JCI, Barton et al. report that GUCY2C is enriched in neuropod cells and is involved with sensory nerve firing. Selective deletion of GUCY2C in mouse models suggests that defective GUCY2C neuropod–cell signaling underlies visceral pain. These studies introduce possibilities for dissociating the secretory and analgesic effects of GUCY2C agonism. Although further work remains, unveiling the role of neuropod cells is a major step in understanding visceral pain.Visceral pain associated with irritable bowel syndrome afflicts 15% of the US population. Although treatments are limited, guanylyl cyclase C (GUCY2C) agonists alleviate pain and constipation. Until now, it was assumed that the activation of GUCY2C and production of cGMP in enterocytes stimulated fluid secretion and reduced visceral sensation. The recent discovery that a subtype of enteroendocrine cells (EECs) known as neuropod cells synapse with submucosal neurons unveiled a pathway for communicating gut signals to the nervous system. In this issue of the  GUCY2C gene on intestinal mucosal cells and stimulate intestinal fluid secretion and motility to treat constipation-predominant IBS. GUCY2C is expressed on the apical surface of enterocytes throughout the intestine, and, upon ligand binding, converts guanosine triphosphate (GTP) to cyclic guanosine monophosphate (cGMP). cGMP increases intestinal fluid secretion by (i) activating cystic fibrosis transmembrane conductance (CFTR) and (ii) inhibiting the apical Na+/H+ exchanger 3 (NHE3). During the development of GUCY2C agonist drugs — e.g., linaclotide and plecanatide — for the treatment of constipation, it was noted that they also reduced visceral pain in both animal models and humans. Since cGMP is released from enterocytes and cGMP reduces the firing of nociceptive neurons, it was naturally assumed that both the secretory and nociceptive effects of GUCY2C agonists were mediated by enterocytes  , 2. IBS erocytes . HoweverAlthough cGMP is indeed released from enterocytes following GUCY2C activation, apical secretion greatly exceeds basolateral release . Thus, tEnteroendocrine cells (EECs) are scattered throughout the gut mucosa with their apical surface exposed to the lumen. In this orientation, EECs detect and respond to ingested nutrients by secreting hormones from their basal surface into the paracellular space, where the hormones are taken up by blood vessels to reach the systemic circulation. Although originally named for their production of hormones, recently, it has been recognized that EECs possess many neuron-like properties . When viJCI, reveal how GUCY2C agonists ameliorate visceral pain (Gucy2c was enriched in a subset of intestinal cells possessing neuropods and that these cells expressed both endocrine and neural markers consistent with neuropod cells, thus providing the initial hint that EECs might be a target for GUCY2C agonists. Using intestinal organoids that contained neuropod cells, the authors demonstrated that sensory neurons isolated from dorsal root ganglia grew toward and connected with GUCY2C enriched cells. In this in vitro model, the GUCY2C agonist linaclotide elevated the DRG-neuron rheobase and reduced action potential firing, whereas cGMP administered to the solution bathing organoids and neurons did not affect excitability. Notably, the effects of linaclotide were not seen in –/–Gucy2c cells. These findings indicated that neuropod cells mediated the linaclotide effects on sensory neurons through a mechanism that could not be reproduced by exogenous cGMP (Capitalizing on these observations, Barton et al., in the current issue of the ral pain . First, ous cGMP .–/–Gucy2c mice exhibited spontaneous visceral pain that was refractory to linaclotide. The selectivity of this phenomenon for neuropod cells was demonstrated by deletion of Gucy2c only in CCK cells. These animals also developed spontaneous visceral pain (Importantly, ral pain .Gucy2c in neuropod cells provided an initial clue, and linking agonist stimulation to a reduction in sensory nerve firing provided mechanistic explanations for both the pain and therapeutic response to GUCY2C-targeted drugs (Thus, GUCY2C agonists ameliorated visceral pain through neuropod cell signaling . Not onled drugs . It is ied drugs , or neurCyclic GMP is the only second messenger signaling pathway known to be activated by GUCY2C. The observation that, in neuropod-connected DRG neurons, action potential firing was suppressed only through GUCY2C activation of neuropod cells and not by exogenous cGMP  raises tGucy2c only in CCK cells was sufficient to induce spontaneous visceral pain (Although CCK cells are a major EEC type, they represent only a subset of all EECs . It is sral pain . This fiCCK cells are also concentrated in the proximal small intestine. Even though the colon is a site of IBS pain, GUCY2C agonists exert their pain-reducing effects by targeting cells in the small intestine , 18. TheGUCY2C agonists ameliorate constipation and IBS pain; however, IBS has multiple manifestations and is commonly associated with diarrhea. Even the same patient may alternate between constipation and diarrhea. Because of their intestinal secretory effects, linaclotide and plecanatide are not used for diarrhea-predominant IBS. It is not known if the pain associated with diarrhea-predominant IBS has the same pathophysiological basis as constipation-predominant IBS, i.e., neuropod GUCY2C–signaling deficiency. However, based on the effects of linaclotide on visceral pain in mice it is tempting to think GUCY2C agonists could reduce pain in IBS regardless of its association with diarrhea or constipation. The discovery by Barton et al. provides a route to dissociate the effects of GUCY2C on pain from intestinal secretion . To avoi


Low platelet reactivity levels are associated with higher risk of bleeding in patients receiving dual antiplatelet therapy relative to patients with optimal platelet blockade. This study set out to evaluate the prevalence of low platelet reactivity in patients with acute myocardial infarction treated with ticagrelor and aspirin.®, PFA-100® with Innovance® PFA-P2Y cartridge and PFA-100® with Collagen/ADP cartridge. Platelet reactivity was assessed in the presence of peak levels of ticagrelor and defined according to previously validated cut-offs for each method . The level of significance was set at p<0.05.Patients admitted with acute myocardial infarction who were already undergoing dual antiplatelet therapy with aspirin and ticagrelor were enrolled. Blood samples were collected 1 hour before and 2 hours after the maintenance dose of ticagrelor to investigate trough and the peak effects of the drug respectively. Platelet reactivity was measured by three methods: Multiplate®/Innovance®PFA-P2Y: 86%; Multiplate®: 74%; PFA-100®/Collagen/ADP: 48%; p<0.001).Fifty patients were enrolled (44% with ST-elevation). Median duration of DAPT was 3 days . On average, peak and trough platelet reactivity were markedly low and did not differ between different methods. Low platelet reactivity was common, but varied according to analytic method (PFA-10012 inhibitors.Low platelet reactivity was very common in patients with acute myocardial infarction submitted to dual antiplatelet therapy with ticagrelor and aspirin. Findings of this study justify the investigation of less intensive platelet inhibition strategies aimed at reducing the risk of bleeding in this population, such as lower dose regimens or monotherapy with P2Y Several clinical trials have shown that DAPT with aspirin and ticagrelor or prasugrel is superior to DAPT with clopidogrel in reducing thrombotic and ischemic events, and is the current recommendation for patients with ACS.,3 However, it is widely recognized that the protective effect of DAPT with newer P2Y12 antagonists against thrombosis is achieved at the expense of an increasing risk of hemorrhagic complications.,5Dual antiplatelet therapy (DAPT) with aspirin and P2Y–8 In patients undergoing DAPT with clopidogrel submitted to percutaneous coronary intervention (PCI), low platelet reactivity  was associated with a two-fold higher risk of major bleedings, with no additional benefits in stent thrombosis reduction. In turn, patients with high platelet reactivity  had a three-fold increase in the risk of stent thrombosis compared to patients within the so-called optimal platelet reactivity window. The introduction of more potent P2Y12 inhibitors in clinical practice stemmed from the fact that DAPT with aspirin may induce insufficient platelet inhibition and have limited clinical benefit. Patients undergoing DAPT with new P2Y12 antagonists have an increased risk of bleeding due to over-inhibition of platelet function. However, the anti-platelet effects of DAPT with new P2Y12 agents are poorly understood, especially in real-world treatment of patients with ACS.Compared to older drugs, such as clopidogrel, the modern prasugrel and ticagrelor have been shown to have faster and more profound anti-platelet effects, with lower intra and interindividual variability.i.e., low platelet reactivity) in a real-world population of individuals with myocardial infarction treated with coronary intervention.To evaluate the therapeutic levels of dual antiplatelet therapy with ticagrelor and aspirin and to determine the frequency of over-inhibition of platelet function  , in compliance with the declaration of Helsinki. A signed informed consent was obtained from all patients. From March 2016 to August 2018, 50 individuals with acute myocardial infarction (MI) diagnosed in the emergency care unit within six hours of symptom initiation were consecutively enrolled. Inclusion criteria included diagnosis of MI according to international guidelines , treatment with a loading dose (180mg) of ticagrelor followed by the standard maintenance dose (90mg bid) combined with aspirin (100mg/day) and invasive coronary intervention. Therapeutic strategies and P2Y12 receptor inhibitors were selected by the medical team, which was unrelated to this study. Exclusion criteria were as follows: previous use of oral anticoagulants, active bleeding, previous hematologic disorder, use of thrombolytic agents, active infection, cardiogenic shock, renal failure requiring dialysis, use of non-ticagrelor P2Y12 and/or GPIIbIIIA inhibitors.This was a prospective, single-center observational study approved by the ethics committee of ® analyzer, Roche, Germany) and the PFA-100® system  with Innovance® PFA®P2Y and PFA®Collagen/ADP cartridges used according to manufacturer instructions. Tests were performed using quality laboratory practices.Platelet function tests of the 50 patients included in this study were carried out using multiple electrode aggregometry (MEA) ADP (adenosine diphosphate)-induced . Patients were only followed while hospitalized. Metabolic and hematological laboratory test results were obtained from medical records.To assess platelet reactivity at 2 time points (highest and lowest bioavailability), whole blood samples were collected by venipuncture 1 hour before and 2 hours after (trough and peak level respectively) the morning maintenance dose of ticagrelor (90mg). Samples were immediately sent to the clinical laboratory and processed within 2 hours of collection, in compliance with platelet stability times in whole blood.® analyzer assays were processed using the ADPtest® agonist, which promotes platelet activation via ADP platelet receptors and is sensitive to P2Y12 receptor inhibitors, using 6.5µM as the final ADP concentration. Low platelet reactivity was defined as platelet reactivity less than 19 AUC in the Multiplate® assay. This cut-off was recommended by two international expert consensus statements,14 and supported by data from a collaborative analysis of more than 20,000 patients treated with DAPT and coronary angioplasty.Multiplate® system, samples were processed using a hematology analyzer for quality assessment (testing is highly influenced by hematocrit level and platelet count). Approved samples were inserted into cartridges without blistering. Platelet function analysis is performed automatically on a closed platform and assessed by occlusion time. There is no consensus regarding therapeutic range or bleeding risk threshold for PFA-100® test results for both cartridges. According to Breet et al. only patients with occlusion time longer than 299 seconds (PFA®P2Y) or 116 seconds (Collagen/ADP cartridge) are defined as LPR patients.For the PFA-100t-test were used to compare continuous variables between groups. Trough and peak of platelet reactivity measured using different methods were compared using the McNemar test. Baseline clinical parameters were compared between normal and LPR patients using the χ2, the Fisher’s or a likelihood ratio test. All statistical tests were bilateral. The level of significance was set at p<0.05. Statistical analyses were performed using IBM-SPSS for Windows, version 22.0.Continuous variables were expressed as mean±standard deviation (SD) or medians and interquartile ranges, as appropriate. Categorical variables were described as absolute and relative frequencies. The Mann-Whitney test and the paired Student’s ®, are shown in A total of 50 patients were enrolled . All patients were submitted to coronary angioplasty and received drug eluting stents. The median time from ticagrelor therapy initiation to blood collection was 3 days . Clinical characteristics of patients with and without LPR at peak drug activity, as measured by Multiplate® , PFA®P2Y  or PFA®Collagen/ADP  .Peak and trough platelet reactivity were markedly low and did not differ significantly when measured using Multiplatep=0.347) . The pre® and PFA®P2Y relative to PFA® Collagen/ADP   .The main finding of this study was that patients with MI undergoing DAPT with ticagrelor and aspirin have a high prevalence of LPR and are therefore at a high risk of bleeding, with no theoretical gain in terms of protection from ischemic events. Bleeding complications are strongly associated with death in ACS patients and the risk of bleeding during treatment with antiplatelet drugs may be higher in more vulnerable subgroups .,18 A collaborative analysis of 17 studies with 20,839 patients submitted to DAPT and coronary angioplasty (97% treated with clopidogrel and 3% with prasugrel) revealed that 20% of patients had LPR . Importantly, when compared to patients with optimal platelet reactivity , this subgroup had a two-fold higher in-hospital PCI-related risk of bleeding  without further reduction of ischemic events.Prior studies have shown that platelet reactivity can be used to stratify the risk of thrombotic and/or hemorrhagic events in ACS patients receiving with DAPT.® or the PFA®P2Y method was similar. However, when measured using PFA® Collagen/ADP, the prevalence was approximately 30% lower. Importantly, different from prior studies,,8 this analysis included a contemporary approach to evaluate P2Y receptor inhibition by ticagrelor (Multiplate®) and adopted an evidence-based cut-off for hemorrhagic adverse event prediction in patients submitted to coronary angioplasty.In this study, the prevalence of LPR measured using the MultiplateµM and ADP 5µM]; vasodilator-stimulated phosphoprotein (VASP) and VerifyNow). Of note, mean values of platelet reactivity measured using the VerifyNow assay in patients treated with ticagrelor (peak drug activity) were approximately 31 PRU. Such values are far lower than the cut-off value (<85 PRU) that was subsequently recommended by two international expert consensus statements.,14 Moreover, in a PEGASUS-TIMI 54 substudy involving stable coronary artery disease patients with history of MI, platelet reactivity was tested after administration of different doses of ticagrelor (60mg and 90mg) and compared with placebo. In that study, patients also received aspirin. Patients treated with ticagrelor had very low levels of platelet reactivity (from 20±19 to 29±39 PRU) regardless of dose.The prevalence of LPR in MI patients treated with ticagrelor and aspirin in this study are in keeping with findings of other small trials using different laboratory methods in patients with acute or stable coronary disease. In a substudy of the PLATO trial, which investigated platelet responses in MI patients treated with clopidogrel or ticagrelor combined with aspirin, platelet reactivity was lower in the ticagrelor arm, regardless of measurement method . Patients treated with ticagrelor had a lower incidence of ischemic cardiovascular events. However, the risk of major bleeding was significantly (2.3 fold) higher in these patients relative to those treated with placebo.12 receptor inhibitors suggests DAPT with ticagrelor may carry an increased risk of bleeding. In a meta-analysis comparing ticagrelor and prasugrel, prasugrel was associated with lower myocardial infarction rates  and significantly lower bleeding rates , in spite of similar impacts of both strategies on other ischemic outcomes and mortality. In an Swedish observational study comparing clopidogrel and ticagrelor use in 45,206 cardiac patients, ticagrelor was associated with a lower risk of combined ischemic outcomes . However, the rate of in-hospital PCI-related bleeding events was significantly higher .From a clinical perspective, evidence from randomized and observational studies comparing ticagrelor with other P2Y Results presented beg the question of whether less intensive platelet inhibition strategies based on modern P2Y12 receptor antagonists might attenuate LPR and possibly reduce the risk of bleeding without increasing thrombotic events in this clinical setting. For instance, findings of this study may inform future clinical trials testing monotherapy with ticagrelor  in patients submitted to coronary interventions.Therefore, in keeping up with existing evidence, this study demonstrated a confirmed marked platelet inhibition by DAPT with ticagrelor and aspirin in MI patients. Findings also revealed similar LPR levels before and after the morning dose of ticagrelor, suggesting a significant residual inhibitory effect even at trough blood concentrations. High levels of platelet aggregation inhibition may be explained by the direct effect and potent reduction of thromboxane A2 and other platelet agonists by ticagrelor combined with the irreversible antiplatelet effect of acetylsalicylic acid (ASA).,23 Combined with current evidence, these findings suggest monotherapy with P2Y12 inhibitors following short-term DAPT is safe and may reduce the risk of bleeding, regardless of patient age.,25 A recent small trial with aspirin-free prasugrel monotherapy immediately after angiography and coronary stent implantation in stable patients also suggested this strategy is safe and not conducive to stent thrombosis.Importantly, a number of recent trials have compared the effects of DAPT with monotherapy with ticagrelor in ACS. As an example, two clinical trials compared ticagrelor monotherapy after short-term DAPT with conventional DAPT after PCI. Patients submitted to monotherapy with ticagrelor had a lower risk of major bleeding, with no evidence of increased ischemic risk relative to patients receiving conventional DAPT.,14 Although current data are derived primarily from trials with clopidogrel and aspirin combination therapy, the existing body of evidence and the expert consensus suggest potent P2Y12 receptor inhibition is associated with an increased risk of bleeding in patients submitted to DAPT. Importantly, this piece of information may not apply to alternative antiplatelet strategies implemented after coronary interventions, such as monotherapy with ticagrelor, for which different cut-off values for outcome prediction remain to be determined.This study has important limitations. It is a single center study with a small sample of patients. Therefore, clinical characteristics associated with LPR may have been underestimated. One example is the numerical difference in age between patients with and without LPR, which lacked statistical significance. A larger cohort of patients might have settled this matter. Two platelet function tests based on different methods and with different particularities were used to detect responsiveness to ADP receptor inhibitors. Results obtained with different methods are expressed in units of measurement that cannot be numerically compared. However, all tests indicated a high rate of LPR in the target population. Cut-off values used to define LPR measured by PFA methods are not supported by clinical evidence regarding bleeding prediction in patients with coronary artery disease. Nevertheless, high levels of LPR were detected not only by PFA, but also by Multiplate testing, according to cut-off values recommended by international expert consensus and supported by robust clinical data in patients submitted to coronary interventions.In conclusion, patients with acute myocardial infarction submitted to invasive management and therapy with ticagrelor and aspirin had a high prevalence of low platelet reactivity. Findings of this study justify future clinical trials testing less intensive platelet inhibition strategies with potentially lower bleeding risk in this clinical setting, such as low dose regimens or monotherapy with ticagrelor.


With the rate of oropharyngeal cancer on the rise, appropriate surgical management is an increasingly important consideration. Much debate currently exists regarding the necessary extent of neck dissections when performing curative surgery for primary oropharyngeal malignancies. Here, we present the case of a 64-year-old patient with p16+ T1N1M0 squamous cell carcinoma (SCC) of the right tonsil. Approximately four years following transoral robotic surgery oropharyngectomy and ipsilateral level II-IV right selective neck dissection, metastatic SCC was discovered on fine-needle aspiration biopsy of a right perifacial lymph node (level Ib). The patient then underwent a revision right neck dissection at levels Ia and Ib. Adjuvant immunotherapy was recommended following revision neck dissection. Postoperative imaging and flexible laryngoscopy three months after surgery were not concerning for cervical lymphadenopathy or oropharyngeal lesions. Although rare, physicians must maintain a healthy level of suspicion for recurrence to level Ib in oropharyngeal primary malignancies. The tonsil is the most common site of oropharyngeal cancer, with 23.1% of oropharyngeal malignancies arising at this site [Management of neck metastasis is an important part of the treatment plan for tonsillar squamous cell carcinoma (SCC) given that the presence of neck metastasis is a significant prognostic factor and is associated with a worsening in survival . CurrentWe present a case report of a patient who was previously treated with transoral robotic surgery (TORS) and SND of levels II-IV with delayed recurrence in a level Ib perifacial lymph node that appeared about four years after the initial surgery.The patient is a 64-year-old male former smoker with a history of p16+ pT1N1M0 right palatine tonsil SCC metastatic to the right neck. The patient underwent a TORS tonsillectomy and right limited oropharyngectomy as well as right SND at levels II-IV after diagnosis. The final pathology of the right opharyngectomy specimen revealed a p16+ 1.9 cm primary tumor without perineural invasion or lymphovascular invasion. Margins of the specimen were focally close (<1 mm) but negative for carcinoma. A single lymph node (2.3 cm) out of 23 submitted from the right neck dissection was positive for disease without extranodal extension. After a multidisciplinary tumor board discussion, observation without adjuvant therapy was recommended, and a subsequent postoperative positron emission tomography-computed tomography (PET-CT) was negative for recurrent disease one year after the operation. The patient was also diagnosed with stage III primary kidney cancer and underwent right radical nephrectomy two years following oropharyngectomy and right neck dissection.The patient had a long-standing palpable perifacial lymph node overlying the mandibular body that was first noticed following a dental infection. Computed tomography (CT) with contrast of the neck revealed a heterogeneous enhancing 1.2 cm nodule adjacent to the right mandible Figure . AlthougAdditionally, the patient completed a course of stereotactic body radiation therapy of his right acetabulum for metastatic renal cell carcinoma in the interval between repeat lymph node biopsy and metastatic lymph node removal.The patient underwent revision right neck dissection at levels Ia and Ib, including resection of the perifacial lymph node, shortly after the biopsy. Complications included the sacrifice of a distal right marginal mandibular nerve branch due to adherence to the tumor without a clear tissue plane. Pathology of the perifacial lymph node returned as poorly differentiated SCC without extranodal extension. All other cervical nodes were negative for malignancy (0/8). After a second tumor board discussion, adjuvant immunotherapy using pembrolizumab without radiation therapy was recommended given the patient’s history of metastatic renal cell carcinoma.Postoperative CT of the neck three months after the revision neck dissection revealed no additional cervical lymphadenopathy, and flexible laryngoscopy was without concerning oropharyngeal findings.While alcohol and tobacco are well-known risk factors for oropharyngeal cancer, HPV can now be identified in more than 70% of new cases of oropharyngeal cancer in the United States . Oral HPAlong with the prevention of oropharyngeal cancer, effective treatment is critical in reducing patient morbidity and mortality. Current National Comprehensive Cancer Network (NCCN) Guidelines include SND of level II-IV when performing surgical treatment with curative intent for lateralized oropharyngeal carcinoma, which is the initial treatment this patient received . The incTo determine whether additional level I dissection needs to be performed, several studies have examined the rate of metastasis to level I from oropharyngeal primary sites. Lim et al. found that only 10% out of 68 patients who underwent therapeutic ipsilateral neck dissection had positive lymph nodes at level I . These fStudies have also examined the rate of recurrence to level I lymph nodes after primary lesion removal and SND in patients with oropharyngeal SCC, which reflects the scenario presented in this case. In a study of 58 patients with tonsillar SCC treated with neck dissections and with/without adjuvant radiation treatment, only one patient who had undergone level I-V dissection for T2N0 tonsillar SCC developed recurrent carcinoma in ipsilateral level I . Lim et This case report of an unusual recurrence to a level Ib perifacial lymph node from a tonsillar primary SCC highlights a unique pattern of metastasis from an oropharyngeal primary site. Several studies have determined that the rate of metastasis and recurrence after treatment to level I lymph nodes from primary oropharyngeal SCC is very rare; therefore, it is likely not necessary to include level Ib in the initial SND when performing curative surgery for oropharyngeal malignancy. Although infrequent, this case shows that physicians must maintain an appropriate level of suspicion for metastasis from oropharyngeal primary malignancies to level Ib lymph nodes.


Megakaryocytes (MKs) are multifunctional hematopoietic cells that produce platelets, serve as components of bone marrow (BM) niches that support the development of hematopoietic stem and progenitor cell (HSPC) and provide inflammatory signals. MKs can dynamically change their activities during homeostasis and following stress, thereby regulating hematopoietic stem cell (HSC) function. Myelofibrosis (MF) is a progressive chronic myeloproliferative neoplasm (MPN) characterized by hyperactivation of JAK/STAT signaling and MK hyperplasia, which is associated with an aberrant inflammatory signature. Since JAK1/2 inhibitor alone is incapable of depleting the malignant HSC clones or reversing BM fibrosis, the identification of mechanisms that cooperate with MF JAK/STAT signaling to promote disease progression might help in developing combination therapies to modify disease outcomes. Chronic inflammation and MK hyperplasia result in an abnormal release of TGFβ1, which plays a critical role in the pathobiology of MF by contributing to the development of BM fibrosis. Dysregulated TGFβ signaling can also alter the hematopoietic microenvironment supporting the predominance of MF-HSCs and enhance the quiescence of the reservoir of wild-type HSCs. Upregulation of TGFβ1 levels is a relatively late event in MF, while during the early pre-fibrotic stage of MF the alarmin S100A8/S100A9 heterocomplex promotes pro-inflammatory responses and sustains the progression of MF-HSCs. In this review, we will discuss the recent advances in our understanding of the roles of abnormal megakaryopoiesis, and the altered microenvironment in MF progression and the development of novel combined targeted therapies to disrupt the aberrant interplay between MKs, the BM microenvironment and malignant HSCs which would potentially limit the expansion of MF-HSC clones. JAK2), calreticulin (CALR), and myeloproliferative leukemia virus oncogene (MPL) resulting in constitutive activation of JAK-STAT signaling (Myelofibrosis (MF) is a clonal blood cancer originating at the level of the hematopoietic stem cell (HSC) which is characterized by the acquisition of specific MPN driver mutations in janus kinase 2  fibrosis (reticulin and/or collagen deposition), progressive splenomegaly, systemic symptoms, anemia and thrombocytopenia. Despite MF has being considered a chronic disease, the median expected survival is 5.9 years .Abnormalities in megakaryocyte (MK) numbers and morphology are considered the common feature of the MPNs. MK morphology plays a critical role in distinguishing MF from other types of Philadelphia-negative MPNs such as polycythemia vera (PV) and essential throbocythemia (ET) , 4. MK iAlterations in MK maturation lead to excessive release of pro-inflammatory cytokines and growth factors, which alter the hematopoietic microenvironment in a manner that supports MF stem/progenitor cell (HSPC) predominance and progression to MPN-blast phase. MKs are an important source of transforming growth factor β (TGFβ1). TGFβ1 has been implicated in the alteration of HSCs regeneration during stress conditions . TGFβ1 iIn this review, we will discuss the contribution of MKs in promoting inflammation by releasing factors that sustain the proliferation of MF malignant clones and alter the hematopoietic microenvironment.Aberrant megakaryopoiesis is a pathological hallmark of each of the MPNs . SeveralJAK2V617F mutant MF HSPCs are biased toward the MK lineage with the CD41 MK marker being expressed by both CD38+ and CD38- fractions of CD34+ cells as compared to the healthy donors  in an MF murine model, have implicated GATA1 as an important player in the progression of the MF (Anemia and thrombocytopenia due to ineffective erythropoiesis and megakaryopoiesis characterize patients with advanced MF . Megakarf the MF  impairinf the MF . Mice thhigh), are characterized by a decrease of GATA1 in MKs and simultaneously by an increase of TGFβ1 content in MKs contributing to the development of marrow fibrosis  signaling leads to a deficiency of GATA1 expression contributing to impaired MK maturation . Similarfibrosis , 23, 37.fibrosis . The mecHSCs are predominantly quiescent but they can be rapidly recruited into cell cycle by stress such as chemotherapy, infection, inflammation or bleeding. In MPNs, HSC mutations induce a selective growth advantage over normal HSCs due to MPN-HSCS being more responsive and sensitive to microenvironmental signals. The expanded abnormal myeloid clones, especially MKs and monocytes perturb the bone marrow (BM) microenvironment promoting a further self-reinforcing malignant niche that favors clonal amplification of MPN-HSCs at the expense of normal HSCs and eventual egress to the spleen and liver .Cytokine overproduction in MPN mouse models has been found to be dependent on maintenance of enhanced NF-κB-dependent gene expression by BET bromodomain proteins  and on Jreg) that inactivate helper T cells by inducing transcription factor FOXP3  that induces T cell exhaustion by binding to the PD-1 receptor , 43. Theor FOXP3  which isfunction . Since Tactivity . This practivity . Accordiactivity . Moreoveactivity –55.MKs are a cellular component of the niche that contributes to the maintenance of the BM HSC pool size by promoting HSCs quiescence due to TGFβ and CXCL4 signaling , 14. TGFlow mice (JAK2 by blocking TGFβ1-induced p57Kip2, GATA2, p21 expression and SMAD2 activation followed by a reduction of JAK2V617F mutated clones, suggesting that the specific TGFβ1/3 inhibition can restore normal hematopoiesis by allowing wild type HPSCs to exit quiescence (+JAK2V167F patients have been reported to be characterized by a high degree of heterogeneity with different genetic sub-clones and sub-fractions of MK progenitors transcriptionally similar to healthy donors that can play a role in the MF evolution and therapy response (JAK2V617F allele burdens and by additional mutations that could have altered the TGFβ response (During HSPCs regeneration, TGFβ1 signaling is transiently activated and experiments in mice indicate that blocking TGFβ1 signaling during stress conditions, accelerates HSC multi-lineage cellular reconstitution and delay the return of cycling HSCs to quiescence . TGFβ, dlow mice . More priescence . While tiescence . HSPCs fresponse . Our sturesponse . TGFβ haresponse . HoweverIn addition,TGFβ activates MSCs which participate in the amplification and differentiation of hematopoietic clones as a consequence of altered HSC-niche cross talk. In human and murine MF, MSCs as fibrosis driving cells are characterized by a significant upregulation of JAK/STAT and TGFβ signaling pathways.MSCs can be activated as well by inflammatory cytokines secreted by malignant HSC clones and once activated can become inflammatory imprinted and their activation causes an altered cross talk between HSCs and stroma within their BM niche . These dUltimately, MSCs are the cell population responsible for initiating BM fibrosis in both murine and human MF. Based on transcriptomic pathway analyses during the course of the disease, MF-MSCs pre-fibrotic can be distinguished from overt fibrosis MF-MSCs. More precisely, two distinct subpopulations in the MSC compartment have been identified with adipogenic and osteogenic signatures respectively during the pre-fibrotic stage and at the time of acquisition of the fibrotic phenotype . The preThe MPNs are clonal hematopoietic disorders with profound degrees of systemic inflammation. The release of inflammatory mediators by clonal hematopoietic cell populations remodels the HSC compartment promoting the emergence of disease and progression. It is crucial that future studies focus on better understanding how inflammation and innate immune pathways regulate the interactions of HSCs with the HSC niche both during normal and malignancy-associated hematopoiesis.Conventional treatments for MF are focused on controlling symptoms and have limited MPN-disease modifying potential. Due to the critical role that MKs play in MF, clinical trials that can more optimally target the dysregulated MKs are needed. Numerous trials are being developed to assess whether targeting the inflammatory pathways can modify MF disease progression. MF with advanced marrow fibrosis is associated with excessive production of fibrogenic, pro-inflammatory cytokines and cell contact mediated inflammatory activators, and therefore interruption of vicious inflammatory cycle with a single agent therapy seems unlikely. Therapeutic approaches targeting MKs might be more successful if used early on in the course of the disease preventing MK/MF HSC interactions as well as differentiation of pro-fibrotic stromal cell populations and their interaction with hematopoietic clones.LV and RH conceptualized and designed the outline for the manuscript, wrote, edited and approved the manuscript.This work was supported by the National Cancer Institute Program Project grant P01CA108671.The authors declare that the research was conducted with the financial support of Forbius who provided AVID200 and research support for the preclinical studies as well as partial support for the clinical trial of AVID200, which was used to assist with regulatory matters. Forbius did not influence the outcomes of these studies.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


SmnB/−2 mouse model of SMA. We have generated a triple mutant (TKO), the SmnB/−2; Ripk3−/−; Casp1−/− mouse. TKO mice displayed a robust increase in survival and improved motor function compared to SmnB/−2 mice. While there was no protection against motor neuron loss or neuromuscular junction pathology, larger muscle fibers were observed in TKO mice compared to SmnB/−2 mice. Our study shows that necroptosis modulates survival, motor behavior and muscle fiber size independent of SMN levels and independent of neurodegeneration. Thus, small-molecule inhibitors of necroptosis as a combinatorial approach together with SMN-restoring drugs could be a future strategy for the treatment of SMA.Spinal muscular atrophy (SMA) is a monogenic neuromuscular disease caused by low levels of the Survival Motor Neuron (SMN) protein. Motor neuron degeneration is the central hallmark of the disease. However, the SMN protein is ubiquitously expressed and depletion of the protein in peripheral tissues results in intrinsic disease manifestations, including muscle defects, independent of neurodegeneration. The approved SMN-restoring therapies have led to remarkable clinical improvements in SMA patients. Yet, the presence of a significant number of non-responders stresses the need for complementary therapeutic strategies targeting processes which do not rely solely on restoring SMN. Dysregulated cell death pathways are candidates for SMN-independent pathomechanisms in SMA. Receptor-interacting protein kinase 1 (RIPK1) and RIPK3 have been widely recognized as critical therapeutic targets of necroptosis, an important form of programmed cell death. In addition, Caspase-1 plays a fundamental role in inflammation and cell death. In this study, we evaluate the role of necroptosis, particularly RIPK3 and Caspase-1, in the  Survival motor neuron 1 (SMN1) gene located on chromosome 5q13  is a devastating neuromuscular disease characterized by low levels of the ubiquitously expressed Survival Motor Neuron (SMN) protein. The SMN protein is produced by the SMN depletion leads to the profound loss of motor neurons of the spinal cord . While we did not observe protection against motor neuron loss nor neuromuscular junction (NMJ) pathology, there was a significant increase in survival and motor function improvement in TKO mice compared to SmnB/−2 mice. Interestingly, we also observed larger muscle fibers without any significant changes in neurogenic muscle atrophy markers. Our study shows that necroptosis plays a role in muscle fiber size indicating an intrinsic mechanistic relevant in skeletal muscle development. Understanding this mechanism is important given the availability of small-molecule inhibitors of necroptosis which could be combined with SMN-restoring drugs as a novel approach in the early treatment of SMA.In seeking to understand the pathways that could lead to neuron and muscle death in SMA, we turned to investigate necroptosis, a disease process in which cell death and inflammation have been linked. Given the strong evidence of programmed cell death and inflammation occurring in SMA, we generated a mouse model to study the pathway by knocking out Smn+/− mice were crossed to SmnB/2B2 mice to obtain SmnB/+2 and SmnB/−2 animals maintained on the C57BL6/J background  were derived after multiple generations of crossing Smn+/−; Casp1−/−; Ripk3−/− and SmnB/2B2; Casp1−/−; Ripk3−/− mice on the C57BL6/J background. Establishment of the line was confirmed by genotyping the DNA extracted from mouse ear biopsies using multiplex PCR (see 2O), a purified dietary supplement. Additional animals were sacrificed at P9 or P19 for tissue collection and analysis.Mice were tattooed by Animal Care Facility staff between P4 and P6 for longitudinal assessment of survival, body weight change and motor function tests. Mice were weighed every 2 days. For longitudinal assessment of survival, mice were weaned at P21 into cages containing DietGel 76A  Righting reflex, (2) Pen test, and (3) Inverted mesh/wire grip test on the same day, every 2 days. A maximum of 30 s on the pen test and 60 s on the inverted mesh grip was considered the maximal threshold. Three measurements were taken in succession and the average was recorded for analysis using the lab's previously reported protocols  muscles were fixed for 24 h while livers, hearts, and spinal cords were fixed for 48 h in 1:10 diluted buffered formalin  at 4°C and then transferred to 70% ethanol at 4°C until processing. All samples were processed, sectioned and stained at the Department of Pathology and Laboratory Medicine (University of Ottawa) as previously described  was dissected and isolated under a dissection microscope according to protocol detailed in  positive motor neurons, lumbar spinal cords were dissected, postfixed in 4% paraformaldehyde overnight at 4°C, then prepared for cryosectioning as previously described  were counted. NMJs were quantified as either normal or displaying neurofilament accumulation, and endplates were noted as either occupied or unoccupied.Quantification of spinal cord and muscle histology was performed in a blinded fashion. N number refers to individual mice and is described in each figure legend. Representative images of H&E-stained sections were acquired using the ZEISS Axio Scan Z1 slide scanner for brightfield at different magnifications. Muscle fiber size was quantified using ImageJ. About 100–200 muscle fibers in randomly selected areas of the muscle were counted to ensure proper coverage. The area of each fiber was measured to calculate a mean fiber size for each animal. Quantification of motor neuron number and size was performed on a total of 5 sections separated by 10 sections each per animal from the H&E-stained lumbar spinal cord. To ensure proper counts, motor neurons were identified by location in the ventral horn, presence of a nucleolus, and cells having cross-sectional area >350 μtibialis anterior (TA), and hindlimb muscles in RIPA lysis buffer  in the presence of PMSF  and proteinase and phosphatase inhibitors (Abcam). Protein concentrations were determined using Pierce BCA Protein Assay kit (ThermoFisher). Thirty ug protein per sample was separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis. Proteins were transferred to a PVDF membrane  and blocked for 1 h at room temperature in 5% (w/v) skim milk in TBST. Membranes were then incubated in primary antibodies and secondary antibodies  according to the manufacturer's protocol. qPCR was performed in technical triplicates for each sample using mouse primers obtained from Bio-Rad targeting Atrogin-1 (Fbxo32), MuRF1 (Trim63) and Gapdh. Each qPCR reaction contained 300 ng of cDNA, 2x SsoFast EvaGreen (Bio-Rad), RNase/DNase-free water, and respective primers in a final volume of 20 μl. Three negative controls containing water instead of cDNA were included in every qPCR plate. Results were quantified using 2ΔCt−Δ method. Results were normalized to Gapdh.Total RNA from the TA muscle was extracted using Qiagen RNeasy Mini kit, quantified and reverse transcribed using RTpost-hoc test was used to distinguish differences between more than two groups when multiple comparisons were necessary. Matched two-way ANOVA with Geisser–Greenhouse correction was used with complete data sets. Matched two-way ANOVA with mixed effects model was performed where values were missing (in case of death) with Sidak's or Tukey's method for multiple comparisons depending on the characteristics of the dataset. Statistical tests were performed using GraphPad Prism V.9.0 . Significance was set at P < 0.05.All data were expressed as mean ± standard error of the mean. Survival data was represented with Kaplan-Meier survival curves and statistically significant differences between groups were evaluated using the Mantel-Cox test. One-way analysis of variance (ANOVA) with Tukey All data will be made available upon contacting the corresponding author.SmnB/−2 mice. This was achieved via genetic crosses of mice to introduce the SmnB2 allele onto the Ripk3−/−; Casp1−/− double knockout  and littermate double knockout  control mice with necroptotic pathway ablation , which is a macroscopic pathological feature   . In fact 45 days . The inc 45 days . To detesurvival .SmnB/−2 mice at P7 and P9 . We therefore employed a battery of age-specific tests tailored to follow motor function in our mice . Spinal 7 and P9 . In cont7 and P9 . However7 and P9 . This teSmnB/−2 animals (via choline acetyltransferase (ChAT) immunostaining, a specific marker of motor neurons . Further, we were unable to detect basal levels of these mediators nor their phosphorylated forms in tissue homogenates through western blotting. Our inability to detect any changes may be due to transient or temporal upregulation that we were unable to pick up given that our tissues were analyzed only at pre-symptomatic and symptomatic time points. Additionally, changes may be occurring at a cell-specific level that are difficult to pick up with analysis of whole tissue homogenates. Despite the lack of direct evidence in this model, it is relevant for the field to understand the impact of targeting necroptosis in a whole-body model.Since there is mounting evidence that p53 plays a role in motor neuron death in different SMA mouse models  mouse of the necroptotic pathway in the Ripk3 ablation in the mdx mouse model improved motor function, which was attributed to a reduction in muscle degeneration, inflammation, and fibrosis  findings of inflammation, (ii) dysregulation of muscle satellite cell homeostasis and (iii) increased apoptotic activity in SMA. While there appears to be no clear signs of muscle-intrinsic inflammatory processes, there is evidence of systemic inflammation. Reported inflammation across different tissues from SMA patients and mice  (grant number 575466), Muscular Dystrophy Canada, and Canadian Institutes of Health Research (CIHR) (grant number PJT-156379). LC was supported by a CIHR Vanier Canada Graduate Scholarship. M-OD was supported by Fredrick Banting and Charles Best CIHR Doctoral Research Award.SmnB/−2 mouse model. This COI is outside the scope of this study. The remaining 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.M-OD received honoraria and travel accommodations from Biogen for speaking engagements at the SMA Summit 2018 held in Montreal, Canada and SMA Academy 2019 held in Toronto, Canada. RK received honoraria and travel accommodations from Roche as an invited speaker at their global and national board meetings in 2019. RK and the Ottawa Hospital Research Institute have a licensing agreement with Biogen for the All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


HOX genes, which have roles in NHL development, as well as PAX5 - a master transcriptional regulator of B-cell fate. Subjects with higher risk scores showed higher regulatory T-cells, memory B-cells, but lower naïve T helper lymphocytes fractions in the blood. Future prospective studies will be required to confirm the utility of our signature for managing patients who are at high risk for developing future NHL.Lack of accurate methods for early lymphoma detection limits the ability to cure patients. Since patients with Non-Hodgkin lymphomas (NHL) who present with advanced disease have worse outcomes, accurate and sensitive methods for early detection are needed to improve patient care. We developed a DNA methylation-based prediction tool for NHL, based on blood samples collected prospectively from 278 apparently healthy patients who were followed for up to 16 years to monitor for NHL development. A predictive score was developed using machine learning methods in a robust training/validation framework. Our predictive score incorporates CpG DNA methylation at 135 genomic positions, with higher scores predicting higher risk. It was 85% and 78% accurate for identifying patients at risk of developing future NHL, in patients with high or low epigenetic mitotic clock respectively, in a validation cohort. It was also sensitive at detecting active NHL (96.3% accuracy) and healthy status (95.6% accuracy) in additional independent cohorts. Scores optimized for specific NHL subtypes showed significant but lower accuracy for predicting other subtypes. Our score incorporates hyper-methylation of Polycomb and  NHL is the 7th most prevalent type of cancer in the U.S. According to the National Institutes of Health, approximately 2.2% of men and women will be diagnosed with NHL at some point during their lifetime. It is estimated that 80,470 NHL cases will be diagnosed and 20,250 deaths will occur in 2022 in the US alone. The overall 5-year survival rate is around 70%1.Non-Hodgkin lymphoma (NHL) is a malignancy of the lymphoid system which can often spread to distal organs. If NHL is detected early, patients are more likely to have a favorable outcome since stage I patients have 84% 5 years survival; whereas Stage IV patients have 64% survival3, motivating the research of early molecular changes associated with cancer initiation. Despite significant efforts, the impact of biomarkers for early detection of cancer has remained limited. The International Lymphoma Epidemiology Consortium (InterLymph)5 aims to identify common as well as distinct risk factors for developing NHL among NHL subtypes. While the Consortium has identified genetic, environmental and demographic risk factors, currently there are no predictive models that can robustly quantify the risk of developing NHL. This limitation is partly due to the heterogeneity of NHL, which is a major challenge for developing such models. While identifying subjects at risk for NHL could aid in early diagnosis, to date there are no recommended screening tests for lymphomas. The typical scenario for early diagnosis of NHL is regular medical check-ups with attention to symptoms and known risk factors for NHL such as first-degree family history of NHL, age and autoimmune disorders. If NHL is suspected of causing patient symptoms, a biopsy of a swollen lymph node or other affected area may be performed.Early treatment intervention is crucial, as it is more difficult to treat patients with advanced disease. Previous studies had observed aberrant global DNA methylation in samples collected close to diagnosis of blood cancers6. Methylation of DNA at genomic CpG sites can regulate gene expression without changing the underlying DNA sequence. Since DNA methylation alterations can reflect genetic background, environmental factors, aging, Epstein-Barr virus, tissue composition, smoking and other lifestyle factors, these alterations could represent surrogate markers of underlying NHL risk factors. The association between aberrant patterns of DNA methylation and cancer development has long been recognized, including in blood malignancies8. In particular, Polycomb group proteins (histone-modifying enzymes involved in maintaining stem cell identity and orchestrating cellular differentiation during development) play a central role in malignant transformation of lymphomas10. In addition, DNA methylation is the only epigenetic modification that is stable enough to be measurable in archival tissue samples, allowing the exploitation of existing biobanks by correlating molecular profiles with health outcomes over the course of a lifetime.Detection of altered DNA methylation in liquid biopsies is a promising approach to detect cancer early11, especially for the study of complex diseases like NHL whose origin is not driven by one variable  but rather by many features (genes and gene-environment interactions)14.Harnessing quantification of genome-wide DNA methylation for developing predictive and prognostic models require mining large volumes of data involving complex interactions. Artificial intelligence algorithms and rigorously-defined statistical learning frameworks have become indispensable tools for biomarker discovery from genomics data generated by high-throughput technologies in population studiesHere we developed a computational framework to identify features associated with future (lead time up to 16 years) development of NHL, defining a predictive signature based on DNA methylation profiles from blood samples  and NFATC1 , MCL1  and the tumor suppressor BCL11B,  were amongst the genes which contain this predictive set of CpG loci  cells. CIBERSORTx is a computational framework that infers, in addition to cell fractions, cell-type-specific transcriptomes from RNA profiles of intact tissues. Here we extended the framework to estimate DNA methylation profiles of individual cell types within bulk profiles. Supplementary Table 19 with an independent signature (see methods section for details), resulting in high correlations between our estimates and those obtained by alternative methods except for eosinophils . Deconvolution using the Salas et al.18, library resulted in more reliable eosinophil estimates than those obtained from other libraries as reported in the original manuscript. Blood samples from the future-NHL group showed few differences from the control group, except for a trend towards higher memory B cells (P = 0.07) and T regulatory cells (P = 0.03) (Supplementary Table High-resolution mode). We used this capability to test if methylation patterns within cell types can identify biological mechanisms associated with increased risk of NHL. We identified significant pathways amongst differentially methylated genes  between future-NHL and controls identified by CIBERSORTx. Among others, pathways related to pluripotency of stem cells (q = 0.002), WNT signaling (q = 0.001) and G protein coupled receptor signaling (q = 0.01) in memory B cells and NOTCH signaling in neutrophils (q = 0.01). We identified lymphoma genes enriched in several cell types  indicating that abnormal DNA methylation from cell-specific DNA methylation profiles occurs at genes implicated in lymphoma prior to diagnosis of disease.We estimated proportions of immune cells in blood samples using CIBERSORTx22. The 385 CpG sites that comprise the EpiTOC clock are unmethylated in many different fetal tissue types, and since their methylation tends to increase with age  , which reflects a mitotic clock-like process encoding the number of cell divisions, has been used to relate DNA methylation alterations arising during cell division to disease riskp ≫ n). For subsequent analyses, we removed B cell chronic lymphocytic leukemia (BCLL) and multiple myeloma (MM), as these are distinct clinical entities from the remaining NHL types, after which 278 samples remained . We did not observe significant differences between misclassified and correctly classified subjects for other variables such as age, sex, memory helper T cells, cytotoxic CD8 memory T cells, and naïve B cells. Interestingly, the observed differences between misclassified subjects in epiTOC estimates (P = 5.7 × 10−4) revealed that true positives had higher epiTOC values than false negatives. We also observed that subjects with high epiTOC showed more significant hyper-methylation in CpGs from genes known to be important regulators of B lymphocyte development than the CpGs from subjects with low epiTOC values  and low epiTOC (epiTOC < = 0.18) within the training set .Most of the loci included in FLYP are found in CpG islands near the transcription start sites of genes, especially those with higher predictive importance (Supplementary Table P = 0.006). The higher memory B cell fractions in future-NHL samples suggests possible higher proliferation of B cells prior to NHL development. In addition, higher levels of NK cells in peripheral blood have been observed in newly diagnosed DLBCL patients as compared to controls26. Together, these analyses identify differences in blood immune composition associated with elevated NHL risk. However, use of artificial intelligence approaches applied to DNA methylation improve predictions above those associated with immune composition, age and sex, supporting the utility of FLYP.Models based on cell fractions, age and sex as features instead of CpGs yielded NHL predictions better than random  but less accurate than the CpG-based model. The future-NHL/high-epiTOC score and the EpiTOC values were positively correlated with inferred fractions (by deconvolution) of cytotoxic memory CD8 T cell, memory B cell, regulatory T cell, and NK cell fractions; and negatively correlated with neutrophil, naïve T helper cell, naïve cytotoxic CD8 T cells and naïve B cell fractions  to evaluate the predicted proportion of subjects at risk of developing NHL in the entire population (details in Supplementary Table P = 4.4 × 10−5). However, EpiTOC estimates alone did not yield accurate NHL predictions (56% accuracy).EpiTOC estimates from all current-NHL cohorts were significantly higher than those from the future-NHL cohort or current healthy controls , DLBCL , LYM  and FL  subtypes showed better performance from models built on other subtypes as training sets than MCL   with the development of the B-cell repertoire in the germinal center and the attributed origin of each NHL subtype  and repressed by polycomb proteins or the “ReprPC” chromatin state (FDR: 1.64 × 10−11) are larger than expected by chance   , BENPORATH_EED_TARGETS (targets of the Polycomb protein EED in human embryonic stem cells), BENPORATH_ES_WITH_H3K27ME3 (genes possessing the trimethylated H3K27 mark in their promoters in human embryonic stem cells), BENPORATH_PRC2_TARGETS (Polycomb Repression Complex 2 (PRC) targets identified in human embryonic stem cells, and TAVAZOIE_METASTASIS   Fig. . Most of−9) Fig. . Polycom−9) Fig. . The sigma) Fig. . OverexpLow levels of molecular signals, small effect sizes, population heterogeneity, and relatively small groups of subjects from long-term prospective studies are major challenges inherent in cancer prediction. Furthermore, some subjects might not have developed NHL-related molecular alterations at time of sample acquisition, hampering the classification task. We developed a peripheral blood-based DNA methylation signature (FLYP) based on 135 key CpG sites that can predict NHL development, with an accuracy of 85% for the future-NHL/high-epiTOC group and 78% for the future-NHL/low-epiTOC group. We built a comprehensive computational framework to condense the original DNA methylation data into a lower-dimensional feature space to develop the prospective signature, by studying differences in DNA methylation levels from blood samples between a future NHL group (lead time of 16 years) and healthy control group. Notably, FLYP performance was improved by stratifying the population into high-epiTOC and low-epiTOC individuals, reflecting accelerated mitosis of cells. Clearly this require validation in an external prospective cohort but nevertheless this observation was crucial to identifying a predictive model. We performed several tests to confirm that FLYP predicts mostly “control” or low-epiTOC in an independent population of 656 healthy subjects; and that it predicts patients who currently have NHL in tissue samples from four independent cohorts.31. It has been suggested that accelerated cell division could lead to epigenetic cellular heterogeneity and predispose the tissue to future neoplastic transformation34. The proliferation of these new clones could be responsible for the increased cellular proliferation and epigenetic changes observed in active NHL subjects. DNA methylation profiles may implicitly capture some of the complexity of cell heterogeneity (both cell abundance and molecular profiles) that is not captured when using cell type fraction estimates, facilitating good model performance.While immune cell fractions, age and gender have some degree of predictive power, the combination of epiTOC and FLYP outperforms the other models or a model based on epiTOC alone. An elastic net baseline model resulted in worse performance (81% accuracy for high epiTOC group and 68% accuracy for low epiTOC group) than Random Forests. The association between NHL risk and epiTOC is consistent with other studies reporting accelerated mitosis based on methylation in cancer tissue samples, cancer cell lines, pre-cancerous lesions and normal epithelial cells exposed to a major carcinogenHOX and PAX5 deregulated at the DNA methylation level in future NHL compared to controls. Overall, our study suggests a biologically informative DNA methylation signature that reflects B-cell deregulation before NHL diagnosis. This also supports the idea that the predictive features represent biological mechanisms leading to increased risk of NHL, rather than being passive markers of neoplasticity.Overrepresentation of the “ReprPC” chromatin state with hyper-methylation CpG sites in our DNA methylation signature indicates polycomb-mediated repression, which usually targets pro-differentiation genes in stem cells and cancer. Hyper-methylation potentially silences differentiation-related genes in effector lymphocytes, inducing a more undifferentiated, stem-like cell state. This hyper-methylation might be mechanistically relevant for NHL initiation, and lead to higher epiTOC values observed in active NHL/future NHL subjects. We also found B-cell lymphoma related key genes such as FLYP has the potential to be a convenient and clinically applicable risk score for predicting NHL using DNA methylation. Future prospective studies will be required to confirm its utility and incorporate these results into clinical practice, for example to recommend that people with high risk have more regular checkups. Since only <2% of CpG sites are covered by the Illumina HumanMethylation450k Beadchip, the predictive value of FLYP would likely be improved by generating new datasets using a sequencing-based methylation method. In addition to a predictive NHL signature, we provide our computational framework as a resource to identify top performing approaches across pre-processing, normalization, feature selection, data transformation, predictive models, deconvolution analyses and omics integration. This should also facilitate reproducible DNA methylation analyses using new data sets.In conclusion, we built a score based on DNA methylation from peripheral blood samples that can predict future (up to 16 years) development of NHL. This study demonstrates the potential for early detection to enable early treatment of NHL that may improve outcomes of patients.27 and the Northern Sweden Health and Disease Study (NSHDS)35. After providing written informed consent, blood samples were prospectively collected from subjects who were healthy at enrolment . Instances of NHL occurring during the study period (16 years) were identified through local Cancer Registries (loss to follow-up < 2%). This study was approved by the committee on research ethics at the relevant institutions in accordance with the Declaration of Helsinki of the World Medical Association. All participants provided written informed consent to take part in the study at recruitment.The study population is based on participants from two existing prospective cohorts: the Italian component of the European Prospective Investigation into Cancer and Nutrition For each NHL case identified within the two cohorts during follow-up, one random control was selected among all cohort subjects free of cancer at the time of diagnosis, matched by cohort, center, gender, date of blood collection (±6 months) and age at recruitment (±2.5 years). Participants diagnosed with disease within less than two years of blood sample collection were excluded.2 in EPIC- Italy and −80 °C in NSHDS). Careful evaluation of the data from a pilot study experimenting with the conditions of sample collection did not reveal any systematic influence of storage time on epigenomic profiles36.Buffy coats were isolated from the collected blood samples within 2 h of blood collection for both cohorts, and placed in long-term cold storage  for visualization and epigenetic clock calculations and M-values  for construction of predictive models. Because SNPs near the CpG site may alter methylation levels, we removed CpG at SNPs with a minor allele frequency >1% from the dbSnp database38. We also filtered out CpGs from sex chromosomes.Genome-wide analysis of CpG methylation was conducted on the Illumina HumanMethylation450k Beadchip. We used the “preprocessQuantile” function from the “minfi 1.32.0” R package to normalize the data. We corrected M values for batch effects  using the function “ComBat”17 in combination with a previously defined signature matrix based on epigenetic profiles to infer the proportions of cell-types present in the blood samples, and estimate the DNA methylation profiles for each immune cell-type. The cell composition was estimated using methylCIBERSORT (“FeatureSelect.V4” function) and a signature obtained from sorted cells from ref. 18, (GSE167998). We used the High-Resolution mode of CIBERSORTx to impute sample-level DNA methylation profiles using CpGs located in CpG islands and Wilcoxon rank sum test to identify differentially methylated CpGs in each imputed cell-specific DNA methylation profiles. Then, we used ToppGene20 and GoMeth39 for pathway analysis.We used the deconvolution algorithm CIBERSORTx19 and different data: purified normal blood cell sub-populations white blood DNA methylation data derived from granulocytes (12), CD8 + (cytotoxic T-lymphocytes) (6), CD4 + (cytotoxic T-lymphocytes), CD19 + (B-lymphocytes) (6), CD56 + (NK cells) (6), CD14 + (monocyte lineage) (6) and eosinophils40 as a sanity check of the first of the deconvolution involving estimation of cell fractions.The immune cell fractions inferred from deconvolution were compared with those generated from a different algorithm called EpiDISH based on Robust Partial Correlations21. EpiTOC correlates with chronological age to a lesser extent than other epigenetic clocks that aim to predict age 41 to further investigate whether pre-defined sets of genes from the MSigDB database (C2-curated gene sets and C7-immunologic gene sets) show statistically significant and concordant differences between the future NHL and control subjects.We identified CpG sites significantly associated with future development of NHL . We fit models using least squares regression on all CpGs separately, and accounted for multiple hypothesis testing using a 2.4 × 10−7 cutoff specific for DNA methylation based on permutations24.We first split the data into 70% training and 30% test, balanced for the three classes  distributions within the split, and then used linear regression models using limma from the ‘missMethyl_1.20.4’ R package44 with the same split as the univariate approach to select a subset of the CpGs. We efficiently maximized the amount of joint information relative to the status of future disease by identifying the top common features with non-zero coefficients.Then, we selected the common CpGs significantly associated with both high and low EpiTOC future NHL groups and performed regularization using elastic net  methodsWe built random forest classification model using the same training data from the split in the initial two-step feature selection. We assessed performance of the final model fit on the left-out test data. We used “Random Forest“ model from the “randomForest 4.6–14” R package, implemented in the R package “caret 6.0–86”. To train the models, three separate 10-fold cross-validations were used as the resampling scheme. We set to 30 the parameter that defines the total number of parameter combinations that will be evaluated (“tuneLength”). We tuned hyper-parameters to assess the model fit on subsets of the training dataset and we reported the results from the test set to assess the performance on an independent set.We assessed the performance of the classifier using the following metrics: accuracy (percent of correct classifications obtained), sensitivity , specificity (ability to correctly rule out the future disease in a disease-free population), F1 Score = 2 * Precision * Recall/ and AUC value of the resulting ROC curves.We also tested if the combination of gender, age, epigenetic age, epiTOC and proportion of cell-types or some of these variables alone had predictive power by training models using these variables as features, with future case/control status as the dependent variable.We evaluated our predictive model in four cohorts of subjects with current NHL, and in a cohort of 656 healthy subjects (at the time of blood sampling – no follow up is available), utilizing genome-wide DNA methylation data from the same platform as our prospective cohort were available (Supplementary Table 27. These include B-cell chronic lymphocytic leukaemia (BCLL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), (lymphoplasmacytic lymphoma) LPL, multiple myeloma (MM) and mantle cell lymphoma (MCL), heterogeneous category of B-cell malignancy and others (LYM) (Table NHL future cases were classified into subtypes according to the SEER ICD-0-3 morphology codesM) Table . We buil29 which were derived from primary mononuclear cells (E062) using 450k arrays.We investigated whether methylation signatures were statistically enriched for a comprehensive set of previously defined DNA chromatin features based on methylation profiles. We used the 15 chromatin states from the 111 epigenomes studyWe studied the location of the CpGs from the signatures and characterized the proportion of CpGs from islands, shores, shelves, and open seas.We calculated significance levels by comparing the distribution of chromatin states and genome location of the CpGs from the FLYP signature with 1000 lists of the same number of CpGs randomly selected from the entire genome.Further information on research design is available in the Supplemental MaterialData Set 1Data Set 2Data Set 3REPORTING SUMMARY


The molecular mechanisms that regulate stem cell pluripotency and differentiation has shown the crucial role that methylation plays in this process. DNA methylation has been shown to be important in the context of developmental pathways, and the role of histone methylation in establishment of the bivalent state of genes is equally important. Recent studies have shed light on the role of RNA methylation changes in stem cell biology. The dynamicity of these methylation changes not only regulates the effective maintenance of pluripotency or differentiation, but also provides an amenable platform for perturbation by cellular stress pathways that are inherent in immune responses such as inflammation or oncogenic programs involving cancer stem cells. We summarize the recent research on the role of methylation dynamics and how it is reset during differentiation and de-differentiation. The field of ‘Epigenetics’ has led to a “paradigm shift” in several domains of biomedical research . WaddingThe stem cells have been excellent cellular models to understand the molecular mechanisms of epigenetics. Stem cells are capable of self-renewal and differentiation to all three lineages, and can be classified as follows: a) Naïve stem cells , b) Primed stem cells/Epiblast stem cells (EpiSCs)  Embryonic stem cells (ESCs)  Adult stem cells (ASCs), found in adult tissues and organs within their respective niche responsible for maintaining tissue homeostasis, repair and regeneration. These stem cells remain in a quiescent state till activation by a signal like cell damage, and capable of self-renewal and multi-lineage differentiation potential, e) Cancer stem cells (CSCs) that are derived from the dedifferentiation of cancer cells or from the malignant transformation of normal stem cells. These cells like any other stem cells have self-renewal abilities and multi-lineage differentiation potential and play a major role in the prognosis of the disease .These unique characteristics of a stem cell are regulated by molecular mechanisms that involve transcription factors, signalling pathways, epigenetics and epitranscriptomics. Transcription factors such as Oct3/4, Sox2, c-Myc and Nanog bind to their target genes and regulate their expression . Many siDNA Methylation: A family of DNA methyltransferases (DNMTs), catalyzes the Methylation of cytosine’s fifth carbon position, leading to 5-methylcytosine (5 mC) formation. DNMT1 copies existing methylation patterns for inheritance during DNA replication, while DNMT3A and DNMT3B act as de novo methyltransferases to create new methylation patterns  (Nucleosomes of stem cells show a higher level of modifications marks that are involved in active gene expression such as histone H3 lysine four trimethylation (H3K4me3), histone H4 lysine 9 and 14 acetylation . The two methyl modifications on H3K4 and H3K27 form a bivalent chromatin mark which is seen in the chromatin of stem cells. In stem cells, the highly conserved non-coding elements (HNCE) were found to be enriched with bivalent histone modifications, an active chromatin mark, H3K4me3 and a repressive chromatin mark, H3K27me3 . These ms (HSCs) .Dnmt genes causes significant developmental defects and embryonic or early postnatal death . proteins . Hematopproteins . DNMT3a proteins . DNMT1 aogenesis . Deletioogenesis . The RNAogenesis .Cancer Stem Cells (CSCs) are a small subpopulation of cells within tumors, which are capable of self-renewal, differentiation, and tumorigenicity when transplanted into an animal host. CSCs can be distinguished from other cells within the tumor by differences in their cell division and gene expression . The firThe dynamicity of the methylation mark on DNA, histones or RNA serves as an important biochemical rheostat for regulating stem cell pluripotency and lineage commitment along with other regulatory factors . The revA meta analysis of the available datasets was done to assess the changes that occur during these stages, as shown in The epigenome modifiers such as Histone methyltransferases/demethylases, DNMTs/demethylases, RNA methyltransferases/demethylases also have dynamic expressions in the different cell types . In ESCsIn this context, the inflammatory pathway has been shown to be recognized as a major component of tumorigenesis in various cancers. Solid tumors are also associated with Tumor Associated Macrophages (TAM) which constitute various immune infiltrating cells. These TAMs and stromal cells secrete cytokines such as Interleukin 1(IL1), IL6 and TNFα acts in paracrine fashion for sustenance and reprogramming of CSCs, by altering epigenetic mechanisms and thereby regulating transduction pathways such as NFκB, STAT3 and SMADs. . These i


Inferior health outcomes for people of African and Afro-Caribbean ancestrycompared to those of European ancestry are well recognised. There is adisproportionate impact within these communities compared to other ethnic groupsincluding pregnancy outcomes, hypertension, kidney disease and diabetes. The‘Black Lives Matter’ movement has highlighted that it is imperative to examineall factors contributing to this inequity and to strive to explore multifacetedways, including social, economic, psychological and biological to improveoverall health equity. It is within this context that we discuss the novelfinding of Apolipoprotein 1 genetic polymorphisms which have been identified insome populations of African ancestry. We will explore the history andevolutionary advantages of Apolipoprotein 1 polymorphisms and thepathophysiology resulting from these adaptations and examine the impact ofApolipoprotein 1 on pregnancy outcomes, the risks and benefits of screening forhigh-risk Apolipoprotein 1 alleles in black communities and potential treatmentscurrently being investigated. Of all the forms of inequality, injustice in health is the most shocking and themost inhuman.Martin Luther King, 1966 hypertension, kidney disease or diabetes.24 The ‘Black Lives Matter’ movement has highlighted that it is imperative to examine all factorscontributing to this inequity and to strive to explore multifaceted ways, includingsocial, economic, psychological and biological to improve overall health equity. Itis within this context that we discuss the novel finding of Apolipoprotein 1(APOL1) genetic polymorphisms which have been identified insome populations of African ancestry. We will explore the history and evolutionary advantages ofAPOL1 polymorphisms and the pathophysiology resulting fromthese adaptations and examine the impact of APOL1 on pregnancyoutcomes, the risks and benefits of screening for high-risk APOL1alleles in black communities and potential treatments currently beinginvestigated.Inferior health outcomes for people of African and Afro-Caribbean ancestry comparedto those of European ancestry are well recognised. There is a disproportionateimpact within these communities compared to other ethnic groups with access to thesame health systems and standards of care for diverse conditions including pregnancy outcomes,APOL1 gene polymorphisms, which are present in people of Africanancestry, were only identified and studied during the last 10 years.8 The APOL gene cluster(APOL1-6) is found on chromosome 22 and is present solely in humans and few higher primates.APOL1 is the only APOL gene with signal peptide enabling bothcirculating and intracellular effects, and is expressed in a number of cell types including liver (the source ofsecreted circulating apolipoprotein-L1), placental and renal parenchymal cells but the absence of expression is not associated with a disease phenotype.APOL1 protein was due toprotection against Trypanosoma brucei species (including one whichcauses African sleeping sickness) through trypanolytic effect. Counter evolution ofserum resistance-associated protein by Trypanosoma bruceirhodesiense and T. b. gambiense-specific glycoprotein(TgsGP) by T. b. gambiense which both block the action ofAPOL1 is thought to have led to the development ofAPOL1 variants G1 (two missense variants: rs73885319 andrs60910145) and G2  to prevent parasitic evasion of apolipoprotein-L1  and are most prevalent in West Africa. Due to historical slavetrading, there is also a high prevalence of APOL1 risk alleles inAfrican-American populations.Homozygotes for G1 and G2 risk alleles or compound heterozygotes have an increasedrisk of kidney disease and progression to end-stage kidney disease (ESKD) see .6 ThesAPOL1 protein in its circulating form does not appear to impact ondevelopment of kidney disease. Studies of kidney transplant patients have demonstrated that recipients withAPOL1 risk alleles do not develop recurrent disease or showreduced graft survival when transplanted with APOL1 G0/G0 kidneys. Conversely, APOL1 G1/G2 donor kidneys transplanted intolow-risk recipients have higher rates of graft failure and reduced graft survivaltime.1618 Whilst exactpathophysiological mechanisms are still being elucidated, the expression ofAPOL1 mRNA in renal parenchymal cells, in particular podocytes,is associated with cell damage from membrane pore formation. This results in reduced podocyte density possibly also due to increasedsenescence and apoptosis mediated by APOL1 expression. However,this process does not invariably lead to kidney disease and it is hypothesised that rather than true Mendelian inheritance thesepolymorphisms cause disease via a ‘second hit’ mechanism.APOL1 related kidney disease presents with albuminuria withhistological changes consistent with focal segmental glomerulosclerosis (FSGS) on biopsy, but APOL1 risk alleles are also considered to underlie‘hypertensive nephrosclerosis’ in people of African or Afro-Caribbean ancestry. Manyof these patients may not receive a kidney biopsy but are known to progress morerapidly to ESKD even in the presence of tightly controlled blood pressure.2224Viral infections , drugs (interferon), low nephron mass (e.g. fromprematurity), diabetes and obesity are all potential ‘second hits’ to the kidneywhich may contribute to the development of clinical disease. MostAPOL1 risk alleles has been strongly associatedwith the development of chronic kidney disease (CKD) in several large cohortstudies.26 The odds ratios for development of FSGS and HIV-associatednephropathy (HIVAN) in those with risk alleles are 17 and 29, respectively, comparedto those without risk alleles . Previous cohort studies have suggested an increasedcardiovascular risk with risk APOL1 alleles, but the US Million Veteran study has demonstrated that augmented cardiovascular risk is likely minimal andmediated through vascular pathology associated with kidney disease rather thanAPOL1 genotype being an independent cardiovascular riskfactor.Conversely, there appears to be a survival advantage of  and APOL1 risk genotypes during pregnancy for some women may be contributory through diverse mechanisms.Pre-eclampsia risk, with associated maternal and fetal morbidity and mortality, isincreased in women of African ancestry,APOL1 G2 model study reported increasedrates of pre-eclampsia and decreased podocyte density with aging, despite notdeveloping overt kidney disease.APOL1 mRNA is strongly expressed in placental tissue and pregnancies with G2 homozygous mouse offspring had higher rates ofpregnancy complications, suggesting a role of an infant rather than maternalgenotype. These findings are aligned with previous clinical observations of paternalassociation with development of pre-eclampsia including in those of Africanancestry.36A novel transgenic mouse APOL1 risk alleles and adverse pregnancyoutcomes (see APOL1 G1 genotype in a dominant model. Two large USA cohorts of black women with pre-eclampsia could not demonstrate an association withmaternal APOL1 genotype due to low case numbers and non-significantresults. This may reflect a phenotypic difference between these black populationswhich has not been fully explored. The prevalence of high-risk fetalAPOL1 genotypes in the USA cohorts was two-fold higher inpregnancies complicated by pre-eclampsia compared to ethnically matched controls whodid not develop pre-eclampsia. Lower rates of maternal high-riskAPOL1 genotypes in women with pre-eclampsia compared to ratesin their fetuses, although not reaching statistical significance, may suggest afurther impact of discordant maternal/fetal genotypes. Prematurity secondary topre-eclampsia in infants carrying high-risk genotypes could be a ‘second hit’leading to a higher incidence of early onset, possibly more severe, kidney diseasein the offspring (40 Identification of these at-risk infants may provide a targetfor early screening and interventions to prevent or delay progression to ESKD.The relationship between omes see has beenffspring .39,40 Id As women more frequently delay pregnancy until older age, there is a furtherincrease in prevalent CKD as well as risk factors for its development, such asdiabetes, obesity and hypertension. Given the asymptomatic nature of CKD in earlystages, routine screening in pregnancy can identify diseases which may not otherwisehave been recognised including APOL1 related kidney disease. EarlyCKD is an independent risk factor for adverse pregnancy outcomes.43APOL1 renal disease is frequently associated with chronichypertension which is itself a risk factor for poor pregnancy outcomes. Furthermore, renal hyperfiltration which occurs as a physiological adaptationin pregnancy may further stress kidneys with previously unrecognised disease leadingto a pathological increase in proteinuria and acceleration of the renal functionaldecline.CKD is one of the most prevalent conditions in women of child-bearing age and isincreasing globally.APOL1 mediated kidneydisease, but several are in development. Standard management includes saltrestriction, fluid management with diuretics and renin-angiotensin-aldosteronesystem blockade. Response to steroids and other immunosuppression varies. Early diagnosis and initiation of highly active antiretroviral therapy forHIV may reduce or delay development of HIVAN in susceptible individuals.There is currently no targeted therapy for APOL1 mediated disease,screening for high-risk genotypes has not been explored. Although earlyidentification of CKD through ongoing surveillance of those at risk could slowprogression to ESKD and improve symptom management, screening is likely to becostly. In addition, there are concerns about impacts on health and life insuranceas well as a perceived sense of own health, particularly for those who do not have a‘second hit’ and develop disease. However, distribution of knowledge is importantand even if widespread screening is not offered potentially affected communities arekeen to raise awareness of the potential role of APOL1 risk allelesin the development of kidney disease.Screening for any genetically mediated health condition is costly, has severalinherent risks and is only justified when the early diagnosis can improve outcomes.In the absence of a direct therapy for APOL1 mediated renal diseaseis a priority that is being investigated in a current commercial Phase 2 trial byVertex Pharmaceuticals of a new medication to block APOL1 mediated podocyte damagein patients with primary FSGS, nephrotic level proteinuria and twoAPOL1 risk alleles. Other investigators are also exploringpotential treatments for FSGS, but without targeting the APOL1pathway.The development of targeted therapy for APOL1polymorphisms. Screening for risk alleles in women of African ancestry could enablebetter risk stratification and the role of aspirin prophylaxis could be explored.Furthermore, knowledge of APOL1 risk alleles in their offspring,particularly in those born pre-term could enable targeted preservation of reducednephron numbers.For maternity research, there remain opportunities for a greater understanding of themechanisms and associated risks in pregnancy from APOL1 there remain many unanswered questions and more studiesare needed to determine the role of APOL1 risk alleles in thepathophysiology, risk stratification and or/therapeutic target for both kidneydisease and management of pre-eclampsia.While the last decade has seen a rapidly advancing understanding of


Laryngeal squamous cell carcinoma (LSCC) is one of the highly aggressive malignancy types of head and neck squamous cell carcinomas; genes involved in the development of LSCC still need exploration.We downloaded expression profiles of 96 (85 in advanced stage and 11 in early stage) LSCC patients from TCGA-HNSC. Function enrichment and protein-protein interactions of genes in significant modules were conducted. Univariate and multivariate Cox regression analyses were performed to explore potential prognostic biomarkers for LSCC. The expression levels of genes at different stages were compared and visualized via boxplots. Immune infiltration was examined by the CIBERSORTx web-based tool and depicted with ggplot2. Gene set enrichment analysis (GSEA) was utilized to analyze functional enrichment terms and pathways. Immunohistochemical staining (IHC) was used to verify the expression of genes in the LSCC samples.UIMC1, NPM1, and DCTN4 in the module ‘cyan’, TARS in the module ‘darkorange’, and COPB2 and RYK in the module ‘lightyellow’ showed statistically significant relation to overall survival. The expression of COPB2, DCTN4, RYK, TARS, and UIMC1 indicated association with the change of fraction of immune cells in LSCC patients; two genes, COPB2 and RYK, indicated different expression in various tumor stages of LSCC. Finally, COPB2 and RYK showed high-expression in tumor tissues of advanced LSCC patients.We identified 25 modules, including 3 modules significantly related to tumor stages of LSCC via weighted gene co-expression network analysis (WGCNA). Our study provided a potential perceptive in analyzing progression of LSCC cells and exploring prognostic genes. Laryngeal squamous cell carcinoma (LSCC) is one of the most frequent types of head and neck squamous cell carcinomas (HNSCC) and is the fifth cause leading to tumor-related death worldwide . PatientBCL2L12 was reported to be a favorable prognostic tissue biomarker in patients with primary advanced-stage LSCC [CDKN2B-AS1 facilitates LSCC by regulating miR-497/CDK6 pathway and is associated with advanced clinical stage [Several publications reported molecular signatures associated with different stages of LSCC patients. age LSCC . The lonal stage . Patiental stage . The proTPX2, MCM2, UHRF1, CDK2, and PRC1 were found to hold a possible association with LSCC using WGCNA [Weighted gene co-expression network analysis (WGCNA) was frequently used to construct networks, detect module, and relate modules and genes to external information . It has ng WGCNA . HoweverIn this study, we aimed to find significant genes networks related to different stages of LSCC and explored novel genes with prognostic significance. Tumor stage related modules was detected using WGCNA; the functional enrichment analysis and protein-protein interaction (PPI) were performed to analyze function and interactions of genes associated with LSCC stages. Univariate Cox regression and multivariate Cox regression analysis were conducted to explore prognostic signature of LSCC; the immune response and gene set enrichment analyses were conducted to characterize the potential mechanism of genes related to various tumor stage of LSCC.https://portal.gdc.cancer.gov/projects/TCGA-HNSC), we downloaded expression profiles using ‘TCGA-HNSC’ as query using GDC data transfer tool (gdc-client); duplicated samples were discarded; HTSeq  counts of samples were downloaded from TCGA-HNSC; LSCC-related expression matrix in tumor tissues (96 samples) was collected for this research. HTSeq counts of RNA-seq data were transformed into the trimmed mean of M-values (TMM) with the edgeR package [limma package [Gene expression profiles were downloaded from the TCGA database; since LSCC patients were involved in HNSC (head-neck squamous cell carcinoma) project in TCGA ; the dissimilarity of TOM (dissTOM) was then calculated with TOM; a hierarchical clustering tree was conducted with ‘hclust’ function of WGCNA to depict the clustering of candidate genes; module of individual tree branches was identified with ‘dynamicTreeCut’ function; considering the potential existence of genes with similar expression among different modules identified by ‘dynamicTreeCut’, we evaluated the whole modules co-expression similarity by calculating the correlation between eigengenes and cluster and merged those potential modules with a correlation of 0.75. To gain larger modules, we set the minimum size of modules as 30. The correlation between identified modules and clinical traits was calculated and depicted through heatmap; the relation of Gene Significance (GS) and module membership (MM) was calculated to evaluate the association between significant genes and tumor stage in identified modules.To explore genes related to the tumor stage of LSCC; we separated LSCC patients into two groups (early: stage I/II and advanced: III/IV); differentially expressed genes (DEGs) analysis between two groups was conducted with the p-value and q value were set to 0.05 for analyzing significantly enriched terms.To explore the function of LSCC-related genes, we performed function enrichment analysis; considering the various roles of genes in different modules, we performed enrichment analysis for genes in each module, respectively. A R package clusterProfiler  was utilhttps://string-db.org/) and Cytoscape software (https://cytoscape.org/), respectively. Genes with similar expression in the same module might participate in certain roles in response to LSCC by forming gene clusters; we predicted gene clusters based on PPI with ‘MCODE’ function of Cytoscape with default parameters; the first cluster in each module was color-labeled.Interactions among genes in each module were predicted and visualized using STRING database  to assess multiple risk factors simultaneously and depicted the result with forest plot; we visualized prognostic significance of candidate genes with Kaplan-Meier (KM) plot.Univariate Cox regression was performed with survival and depicted with survminer package  to analyhttps://cibersortx.stanford.edu/) is a web-based tool for analyzing the proportion of different types of immune cells. The gene expression matrix of LSCC patients was submitted to CIBERSORTx to predict the fraction of immune cells; the distribution of immune cells among all samples was clustered and depicted via heatmap; we evaluated the potential distribution of immune cells in isolated LSCC patients in the early and advanced stages through PCA plots.CIBERSORTx  with a T-test; the variety of gene expression among relatively exact stages  was tested with Kruskal-Wallis method. We visualized the comparison with ggplot2.P-value was 0.05; top 3 enriched pathways were depicted with ‘gseaplot2’ function.In order to explore function of candidate genes, we calculated median of gene expression and separated LSCC patients into two groups according to the expression of candidate genes ; GSEA was performed with clusterProfiler package to analyze enriched pathways between two groups with different expression levels of candidate genes; the minimal size of the gene set was set to 50; the cut off of A total of 40 samples were collected from LSCC tissues removed from hospitalized patients at the Sir Run Shaw Hospital affiliated to Zhejiang University Medical College, China, from January 2019 to October 2021. All the patients were informed about the experiments and signed informed consent. The tissue-associated experiments were approved by the Medical Ethics Committee of Sir Run Shaw Hospital affiliated to Zhejiang University Medical College. The 40 samples comprised 12 samples of early LSCC patients and 28 samples of advanced LSCC patients. No patient received chemotherapy or hormone therapy before surgery .The expression of COPB2 and RYK in the tissue samples was explored by using immunohistochemical staining (IHC). IHC staining was performed according to the manufacturer’s instructions . Tissue P-value < 0.5 for network construction. The dendrogram of clustering samples indicated no obvious outliers. The correlation of samples and clinical traits was depicted in the clustering dendrogram; no significant separation of clustering samples was observed from the image according to gender or tumor stage , module ‘darkorange’ , and module ‘lightyellow’ —were related to tumor stage of LSCC   and 60483 genes by collecting and integrating expression matrix associated with LSCC; clinical summary of LSCC patients was descripted in Table Genes in module ‘cyan’ were enriched in 27 GO terms , mainly focused on the cellular components such as mitochondrial inner membrane, mitochondrial protein complex Fig. a. ModuleSOD1 and SKP1 in module ‘cyan’  in module ‘cyan’, one gene (TARS) in module ‘darkorange’, and two genes (COPB2 and RYK) in module ‘lightyellow’ showed statistically significant relation to overall survival with P-value < 0.05 with univariate Cox regression  showed independent prognostic significance with HR = 2.40 and P = 0.02 , LSCC patients with higher expression (red boxes) represented significantly different fraction of immune cells  was statistically increased in LSCC patients with higher expression; increasing expression of RYK showed association with downregulation of macrophages (M2) and Dendritic cells resting; the TARS1 expression presented association with the upregulation of fraction of mast cells (activated); the proportion of plasma cells was statistically decreased in LSCC patients with higher expression.Compared with lower (blue boxes) expression of five genes  between two stages    Fig. b. RYK ha01) Fig. c. In a wP value < 0.5 were clustered into 25 modules  with different expression in various tumor stages of larynx squamous cell carcinoma, in modules related to tumor stage  of COPB2 expression from stage I to stage III. GSEA showed that expression of COPB2 was associated with genes involved in the PI3K/Akt signaling pathway  and Dendritic cells resting (Fig. RYK, a receptor of noncanonical Wnt ligand Wnt5a, was actively involved in WNT signaling. WNT/RYK signaling restricts goblet cell differentiation during lung development and repair . RYK wasnts Fig. a. RYK hasis Fig. c; these ing Fig. , which iing Fig. d. ECM-reing Fig. a.COPB2 and RYK were found to be significantly correlated with tumor stages of LSCC and represented negative correlation with overall survival of LSCC patients.In conclusion, two genes,