%) and anemia (10.5%) .The LG− included 143 patients aged 86.8 ± 5.7 years and was 53.1% male. Most of these patients lived at home (85%), had polypharmacy (78%), and were ambulating independently (54%).A total of 16 OPs (11.1%) were found to have positive BLI: seven MZL (44%), three monoclonal B-cell lymphocytosis (MBL) (19%), two small lymphocytic lymphoma (SLL) (12.5%), two T-cell lymphoid neoplasms (12.5%), one lymphoplasmacytic lymphoma (LPL) (6%), and one follicular lymphoma (FL) (6%).p = 0.03), and cognitive disorders according to MMSE score (p < 0.01). Among the biological markers, albuminemia was significantly higher in this subgroup (p = 0.02).OPs in the LG− with positive BLI had significantly more comorbidities according to ACCI (p = 0.05) and splenomegaly (p = 0.02).As concerns clinical characteristics, a positive BLI (identifying a circulating lymphoid neoplasm blood phase) was significantly more frequent among OPs with lymphadenopathy(ies) (p < 0.01 for both). However, the presence of strict thrombocytopenia (< 150 G/L) was not significantly associated with positive BLI (p = 0.06).For complete blood count, LG−/BLI positive patients had significantly lower platelet count and higher lymphocyte count compared to those with negative BLI  and one high-grade follicular lymphoma (FL) was diagnosed on skin biopsy (BLI positive).p = 0.02 and p = 0.04, respectively).Hematological and oncogeriatric evaluations were requested in, respectively, 25% and 31% of cases in the subgroup with positive BLI vs. 5.5% and 11% in the subgroup with negative BLI, resulting in a significant difference between the two groups , and frequently cognitively impaired (mean MMSE score of 18.7/30) and dependent (only 38% of the population had an ADL score ≥ 5).p < 0.01). Even if lymphocytosis is not greater than 5 G/L, if it is found to be in the upper limit of the norm, it is a relevant factor in the decision to prescribe a BLI.This paper shows that lymphocytosis remains a valid indication for the prescription of BLI in case of lymphoid neoplasm suspicion, especially if it is significant . Our results suggest a high profitability in this indication, with a likelihood of detecting a lymphoid neoplasm of more than 60% . However, because of the modest size of the LG+, this result should be considered with caution. In addition, a higher mean lymphocyte count was significantly associated with positive BLI in the LG− (p < 0.01). However, despite a trend (p = 0.06), real thrombocytopenia (<150 G/L) was not associated with this gain in profitability, probably due to an insufficient number of included patients. According to the literature, thrombocytopenia is common in lymphoid neoplasm, but not specific [BLI was more frequently prescribed in isolated anemia 10.5%) than in isolated thrombocytopenia (7.7%). However, anemia was not significantly associated with BLI positivity in the LG−. This could be explained by the frequently multifactorial nature of anemia in older adults. This result raises the question of whether the value of BLI should be evaluated in the event of anemia, when the first-line etiological assessment turns out to be negative. A platelet count close to the lower bound of the norm was associated with more profitable BLI results in the LG− (0.5% thanspecific . A trendWe can underline the high rate of BLI prescription in a context of monocytosis (9.1% of prescriptions). In our sample, 44 patients from the LG− (30%) had monocytosis, but no significant association was shown between this criterion (monocytosis) and positive BLI in LG−. Monocytosis can be found in cases of lymphoid neoplasm, but with no specificity for the latter. Tadmor et al., showed that 18.7% of patients suffering from DLBCL had monocytosis at the time of diagnosis and that its presence seemed to suggest a poor prognosis for hematologic malignancy . Howeverp = 0.9). This could be explained by the fact that in OPs, general weakness is frequent, unspecific, rarely isolated, and sometimes multifactorial [Clinically, general weakness was the most common indication for BLI (19.6%) in the LG−, while it was not profitable for BLI positivity  was the second most frequent indication for BLI prescription 11%) and was significantly associated with positive BLI. This seems consistent with the typical presentation of the main lymphoid neoplasm in which lymphadenopathy is often described 1% and wa. We did p = 0.02), splenomegaly was not a frequent indication for BLI (2%). lymphoid neoplasms are a frequent cause of unexplained splenomegaly in patients who have a histological analysis following a diagnostic splenectomy [Though the profitability of BLI in a context of splenomegaly was high in LG−  appears to be of additional interest. CTCL classically includes either mycosis fungoides, less aggressive and primarily cutaneous at the start, or Sezary syndrome, which is rarer, more aggressive, and often with an immediate circulating blood form . TherefoIn our study, the details of the cases with positive BLI showed a predominance of small B-cell lymphoid neoplasms, which is consistent with the literature . Small BFor FL, we identified few positive BLIs (around 5%), yet it is relatively common small B-cell lymphoid neoplasm. This may in part be explained by a younger median age of onset, around 65 years in men .Positive BLI was not found in association with mantle cell lymphoma (MCL). Although MCL has a fairly common circulating blood phase compared to other lymphoid neoplasms, its incidence is lower ,24.In our sample, only one patient had histologically confirmed DLBCL and no circulating blood phase was found, which was surprising since epidemiological data report a high preponderance of DLBCL among lymphomas. However, DLBCL has poor blood circulation due to its large B cells, which may explain its poor representation in our study ,21. In aThe population studied here mainly showed low-grade mature B-cell lymphoid neoplasm, which are generally non-aggressive. The slow progression of most of these mature B-cell lymphoid neoplasms (excluding mantle cell lymphoma) can justify, especially in OPs with comorbidities, a reinforced surveillance strategy without curative treatment as a first step ,26,27. WIt is estimated that approximately 30% of people with DLBCL are at least 75 years of age, yet therapeutic trials for DLBCL rarely include octogenarians . The fewIt is interesting to note that the geriatric assessment made it possible to reduce the chemotherapy protocols according to the degree of frailty, with relatively good therapeutic results .Among the three OPs with histological confirmation of lymphoid neoplasm, the one DLBCL patient was frail and had a major neurocognitive disorder and an ADL score of 3/6. This patient received pre-phase treatment with vincristine, but then deteriorated until death. The other two patients, one with high grade FL and one with MZL, were more “robust” (ACCI < 6 and ADL score > 5) and obtained complete response with specific treatment .Thus, when a blood lymphocyte clone is identified in BLI, excluding CLL, and there is an evocative clinical and possibly paraclinical picture, lymphoid neoplasm can be considered with virtual certainty. Histological confirmation would then be “reserved” for OPs who may benefit most from therapeutic management. Unfortunately, the most common lymphoma, DLBCL, is often aggressive and diagnosis is rarely helped by BLI, which tends to be negative.Hematological and oncogeriatric evaluations were significantly more frequently requested following positive BLI, illustrating a desire to implement appropriate multidisciplinary management, highlighting the importance of the BLI result. Again, this study reinforces the importance of early oncogeriatric assessments in order to avoid unnecessary invasive diagnostic testing with potentially no therapeutic outcome.Our study has some limitations. The first is linked to its retrospective nature and the potentially non-exhaustive description of the criteria that led the physician to prescribe BLI. However, the initial cause for prescribing BLI was almost always clearly mentioned in the medical record. Another limitation of this study is the lack of multivariate analysis due to the low number of included patients, particularly in the subgroup of LG− with positive BLI.This work shows that small mature B-cell lymphoid neoplasms were most commonly diagnosed in the study population in the event of positive BLI findings. Unfortunately, the diagnostic process for DLBCL, the most common lymphoma type, does not appear to be helped by BLI.The reasons for performing a BLI in the event of clinical suspicion of lymphoid neoplasm in the geriatric population are based on various clinical or biological criteria. The discovery of lymphadenopathy, splenomegaly, lymphocytosis, and a low platelet count provides profitable clinical and biological criteria that suggest the need for a subsequent BLI in current practice.The patients included in this study had comorbidities, polypharmacy, low functional independence, and cognitive disorders. The diagnostic and therapeutic approach is more complex for this type of patient; hence, the desire to strengthen the set of diagnostic arguments by first using techniques such as BLI, which are admittedly indirect but noninvasive. In the event of positive BLI, the combined contribution of the oncogeriatrician and hematologist is more often required, highlighting the need for appropriate and multidisciplinary management in OP with lymphoid neoplasm.


Percutaneous magnetic resonance-guided (MR-guided) MWA procedures have traditionally been performed under local anesthesia (LA) and sedation. However, pain control is often difficult to manage, especially in some cases when the tumor is large or in a specific location, such as near the abdominal wall or close to the hepatic dome. This study retrospectively compared the results of general anesthesia (GA) and local anesthesia (LA) for MR-guided microwave ablation (MWA) in patients with hepatocellular carcinoma (HCC ≤ 5.0 cm) to investigate whether different anesthesia methods lead to different clinical outcomes.The results of the analysis include procedure-related complications, imaging response, and the time to complete two sets of procedures. According to the type of anesthesia, the Kaplan-Meier method was used to compare the local tumor progression (LTP) of the two groups who underwent MR-guided MWA.P = 0.012). Both groups had no difference in complications or LTP (both P > 0.05). Notably, the tumor location  and the number of lesions (2–3 lesions) could be the main factors affecting LTP . Univariate Cox proportional hazard regression indicated that using different anesthesia methods (GA and LA) was not associated with longer LTP (P = 0.237), while tumor location  and the number of lesions (2–3 lesions) were both related to shorter LTP . Additionally, multivariate Cox regression further revealed that the tumor location (regular locations) and the number of lesions (single) could independently predict better LTP .All patients achieved technical success. The mean ablation duration of each patient in the GA group and LA group was remarkably different  appear to be the main factors affecting LTP. Until a few decades ago, surgical resection was the only effective choice for the treatment of hepatocellular carcinoma (HCC). However, various effective modalities have been shown, including local ablation and liver transplantation , 2. Due MR-guided thermal ablation has been used in all aspects of tumor treatment and has shown favorable technical feasibility and safety –9. Most Currently, no data have been presented that compare the tumor recurrence rate of GA or LA in MR-guided microwave ablation (MWA) treatment. This study explored the relationship between anesthesia techniques (GA and LA) and local tumor progression and attempted to establish a regression model to further determine the effect of GA on tumor prognosis.This was a retrospective cohort study conducted in a single center approved by the institutional review board. We included 34 patients  who received GA for MR-guided MWA and 38 patients  who received LA for MR-guided MWA  and was treated with sufentanil or nonsteroidal anti-inflammatory drugs (NSAIDs) for postoperative pain. In the LA group, less than 10 mL of 2% lidocaine was injected subcutaneously at the puncture point. When the patient experienced unbearable pain during the treatment process, NSAIDs could be used to complete intraoperative pain management. The patient was awake and breathed spontaneously during the operation. The placement of MR and anesthesia equipment during treatment is shown in Fig. The ablation path was determined according to the preoperative CT / MR. All procedures were performed alternately by two interventional radiologists with 6–10 years of experience in ablation. Routine electrocardiogram and oxygen saturation monitoring  and MR-compatible MWA apparatus  were placed at a distance of 2.5–3 m beside the MR compatible operating table. After using the cod liver oil capsule matrix to mark the surface of the body, a standard MR protocol was completed to locate intrahepatic lesions. Then, insert the microwave probe  into the center of the tumor under the guidance of MR, and perform multiple scans to confirm that the tip of the applicator was 0.5–1 cm beyond the distal tumor. Besides, the tumor at each site was ablated with 45-65wt for 4–9 min  . InterveElectronic medical records were reviewed to collect pre-and post-treatment laboratory results and information on treatment-related complications. Imaging follow-up was performed with contrast-enhanced MR or CT at intervals of 1, 3, and 6 months with a 6-month interval for follow-up thereafter. All post-procedure and follow-up images were reviewed for consensus between a senior radiology resident and a board-certified interventional radiology faculty member with 5 years of experience in oncologic imaging and interventions. If tumor recurrence was found during the period, a second TACE-MWA or MWA procedure was performed separately depending on the patient’s condition. Patients who died early or lost to follow-up were excluded, and each patient met the follow-up time of 36 months.P < 0.05 was considered to indicate a significant difference. To determine the significant difference between the two groups, continuity correction, independent sample t-test, Pearson χ2 test and Fisher exact test were used. Categorical variables are represented as numbers or percentages (%), and continuous variables are represented as means ± Standard deviation (SD). Chi-square test or Wilcoxon rank-sum test was used to compare the two groups. Kaplan-Meier survival curves were used for survival analysis. Univariate and multivariate Cox proportional hazards regression were used to predict the prognostic factors of LTP.Statistical software SPSS 22.0 was used , n = 34; LA-group, n = 38). There were no significant differences in age, gender, ECOG score, etiology, Child-Pugh classification, tumor location, or tumor diameter between the two types of anesthesia. Patients in the GA-group and the LA-group, the mean energy of each tumor was 55.5 ± 5.6wt and 50.7 ± 6.1wt, respectively. The mean ablation duration of each patient in the GA-group and LA-group were 128.7 ± 40.3 and 90.8 ± 33.3, respectively, and the difference between the two groups was statistically significant (P = 0.012).A total of 72 patients with HCC ≤ 5.0 cm were included in the present study . The 12-, 24-, and 36-month LTP rates in GA were 94.1, 87.9 and 74.4%, respectively, and the 12-, 24-, and 36-month LTP rates in LA were 94.7, 84.2 and 62.1%, respectively . The 12-, 24-, and 36-month LTP rates in GA were 100.0, 76.0 and 48.4%, respectively, and the 12-, 24-, and 36-month LTP rates in LA were 88.9, 66.7 and 35.4%, respectively  after MWA under local anesthesia (LA) and general anesthesia (GA) was compared. The mean LTP was 33.434 months  in GA versus 31.132 months  in LA (P = 0.237), while tumor location  and the number of lesions (2–3 lesions) were all related to shorter LTP . Additionally, multivariate Cox regression further revealed that the tumor location (regular locations) and the number of lesions (single) could independently predict better LTP (both P < 0.005) . The 12-, 24-, and 36-month LTP rates in tumors with regular locations were 100.0, 100.0 and 91.6%, respectively, and the 12-, 24-, and 36-month LTP rates in tumors with challenging locations were 88.6, 71.2 and 40.2%, respectively . The 12-, 24-, and 36-month LTP-free survival rates in patients with a single lesion were 97.9, 87.0 and 77.8%, respectively, and the 12-, 24-, and 36-month LTP-free survival rates in patients with 2–3 lesions were 88.0, 84.0 and 47.6%, respectively  was not associated with longer LTP (5) Table . More spRadiofrequency ablation (RFA) and microwave ablation (MWA) are the most commonly used as alternative therapeutic options for HCC. Although the technical characteristics of RFA and MWA are quite similar, there are differences in the thermogenesis mechanisms of the two therapies. During the RFA procedure, the heat is restricted to zones of high current density, while during the MWA, it is produced in the fixed space around the microwave probe. The low energy density in the ablative zone under RFA conditions cannot reach the thermally toxic temperature in the nodules close to the cooling vasculature. Fortunately, MWA can bring the target lesion to a higher temperature in a shorter period and produce a larger ablation zone, thereby reducing the heat sink effect on the MWA treatment result , 17..17.Currently, most institutions perform percutaneous MR-guided MWA procedures under local anesthesia (LA) and sedation. However, pain control has always been an important issue in the ablation process. When the tumor is adjacent to the abdominal wall or near the hepatic dome, the pain caused by the ablation is more severe than other tumors in regular locations. If intraoperative pain cannot be well controlled, interventional procedures will undoubtedly be affected. Besides, the use of low-efficacy LA is suboptimal for intense intraoperative pain management, which may affect the patient’s respiratory activity and lead to respiratory depression or respiratory arrest. Moreover, due to the claustrophobic environment in the MRI room and the intense noise generated during the procedures, some patients could experience severe anxiety and insecurity and cannot complete the treatment process, resulting in the insufficient tumor ablation area. Notably, there may be a remarkable difference in the rate of local recurrence between patients who have reached a sufficient ablation area and those who have not. Therefore, pain management in MR-guided MWA is a critical condition to ensure adequate tumor ablation. As the preferred alternative to LA, general anesthesia (GA) can produce deeper sedation and better analgesia for patients undergoing MWA procedures in the MRI room, thereby ensuring efficient and safe completion of ablation treatment. In the course of this study, we found that intravenous GA could ensure more stable hemodynamics, but there was no significant difference in postoperative complications between the two anesthesia regimens.P = 0.230). Moreover, we conducted a stratified study on the LTP of HCC in challenging sites and reached a similar conclusion (P = 0.180). Multivariate Cox regression revealed that the tumor location (regular locations) and the number of lesions (single) could independently predict better LTP (P < 0.005). Therefore, during the limited follow-up period, MR-guided MWA under different anesthesia methods did not seem to have a significant effect on LTP.Some studies have shown that different anesthesia may affect the long-term results of cancer treatment , 19. TheThere are several shortcomings of our research. Due to software limitations, this study did not use the real-time MR thermometry technique. Second, the cost of MRI-guided ablation treatment under general anesthesia is considerably higher than that of local anesthesia, making it inaccessible for certain patients. Third, although there were fewer local complications of the ablation process in patients under GA, the rate of LTP between the two groups was equivalent, which should be an acceptable result. This study could provide a reference for the selection of anesthesia methods for ablation therapy under the guidance of magnetic resonance in the future. Finally, this is a single-center retrospective control study relating to a small number of cases. In the future, this study needs to be combined with a prospective multicenter study, extend the follow-up time, and increase the overall survival data to reduce the risk of bias.Different anesthesia methods seem to have no significant effect on treatment-related complications and LTP in HCC (≤5.0 cm). Secondly, the number of lesions (Single) and tumor location (regular locations) may be associated with favorable LTP. However, Prospective trials exploring the effects of different anesthetic methods on cancer outcomes in these patients are warranted.


Histological subtypes of colorectal cancer may be associated with varied prognostic features. This systematic review and meta-analysis aimed to compare clinicopathological characteristics, recurrence and overall survival between colorectal signet-ring cell (SC) and mucinous carcinoma (MC) to conventional adenocarcinoma (AC).A literature search of MEDLINE, EMBASE, Ovid and Cochrane Library was performed for studies that reported data on clinicopathological and survival outcomes on SC and/or MC versus AC from January 1985 to May 2020. Meta-analysis was performed using random-effect models and between-study heterogeneity was assessed.I2 88.6%; p = 0.014) and more likely to have right-sided disease . Locoregional recurrence at 5 years was more frequent in patients with SC  and MC . 5-year overall survival was significantly reduced when comparing SC and MC to AC .Thirty studies of 1,087,055 patients were included: 11,510 (1.06%) with SC, 110,179 (10.13%) with MC and 965,366 (88.81%) with AC. Patients with SC were younger than patients with AC (WMD − 0.47; 95% CI − 0.84 to –0.10; SC and MC are associated with right-sided lesions, advanced stage at presentation, higher rates of recurrence and poorer overall survival. This has strong implications towards surgical and oncological management and surveillance of colorectal cancer.The online version contains supplementary material available at 10.1007/s12672-021-00398-6. Colorectal cancer is the third most common diagnosed malignancy and the fourth leading cause of cancer death in the world –3. ThereThe majority of colorectal cancers are adenocarcinomas with three major subtypes: conventional adenocarcinoma (AC), mucinous (MC) and signet-ring cell (SC) adenocarcinomas . MC accoThe histological subtypes of colorectal cancer are believed to have prognostic relevance. It has been suggested that SC and MC tumours are associated with advanced stage at presentation  and a poWe conducted this systematic review and meta-analysis according to the Preferred Reporting Items for Systematic reviews and Meta-Analysis group (PRISMA) guidelines  and the signet cell-ring adenocarcinoma/cancer, mucinous adenocarcinoma/cancer, mucin, colorectal adenocarcinoma/cancer, comparative study, local/locoregional recurrence, metastasis and survival. We retrieved articles published in English from January 1, 1985, to May 30, 2020, that reported outcomes on SC and MC versus AC. The reference lists from the selected studies were reviewed to identify additional relevant studies.A literature search of MEDLINE, EMBASE, Ovid and The Cochrane library was performed. Specific research equations were formulated for each database using the following Medical Subject Headings (MeSH) terms: Studies were included if they met the following criteria: (1) studies were comparative between SC and/or MC with AC, (2) at least one outcome of recurrence and survival pattern was reported, (3) studies reported outcomes at least at yearly intervals throughout the duration of follow-up to enable hazard ratio (HR) calculations, (4) the site of the cancer had to be specified as ‘colon’ or ‘rectum’, (5) definitions of SC and MC had to be reported according to the World Health Organisation criteria or equivalent . ExclusiTwo authors conducted the search and identification independently, and a third author confirmed that the final selected manuscripts met the inclusion criteria. The two authors independently extracted the following information from the included studies: first author, year of publication, country, patient number, study design, age, gender, tumour location, stage   and tumoThe weighted mean difference (WMD) was used to analyse continuous variables, such as patient age and tumour size, accounting for sample size and event rate. Standard deviation (SD) was calculated using statistical algorithms. The odds ratio (OR) was used as the statistical measure for all other dichotomous outcomes. The OR represents the odds of an adverse event (e.g. recurrence) occurring in the experimental group (SC and MC) versus the control group (AC) and approximates the relative risk. An OR of greater than one indicated greater risk of an adverse event happening in the SC or MC groups.2-statistic and the I2 value to measure the degree of variation not attributable to chance alone. This was graded as low (I2 < 25%), moderate (I2 = 25–75%) or high (I2 > 75%). This study was performed in line with Cochrane recommendations and following PRISMA guidelines and using the statistical software STATA V 12.The logarithm of the HR with 95% confidence intervals (CI) was used as the primary summary statistic as described by Parmar et al. . The estThe quality of all observational studies was assessed using the Newcastle–Ottawa Scale (NOS) . This waA total of 799 published articles were identified from the initial search, of which 700 were excluded based on title and abstract review. The remaining 99 articles underwent full-text evaluation and 69 studies were further excluded. The remaining 30 comparative studies, comprising a total of 1,087,055 patients, were eligible and were included in the qualitative and quantitative analyses , 24–49. Fifteen studies evaluated outcomes for all three types of cancer –45, 47. 4445Age, gender, location, stage and tumour size differences between SC and MC with AC were suitable for analysis as shown in Table Stage at presentation was significantly more advanced in SC  and MC  than AC  . Survival analysis was also subject to great between-study heterogeneity in the case of both SC and MC . Begg’s and Egger’s tests were performed for all outcomes with significant heterogeneity and funnel plots were inspected without significant evidence of publication bias.Five-year overall survival was significantly poorer for both SC and MC when compared to AC  . Figure S1. Meta-analysis and forest plot comparing (a) Age, (b) Gender, (c) Tumour diameter (cm) of signet-ring cell cancer and mucinous cancer to conventional adenocarcinoma. SC = signet-ring cell cancer, MC = mucinous cancer, AC = conventional adenocarcinoma, OR = odds ratio, CI = confidence interval.Additional file 1: Below is the link to the electronic supplementary material.


Alzheimer’s disease (AD) is a heterogeneous disease and exhibits diverse clinical presentations and disease progression. Some pathological and anatomical subtypes have been proposed. However, these subtypes provide a limited mechanistic understanding for AD. Leveraging gene expression data of 222 AD patients from The Religious Orders Study and Memory and Aging Project (ROSMAP) Study, we identified two AD molecular subtypes (synaptic type and inflammatory type) using consensus non-negative matrix factorization (NMF). Synaptic type is characterized by disrupted synaptic vesicle priming and recycling and synaptic plasticity. Inflammatory type is characterized by disrupted IL2, interferon alpha and gamma pathways. The two AD molecular subtypes were validated using independent data from Gene Expression Omnibus. We further demonstrated that the two molecular subtypes are associated with APOE genotypes, with synaptic type more prevalent in AD patients with E3E4 genotype and inflammatory type more prevalent in AD patients with E3E3 genotype (p = 0.031). In addition, two molecular subtypes are differentially represented in male and female AD, with synaptic type more prevalent in male and inflammatory type in female patients (p = 0.051). Identification of AD molecular subtypes has potential in facilitating disease mechanism understanding, clinical trial design, drug discovery, and precision medicine for AD. Alzheimer’s disease (AD) is the most common neurodegenerative disease in elderly population, characterized by pathological extracellular deposition of beta-amyloid (Aβ) peptides and intracellular tau protein fibers in the brain . AD is aNeuroimaging, beta amyloid and tau have been used for AD subtyping –13, howeThe Religious Orders Study and Memory and Aging Project (ROSMAP) is a longitudinal clinical-pathologic cohort study of aging and AD . CurrentThe overall methods were illustrated in ROSMAP contains 222 participants with clinical consensus diagnosis of AD at time of death. Raw gene expression data from frontal cortex and corresponding clinical data were downloaded from synapse.org (syn3219045). Raw count data were normalized and processed according to commonly used procedure described in edgeR (version: 3.28.0) , 30. Datn × m (n genes and m samples) and desired number of clusters k, the NMF decomposes A into two non-negative matrices W (n × k) and H (k × m)  .W and H matrix are computed using iterative method to minimize the following cost function.In each iteration, W and H are updated using following multiplicative updating rules,Cluster membership of each sample is assigned based on the row index of maximal number in the column of H matrix.C (n × n). Each element of consensus matrix represents the probability that two samples cluster together. Then, the cophenetic correlation coefficient ρk was computed as the Pearson correlation of the distance matrix between samples induced by the consensus matrix, i.e., I − C, and the distance matrix induced by the hierarchical clustering of I − C. ρk measures how faithfully a dendrogram preserves the pairwise distances in the consensus matrix and was calculated using the cophenet function in the scikit-learn library [k.We used consensus matrix-base model selection strategy to select best number of clusters . For a g library . The besa(i) and b(i) were computed as following,a(i) is the mean distance of a sample to all other samples in the same cluster. It measures how well a sample is assigned to its own cluster. The smaller the value is, the better the assignment is. b(i) is the smallest mean distance of a sample to all samples in any other cluster. |Ci| is the number of samples in its own cluster, |Ck| is the number of samples in any other cluster, and d is the distance of two samples computed with Euclidean distance.To identify molecular subtype-specific signatures, we first computed the silhouette for each sample using following equation.The silhouette is a measure of how similar a sample is to its own cluster compared to other clusters. After removing outlier samples with negative silhouette width from each subtype, we applied statistical package edgeR (version: 3.28.0) to obtain pairwise differentially expressed genes (DEGs) between molecular subtypes. To facilitate downstream analysis of molecular subtypes, we used fold change of 1.5 and false discover rate (FDR) of 0.05 as cutoffs. We define the gene signature of each subtype as DEGs that have the highest value in each molecular subtype.A Bioconductor package clusterProfiler (Version 3.14.3)  was usedTwo independent datasets from Gene Expression Omnibus  were used for validation of AD molecular subtypes. GSE44770 includes gene expression data from frontal cortex of 230 subjects, 128 of which are late-onset Alzheimer´s disease (LOAD) patients. GSE118553 includes gene expression data from frontal cortex of 112 subjects, including 52 AD patients. We used normalized data from GSE44770 and GSE118553 to validate molecular subtypes identified based on ROSMAP data.Since ground truth of clusters in a dataset is unknown, there are no quantitative method to formally validate clusters in an independent dataset. Therefore, visualization is suggested as a valid approach . A discoWe examined the demographic distributions of AD molecular subtype, including age, sex, race and education, and assessed the associations of AD molecular subtype with APOE genotype and clinical variables, including Braak stage, The Consortium to Establish a Registry for Alzheimer’s Disease (CERAD) diagnosis, and Mini-Mental State Examination (MMSE) score. The Braak stage is a semiquantitative measure of severity of neurofibrillary tangle (NFT) pathology , 36. BraFor categorical variables, including Braak stage, CERAD, and APOE, Fisher’s exact test was used to assess their associations with AD molecular subtype. For continuous variables, such as MMSE and education, student’s t-test was used. All statistical analysis was performed using R (version: 3.6.2). Significance level was defined as p value less than 0.05.This is a secondary research use for ROSMAP data and patient information is not identifiable. The IRB at Case Western Reserve University determined that the proposed activity is not research involving human subjects and IRB review and approval is not required (STUDY20190935). Therefore, patient consent is not applicable or not required.We used consensus NMF to cluster gene expression data of 222 AD patients from ROSMAP. Compared with three and four clusters, consensus matrix from two clusters are more stable . In addiWe named the molecular subtypes according to signature genes up-regulated in each cluster. For synaptic type, highly expressed genes are associated with synapse function, such as SNAP25, RAB3A, VAMP1, SYNJ1, and STXBP1. A total of 37 pathways were significantly enriched and 23 of 37 (62.2%) are related to synapse function . The topFor inflammatory type, highly expressed genes are related to inflammatory pathways, such as BST2, GBP4, IFI44L, IFITM2, IFITM3, IL4R, IRF, MT2A, PSMB9, and TXNIP. A total of 3 pathways were significantly enriched using the signature genes. This subtype is characterized with dysfunction of inflammatory responses, including interferon alpha (IFN-α), interferon gamma (INF-γ) and IL2 pathways .We validated the two AD molecular subtypes using two independent datasets from GEO . Using consensus NMF, we identified clusters based on these two independent datasets from GEO Figs . MajoritTo further validate the AD molecular subtypes in these two datasets, we performed pathway enrichment for each cluster in each dataset. For GSE44770 dataset, a total of 30 pathways were significantly enriched in first cluster . Seven oWe investigated whether AD subtypes are associated with demographic and clinical variables using the core samples from ROSMAP dataset (197 patients). The distributions of AD molecular subtype in demographic variables, including age, race and education, show no significant difference . InteresWe then examined whether these associations can also be observed in the two validation datasets. Although we didn’t see a significant association of sex with molecular subtype, we observed that the synaptic type is more prevalent in male patients than in females in both datasets. In the GSE44770, 37 of 60 (61.7%) are synaptic type in male patients, while it is 33 of 66 (50.0%) in female patients. In the GSE118553, the prevalence of synaptic type in male and female patients are 10 of 14 (71.4%) and 17 of 25 (68.0%) respectively. Due to the lack of APOE genotype in these two datasets, we were unable to investigate the association of APOE with molecular subtype .In this study, we applied non-negative matrix factorization combined with consensus matrix-based cluster selection and identified two molecular subtypes based on gene expression data of AD. Synaptic type is characterized by dysfunction of synaptic pathways. Substantial loss of neurons and synapses is a hallmark in late stage AD. Recent studies also show synaptic dysfunction was observed in mild cognitive impairment patients –40, suggGWAS has identified more than 40 genes/loci as the genetic risk factors of AD, which greatly expands our mechanistic understanding of the etiology of AD. While some of these genes/loci have been mapped to Aβ pathology, including amyloid precursor protein (APP) metabolism, Aβ aggregation, clearance, toxicity, and Tau pathology, a large amount of these genes is related to non-Aβ and -Tau pathways . LambertUsing available patient clinical information, we evaluated their associations with molecular subtypes. We didn’t find significant correlation of molecular subtype with severity of cognitive impairment. However, we were unable to control potential confounders due to very limited information available in the dataset. We show that AD molecular subtype is significantly associated with APOE genotype. APOE has three alleles, including E2, E3 and E4. APOE4 is the main genetic determinant for late-onset AD and individual with APOE4 significantly increases the risk of AD , 51. WhiWe observed that inflammatory type of AD is more prevalent in women. On the other hand, synaptic type of AD is more prevalent in men. Sex differences in both synaptic plasticity and inflammatory response have been observed , 55. FemIdentification of AD molecular subtype has an implication for better design in clinical trials. Currently, clinical trials for AD are based on different cognitive groups from mild, moderate, and severe AD. However, most of this symptom-based clinical trials for AD fails, reflecting a lack of mechanistic understanding of AD. A recent clinical trial about a monoclonal antibody solanezumab failed the phase III trials for mild to moderate AD , but latOne limitation of this study is that molecular subtyping is based on gene expression data from post-mortem brain tissue, which limits its clinical usage. Nevertheless, identified molecular subtypes will help to understand the mechanism of AD. In the future, developing a practical molecular subtyping system for AD is demanded. Proteomic data from cerebrospinal fluid and genotype data from blood could be useful for such purpose.In this study, we reported the first gene expression-based molecular subtyping of AD. Using consensus NMF, we identified two robust molecular subtypes-synaptic type and inflammatory type-that represent two fundamental mechanisms of AD. These molecular subtypes are associated with APOE genotype and exhibit sex difference in distribution. Identification of molecular subtypes may have an implication in better clinical trial design and personalized medicine for AD.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.


The oral microbiome is likely a key element of homeostasis in the oral cavity. With >600 bacterial species and >160 fungal species comprising the oral microbiome, influences on its composition can have an impact on both local and systemic health. Candida albicans, Candida dubliniensis, Malassezia restricta, or Saccharomyces cerevisiae. Quantitative PCR analysis showed the Candida-dominated sample clusters had significantly higher total fungal abundance than the Malassezia or Saccharomyces species. Of the 25 clinical variables evaluated for potential influences on the oral mycobiome, significant effects were associated with caries status, geographical site of sampling, sex, HIV under highly active antiretroviral therapy (HAART), and missing teeth, in rank order of statistical significance. Investigating specific interactions between fungi and bacteria in the samples often showed Candida species positively correlated with Firmicutes or Actinobacteria and negatively correlated with Fusobacteria, Proteobacteria, and Bacteroidetes. Our data suggest that the oral mycobiome, while diverse, is often dominated by a limited number of species per individual; is affected by several clinical variables, including HIV positivity and HAART; and shows genera-specific associations with bacterial groups.The oral microbiome is considered an important factor in health and disease. We recently reported significant effects of HIV and several other clinical variables on the oral bacterial communities in a large cohort of HIV-positive and -negative individuals. The purpose of the present study was to similarly analyze the oral mycobiome in the same cohort. To identify fungi, the internal transcribed spacer 2 (ITS2) of the fungal rRNA genes was sequenced using oral rinse samples from 149 HIV-positive and 88 HIV-negative subjects that had previously undergone bacterial amplicon sequencing. Quantitative PCR was performed for total fungal content and total bacterial content. Interestingly, samples often showed predominance of a single fungal species with four major clusters predominated by  HIV disease continues to be an important infection worldwide, with impacts on health and the economy. Persons infected with HIV have been particularly vulnerable to a variety of oral diseases, including gingivitis, periodontitis, dental caries, endodontic infections, oropharyngeal candidiasis (OPC), oral warts, oral hairy leukoplakia, and Kaposi’s sarcoma –3. Howev–The oral microbiome is considered an important factor in health and disease. For example, several studies have shown changes in the composition and diversity of the oral microbiome during periodontal disease –8 and de–10–The fungal community (mycobiome) represents another prominent component of the oral microbiome. The mycobiome has been much less studied than the bacterial community at any anatomical site, including the oral cavity. However, published studies show a more limited fungal representation in the oral cavity, with suggestions of possible antagonism leading to dominance of genera/species –20. Thes–22–There are a multitude of clinical factors or conditions that could be potential modulators of the oral microbiome and thus are considered confounders when examining the effects of HAART in HIV infection. Examples include variables such as antimicrobials; other prescribed medications; oral hygiene practices, including dental cleaning; cigarette smoking; xerostomia; diet; beverage intake, including alcohol; illicit drug use; periodontal disease; gingivitis; caries status and restorations; or remaining teeth. While a limited number of variables have been evaluated as an influence on the oral microbiome in previous studies of HIV-positive persons , , 26–29, 26–The purpose of the present study was to characterize the oral mycobiome in our large cohort of well-documented HIV-positive and HIV-negative persons using fungal internal transcribed spacer 2 (ITS2) amplification and sequencing and to identify clinical variables that modulate the oral mycobiome.We recently reported the distribution of secondary clinical variables for this cohort . The HIV10.1128/mBio.00294-21.1FIG S1+(HAART) and HIV− groups. Dichotomous categories are shown to the right of the labels. Significance was determined by Fisher’s exact test. Box and whisker plots of distributions for the HIV groups are shown for continuous variables. Significance was determined by Wilcoxon signed-rank test. Asterisks indicate levels of significance, as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Group labels are HIV− for HIV negative and HIV+HAART for HIV positive under HAART. HIV+PostART, presence of HIV under HAART; OPC, oropharyngeal candidiasis; clinic; site of sampling; LSU, Louisiana State University; MCG, Medical College of Georgia. Reproduced with permission from Sci Rep. , Candida albicans (65 samples), Candida dubliniensis (48 samples), and Malassezia restricta (43 samples). The tree was split by stratifying the data into 8 clusters, which was required to preserve the 4 major groups; the other 4 minor clusters were dominated by Debaryomyces hansenii (1 sample), Epicoccum nigrum (2 samples), Candida tropicalis (9 samples), and Candida glabrata (12 samples). Candida culture on these samples , followed by C. glabrata (24%), C. parapsilosis (7%), C. krusei (6%), C. dubliniensis (5%), C. tropicalis (4%), and others (7%), with mixed species and non-C. albicans exclusively identified in 13% and 4% of subjects, respectively. The species breakdown was similar for both HIV-negative and -positive subjects. Of note, Chromagar does not definitively differentiate C. albicans and C. dubliniensis. Hence, compared with the DNA sequence, C. albicans and C. dubliniensis culture results were disparate , nonalcoholic beverage intake, diet, marijuana usage, illicit drugs, and frequency of oral sex.2 and a constrained ordination method  was used. This multivariate model identified five of the six variables as influencing the fungal community structure; age was excluded. 2 values with P values. It is notable that HIV-positive samples are shifted toward the C. albicans and M. restricta predominant clusters; samples from females, more missing teeth, and higher caries index are shifted toward the Candida-dominated clusters; and samples collected in the Medical College of Georgia clinic are shifted toward the C. dubliniensis and S. cerevisiae-dominated clusters. Within the HIV parameters, neither HIV load, CD4 cell count, nor HAART regimen  significantly affected the fungal community composition (data not shown).To determine the marginal contributions of the six potentially confounding factors, a stepwise model selection using adjusted RCandida-predominant clusters had higher fungal amounts than either the M. restricta- or S. cerevisiae-predominant clusters, and this result was confirmed by statistical testing . M. restricta or C. albicans predominant fungal composition . M. restricta and each of the other three clusters, but not between any of the other pairs. Significant PERMANOVA results can be caused by differences in beta dispersion, which was significantly lower for the M. restricta cluster (0.42) than the other 3 clusters .Further analyses of the major fungal clusters are shown in Candida species positively correlated with Firmicutes or Actinobacteria and negatively correlated with Fusobacteria, Proteobacteria, and Bacteroidetes.To determine associations between the fungal community composition and the bacterial community composition, Spearman correlation coefficients were calculated for fungal species and bacterial species that were more than 1% mean relative abundance. The calculations were done using both the relative abundance  or the aWe previously reported on the oral bacterial community in this present cohort using whole-mouth gargle/rinse sampling . The priSaccharomyces cerevisiae, Candida albicans, Candida dubliniensis, and Malassezia restricta, together with four minor clusters dominated by other species.In contrast to the number of bacterial species identified in any 1 individual  , the funC. dubliniensis-predominant cluster with the HIV-negative group. This finding is in contrast to previous culture data showing C. dubliniensis is more prevalent in HIV-positive individuals , only 5 had a significant impact on the oral mycobiome as a whole. These variables included the HIV under HAART, geographical site of sampling, sex, missing teeth, and caries index (determined by decayed and restored surfaces). All were also significant variables in the bacterial community analyses  and thusanalyses , is thatanalyses . RecogniEpicoccum and Alternaria more in HIV-positive versus Pichia and Fusarium more in HIV-negative individuals)  . The latviduals) , 23–25. uals) 23–. Likewisuals) 23–. Yet stuMalassezia restricta-dominated cluster showed significant differences from the other 3 clusters. As another method of studying bacterial-fungal interactions, we calculated correlations between the most abundant bacterial and fungal species. Fusobacteria, Proteobacteria, and most Bacteroidetes sp. were positively correlated with Malassezia sp. and negatively correlated with Candida sp. using either relative or absolute abundances. Actinobacteria and Firmicutes were positively correlated with Candida species, and their correlation with Malassezia sp. depended on which abundance measurement was used. Firmicutes and Actinobacteria are the major Gram-positive phyla, although Veillonella spp. are Gram negative, as a genus of the Negativicutes class. Hong et al. show a clear association of oral Candida sp. with Gram-positive aciduric bacteria and an association with dental caries, while oral Malassezia sp. showed an association with inflammophilic bacteria  in the microbiome of palatine tonsil of HIV-positive persons  served as the positive control. For subjects with OPC (n = 16), the lesions were dominated by C. albicans (94%) as expected, although mixed species were also identified 25% of the time and included C. glabrata, C. krusei, and/or C. tropicalis. Only one case was identified as non-C. albicans exclusively.Yeast were identified by plating samples onto Chromagar (CHROMagar Microbiology) and incubating them for 48 h at 37°C to observe colony growth. Initial species identification was assessed according to colony color. All rinse samples were screened for asymptomatic yeast colonization by plating 50 μl of each sample. Some patients were resampled and cultured for DNA was prepared from oral rinse samples with slight modifications of the QIAamp DNA blood minikit   InitiallTCG ATG AAG ARC GCA GC) and 28S_Ra (GTC TCG TGG GCT CGG AGA TGT GTA TAA GAG ACA GTA TGC TTA AGT TCA GCG GGT A), with the gene-specific portions of the sequences underlined. The primers contained a phosphorothioate bond at the 3′ end. The liquid handling was performed in a Biomek 4000 instrument (Beckman Coulter). In the procedure, 5.8S and 28S gene-specific primers with 5′ tails were used in an initial amplicon PCR, while dual indices and specific adapters were added by a second index PCR with a small number of cycles. Oral rinse DNA was adjusted to 5-ng/μl concentration and 2 μl of DNA was amplified in a 25-μl reaction with Accuprime Taq high-fidelity DNA polymerase . The cycling conditions were initial denaturation at 94°C for 2 min; 35 cycles of 94°C for 30 sec, 55°C for 30 sec, and 68°C for 1 min; and a final extension of 72°C for 5 min. Amplicons were purified with 20-μl AMPure XP magnetic beads (Beckman Coulter) and used as the template in the index PCR which used identical times and temperatures but for only 8 cycles. Sequencing was performed on the MiSeq platform with 2 × 300-base pair reads.Sequencing libraries were prepared by a variant of the Illumina 16S protocol  modifiedmake.contigs, mothur screen.seqs was used to select fragments larger than 150 bases with less than 10 ambiguous bases, and mothur trim.seqs was used to remove primer sequences  and primers Eub338F (ACT CCT ACG GGA GGC AGC AG) and Eub518R (ATT ACC GCG GCT GCT GG). The thermal steps were 98°C for 2 min followed by 45 cycles of 98°C for 5 sec and 64°C for 5 sec. Purified Candida albicans genomic DNA was used as the standard.Fungal DNA was measured by qPCR of the ITS2 region. The primers used were 5.8s-F (TCG ATG AAG ARC GCA GC) and the 28S reverse primer that was used for sequencing. The cycling conditions and instrument settings were the same as used for the bacterial qPCR. Purified Clinical data were collected and calculated as described .2) determined by PERMANOVA. Spearman correlations were determined with the rcorr function of the Hmisc package in R  . Clusterage in R . Signifihttps://code.osu.edu/beall-3/hiv-mycobiome.git.Distance-based redundancy analysis (db-RDA) was done using capscale (vegan). Variables were included in a stepwise db-RDA model selection process . The marginal significance of the remaining variables was tested by a permutation-based ANOVA for constrained ordinations . Only the clinical variables that were significant contributors to the variation in microbial community composition after adjusting for multiple comparisons  were entPRJNA530161.The sequence files generated in this study have been deposited in the NCBI Sequence Read Archive , associa