Hey1) and rate of migration of monolayer cultures of lymphatic endothelial cells (hLECs) and blood endothelial cells (HUVEC). GSIs also inhibit HUVEC network formation. hLECs are more sensitive to GSIs compared to HUVEC. GSIs have been found to be safe in clinical trials in patients with Alzheimer’s disease or cancer. Our results provide further rationale to support testing of Notch inhibitors in patients with extracranial vascular malformations.Notch expression has been shown to be aberrant in brain arteriovenous malformations (AVM), and targeting Notch has been suggested as an approach to their treatment. It is unclear whether extracranial vascular malformations follow the same patterning and Notch pathway defects. In this study, we examined human extracranial venous (VM) (n = 3), lymphatic (LM) (n = 10), and AV (n = 6) malformations, as well as sporadic brain AVMs (n = 3). In addition to showing that extracranial AVMs demonstrate interrupted elastin and that AVMs and LMs demonstrate abnormal α-smooth muscle actin just as brain AVMS do, our results demonstrate that NOTCH1, 2, 3 and 4 proteins are overexpressed to varying degrees in both the endothelial and mural lining of the malformed vessels in all types of malformations. We further show that two gamma secretase inhibitors (GSIs), DAPT (GSI-IX) and RO4929097, cause dose-dependent inhibition of Notch target gene expression ( Morbidities are significant such as pain, infection, pulmonary emboli, bleeding, cosmetic concerns, psychosocial issues, and even death. Current treatments include supportive care with compression garments, and interventions such as sclerotherapy, embolization, and surgical debulking or resection. There has been a growing interest in medical management of vascular malformations2. The mammalian target of rapamycin (mTOR) inhibitor, rapamycin (Sirolimus), is the most commonly used drug, but does not result in complete resolution of lesions3. While generally well-tolerated, its results often are not permanent, and life-long treatment may be needed. Thus, there is a need for other medications.Vascular malformations are developmental abnormalities of venous, arterial, capillary or lymphatic vessels which grow with an individual. These can be purely of one type of vessel  or can involve more than one type  has been found to decrease LEC proliferation and migration12. Different results may reflect tissue-, species-, and developmental stage-specific differences in the role of Notch signaling, but are all consistent with a role for Notch in aberrant angiogenesis. Several investigators have suggested targeting Notch as an approach to treating AVMs.Notch proteins (Notch 1–4) are a family of receptors that are important in cell differentiation in virtually all tissues. The Notch pathway governs arteriovenous specification leading to distinction of arteries from veins13. This is of particular interest because a number of gamma secretase inhibitors (GSI) have undergone phase I and II clinical trials in adults with Alzheimer’s disease15 and in children and adults with cancer and related disorders18. That these drugs have known dosing and acceptable safety profiles makes them good candidates for drug repurposing for patients with other disorders such as vascular malformations.In mammals, the Notch signaling pathway is triggered by binding of Notch proteins to any of four activating ligands  which ultimately trigger transmembrane cleavage by a gamma-secretase complex, resulting in release of the Notch intracellular domain (NICD), which activates transcription of downstream target genes such as EphrinB2Hey1) and angiogenesis of both lymphatic and blood endothelial cells in vitro. These results provide additional rationale to support clinical trials of GSIs in patients with vascular malformations.In this study, we show that extracranial vascular malformations, like brain AVMs, are characterized by diminished and incomplete smooth muscle actin (αSMA) in the vascular smooth muscle cells (VSMC) of AVM, and diminished elastin coverage in the internal elastin lamina (IEL) of AVMs. NOTCH1–4 proteins are variably expressed in a range of these extracranial vascular malformations involving lymphatic, venous and arterial vessels. Moreover, two GSIs–DAPT and RO4929097–caused dose-dependent inhibition of Notch target gene expression  and AVMs in mice23. Therefore, we examined NOTCH2 and NOTCH3 expression in extracranial LM and VM. Our results show NOTCH2 and NOTCH3 clearly are expressed in endothelial and mural cells lining both VMs and LMs, and appear to be increased compared to normal vessels (N) within the same field  demonstrated varied expression across different types of malformations. Lymphatic malformation sections showed strong NOTCH4-ICD expression in CCD31+ endothelial cells lining the abnormal vascular channels consistent with active NOTCH4-ICD signaling Fig. . NOTCH4-Hey1 was significantly downregulated in both cell lines. DAPT downregulated Hey1 expression in HUVECs beginning at 4 μM, while 2 μM of RO4929097 was sufficient to downregulate Hey1  and lymphatic endothelial cells (hLECs) without altering their viability. Cell viability was not significantly different compared to untreated controls at concentrations less than 20 μM on either cell type. 20 μM of DAPT and RO4929097 reduced hLEC viability by almost 30% without significant changes observed at lower concentrations Fig. . Neitherey1 Fig. . hLECs aey1 Fig. .Figure 519, to our knowledge there are no studies for the use of DAPT or RO4929097 on lymphatic endothelial cells. Therefore, we investigated the effectiveness of both these GSIs on the rate of migration of monolayer cultures of hLEC and HUVEC cells  of the GSIs Fig. . The numIn this study, we examined a wide range of human extracranial vascular malformations involving lymphatic, venous and arterial vessels and demonstrate that Notch proteins are expressed in each type. Although our results are qualitative, activated NOTCH1-ICD is more apparent in endothelial cells lining extracranial LM, VM and AVM compared to normal vessels within the same tissue. Full length NOTCH1 expression is observed in the endothelium of a majority of the LMs independent of whether the lesion was a focal (cystic LM) or multi-focal . The broader expression of full length NOTCH1 than NOTCH1-ICD in the LM endothelium is consistent with the mechanism of NOTCH signaling, where Notch active cells are surrounded by ligand-expressing Notch inactive cells. NOTCH2 and NOTCH3 were expressed in endothelial and mural cells lining both VMs and LMs, and appeared to be increased compared to what we observed in normal vessels. NOTCH4-ICD was present in LM, but was sparsely expressed in both brain and extracranial AVM and VM. Our results suggest that Notch signaling plays a key role in human extracranial lymphatic, venous, and arteriovenous malformations and adds to the previously focused information on NOTCH overexpression in brain AVMs. We also found that, similar to those in the brain, extracranial AVMs showed interrupted αSMA and elastin layer surrounding malformed vessels.17, was more potent on a molar basis than DAPT, commonly used in vitro. Furthermore, hLECs were more susceptible to Notch inhibition than HUVECs. Our results are consistent with previous findings of GSI inhibition of HUVEC migration and tube formation26 and add to the information with comparative analysis between two GSIs across two different endothelial cells lines. Although GSIs are known to be direct inhibitors of Notch, it also is possible that these drugs have effects downstream of Notch, e.g., on EphrinB2, which also is gamma secretase-processed when activated. Nonetheless, taken together our results present a strong impetus for targeting the Notch signaling pathway in the management and treatment of extracranial lymphatic, venous and arteriovenous malformations with GSIs. Combined loss of one allele of Notch1 and global deletion of Notch3 has been linked with retinal AVMs in mice23. Notch2, while not described to be expressed in normal peripheral vascular cells, has a role in renal and eye capillary development27. Notch-mediated communication between vascular endothelial cells and smooth muscle cells is critical during normal vascular development. Notch signaling is also important to the pathogenesis of vascular disease, and lies both upstream and downstream of critical vascular and lymphatic growth signals such as VEGF, EphB4/ephrinB2 and adrenomedullin/CLR signaling30. Data on the role of Notch in human vasculature are limited, but patients with CADASIL are known to have missense mutations in Notch3 or Jag131. Patients with Alagille syndrome, a pleiotropic developmental disorder involving multiple organs, have demonstrated mutations in the Notch ligand Jag132. Our results support and extend several studies which have identified NOTCH1, 3 and 4 overexpression in AVMs of the central nervous system (CNS) in humans33. Possible tissue-, species-, and developmental stage-specific differences in the role of Notch signaling underline the need to study both inhibition and up-regulation in human vascular malformations.The GSIs, DAPT and RO4929097, were effective in inhibiting angiogenesis in Notch-expressing HUVECs. RO4929097, which has been used in human clinical trials18. The maximum concentrations observed in pharmacokinetic studies of RO4929097 in patients with cancer were in the range of concentrations which we studied34. Renewed interest in making drug available for one or more of these indications or for other rare disorders seems likely in the near future. We studied only one of 5 GSIs which have been used in humans. Preclinical testing of the other four drugs of this class will be of interest. Additional preclinical studies will be needed to detail the signaling pathways involved in development and progression of vascular malformations. However, we suggest that these can proceed concurrent with pilot phase II clinical trials of Notch inhibitors in patients with these disorders.Our observations are of particular interest because several GSIs already have been through clinical trials, both in children and adults. Tolerated doses and schedules for oral administration have been well defined, particularly in older patients. Although lack of efficacy in Alzheimer’s disease, the initial target population, led to shut-down of development of this class of drugs by the pharmaceutical industry, some efficacy was suggested in leukemias, solid tumors and in benign but locally aggressive desmoid tumorsUNC IRB (IRB 15-1423). For human studies at Columbia, de-identified LM tissues were collected from discarded surgical specimens. As protected health information (PHI) was neither stored nor disclosed to the researchers, tissues were collected under exemption by Columbia University IRB (AAAA7338) and thus, there was no verbal or written consent required.We confirm that all experimental protocols were carried out in accordance with relevant guidelines and regulations. All experimental protocols were approved by the following IRB committee: A database  of all patients with vascular anomalies  seen at UNC January 2000-January 2016 by members of our multi-disciplinary Vascular Anomalies Clinic was compiled . Entries were reviewed for diagnostic accuracy, patient demographics, the availability of relevant archived biopsies, location of the anomaly, and treatment prior to biopsy. For human studies at UNC, LM, VM, and AVM tissue samples were collected from archived surgical specimens and samples were de-identified. A waiver of consent and waiver of HIPPA authorization were approved by the After obtaining an IRB waiver of approval, de-identified samples based on the database  were obtained in paraffin blocks from UNC’s Department of Surgical Pathology. The extra-CNS biopsies came from variable locations including head and neck, chest, abdomen and extremities. An attempt was made to select the most recent specimens from patients who had not had prior sclerotherapy or malformation-directed medications. No patient had received GSIs for any disease. Because of the heterogeneity typical of histologic samples of vascular malformations, archival blocks were reviewed (SVS) to optimize sectioning of the part of the block most involved by the vascular malformation. An additional 8 LM samples were available from Columbia P&S.in vitro assays (below) within 8 passages and maintained in EGM-2MV bullet kit (Lonza CC3202) and EGM bullet kit media respectively.Dermal human lymphatic endothelial cells (hLECs) from human neonates  and human umbilical venous endothelial cells  were used for To examine the effect of GSI on cell viability, hLEC and HUVEC cultures were incubated with effects of graded concentrations  of DAPT  or RO4929097  [2–20 uM] for 24–72 hours. The percentage of viable cells was determined using the Countess automated cell counter . Briefly, 80–90% confluent HUVEC and hLEC cultures were trypsinized, washed, and 10 μL of cell sample was mixed with an equal amount of 0.4% trypan blue stain and loaded onto the counting chamber.et al.35. HUVECs and hLECs were grown to confluence in 24 well dishes, and then scratched with a 200 ul pipette tip. After scratching, the wells were rinsed with 1XPBS to remove non-adherent cells and then treated with control DMSO or increasing concentrations  of DAPT or RO4929097. Four fields per well were imaged at T = 0 hrs and at T = 24 hrs post-scratch or T = 72 hrs for HUVECs and hLECs respectively using an Olympus IX-81 inverted microscope equipped with a QImaging Retiga 4000 R camera at 4X magnification. The percent change in migration was calculated by measuring the open area of the scratch at the above mentioned time-points (ImageJ). Results shown are representative of four independent experiments with HUVECs and three for hLECs.Migration assay was performed as per Liang 36. Briefly, HUVEC cells were serum-starved after reaching 70–80% confluence, trypsinized, washed and plated at similar concentrations in a Growth Factor Reduced Matrigel Matrix (BD Biosciences 356230) coated 96-well plate and submerged in growth medium containing either control DMSO or increasing concentrations  of DAPT or RO4929097.This assay was performed based on the protocol by Arnaoutova and KleinmanGAPDH (4310884E), ACTB (Hs99999903_m1) and HEY1 (Hs01114113_m1). Comparative ΔΔCT method was used to analyze relative gene expression with ExpressionSuite Software (Thermo Fisher Scientific). Expression was normalized to housekeeping controls GAPDH and ACTB.RNA was extracted from cultured HUVEC or hLEC cells either 24 hrs or 72 hrs post treatment respectively using TRIzol reagent (Ambion 15596026) followed by DNase (Promega M6101) treatment and cDNA prepared using iScript (BioRad 170-8890). Quantitative RT-PCR was done on StepOnePlus (ABI) using TaqMan Gene Expression Master Mix (TheroFisher Scientific 4369016). Gene expression was assessed using human Single-tube assays (Thermo Fisher Scientific/Applied Biosystems): et al.28. Briefly, they were rehydrated first followed by 20 minutes boiling for antigen retrieval in 10 mM Sodium citrate, 0.05% Tween 20, pH 6.0. They were then permeabilized in 1% triton X-100/PBS for 20 minutes, and blocked in 5% NDS. Sections were incubated with primary antibodies, including mouse anti-smooth muscle alpha actin  and rabbit anti-activated NOTCH1  and NOTCH4  primary antibodies for 2 hours at room temperature. Sections were washed and incubated in secondary antibodies including donkey anti-rabbit Cy3 (Jackson ImmunoResearch 711-225-152), donkey anti-mouse Cy2 (Jackson ImmunoResearch 715-545-151), and Bisbenzimide H 33258 Hoechst (Sigma-Aldrich B1155) at 1:250 at room temperature for 1 hour. The tissue sections were mounted in Prolong gold (Life technologies P36934). Rabbit polyclonal antibodies against NOTCH2 (ab8926) and NOTCH3 (ab60087) were from Abcam . Immunohistochemistry (IHC) carried in the Bond fully-automated slide staining system . Slides were deparaffinized in Bond dewax solution (AR9222) and hydrated in Bond wash solution (AR9590). Antigen retrieval for all targets was performed at 1000 C in Bond-epitope retrieval solution 1 pH6.0 (AR9961) for 20 min. After pretreatment NOTCH2 (1:500) and NOTCH3 (1:200) were applied for 30 min. Detection was performed using Bond polymer refine detection system (DS9800). Stained slides were dehydrated and coverslipped. Positive and negative controls (no primary antibody) were included for each antibody. Elastin special stain was done at the UNC Animal Histopathology Core (AHC). Photomicrographs were reviewed  to confirm the types of cells  which were stained by each antibody. NOTCH staining was evaluated semi-quantitatively by comparing that of abnormal vessels within the malformation with positive and negative control tissues and with normal intra-lesional vessels. Abnormal vessels were categorized as showing more, less, or the same amount and intensity of staining than the controls. A rabbit polyclonal NOTCH4-ICD generated at Columbia P&S and goat polyclonal full-length NOTCH1 (1:100 R&D Systems AF1057) staining were previously described37.Paraffin sections were stained as per Davis Images for the assays were taken on an Olympus microscope with cellSens software. Immunofluorescence images were acquired on a Nikon E800 fluorescence microscope with a Hammamatsu Orca CCD camera with Metamorph software (Molecular Devices Corp.). NOTCH2, 3, and elastin stained sections were digitally imaged (20x objective) in the Aperio ScanScope XT using line-scan camera technology (Leica Biosystems). Digital images were stored in the Aperio eSlide Manager software.t test  with *P < 0.05, **P < 0.01 and ***P < 0.001 considered significant.All experiments were performed 3 or more times with 3 technical triplicates per assay and data are represented as a mean with SD or SEM. Significance was determined by Student Notch signaling pathway is a potential therapeutic target for extracranial vascular malformations


MenB-FHbp is a recombinant meningococcal serogroup B (MenB) vaccine composed of 2 factor H binding proteins (FHbps). Meningococcal vaccines targeting polysaccharide serogroup A, C, Y, and W capsules were licensed upon confirmation of bactericidal antibody induction after initial efficacy studies with serogroup A and C vaccines. Unlike meningococcal polysaccharide vaccines, wherein single strains demonstrated bactericidal antibodies per serogroup for each vaccine, MenB-FHbp required a more robust approach to demonstrate that bactericidal antibody induction could kill strains with diverse FHbp sequences. Serum bactericidal assays using human complement were developed for 14 MenB strains, representing breadth of meningococcal FHbp diversity of ~80% of circulating MenB strains. This work represents an innovative approach to license a non-toxin protein vaccine with 2 antigens representing a single virulence factor by an immune correlate, and uniquely demonstrates that such a vaccine provides coverage across bacterial strains by inducing broadly protective antibodies. Neisseria meningitidis is an important cause of invasive meningococcal disease, effective vaccines exist for some serogroups but immunogenicity to the MenB group is poor. Thomas R. Jones and colleagues examine serum bactericidal responses from volunteers challenged with MenB-FHbp – a recombinant MenB vaccine containing two Factor H (FH)-binding proteins. Serum bactericidal responses are tested against 14 MenB clinical isolates selected in an unbiased manner to cover the vast breadth of FHbp antigen and epidemiological diversity. This work demonstrates the broad efficacy of the MenB-FHbp vaccine using a serum bactericidal activity as a surrogate of protection. Neisseria meningitidis usually results in asymptomatic colonization of the upper respiratory tract, in some individuals, bacteremia and invasive meningococcal disease (IMD) occur.3 IMD commonly presents as meningitis and/or septicemia; pneumonia, septic arthritis, epiglottitis, and otitis media are less frequently observed.5 A high case fatality rate is associated with IMD (10–15%),7 and ~20% of survivors have serious life-long sequelae such as limb amputation, hearing loss, and neurologic impairment.9Although transmission of 12 Effective vaccines based on capsular polysaccharides have been developed for serogroups A, C, W, and Y.2 However, immunogenicity of the MenB polysaccharide is poor because of similarity to polysialic acid structures present on human neuronal cells.14 During recent years, meningococcal serogroup B (MenB) in particular has been associated with a large proportion of IMD in Europe,15 the United States,16 Canada,17 Australia,18 and New Zealand.19 Although vaccines based on outer membrane vesicles (OMVs) have been successfully used to control epidemics caused by a single MenB outbreak strain,21 the generated immune response is predominantly against the highly variable porin A protein (PorA).25 Therefore, effectiveness is generally limited to the target strain. Consequently, surface-exposed proteins capable of inducing protective bactericidal antibodies across diverse MenB strains have been sought for the development of a broadly effective MenB vaccine.Nearly all meningococcal disease worldwide is caused by 6 of the 12 characterized meningococcal serogroups .31 Based on amino acid sequence, FHbp variants segregate into 2 immunologically distinct subfamilies (termed subfamily A and subfamily B); each MenB strain expresses a single subfamily variant , a conserved surface-exposed lipoprotein expressed on nearly all strains of MenB, was identified as such a target.ant Fig. .32,33Fig34 Importantly, it is predicted that this combination of FHbp variants is capable of providing protection against diverse MenB strains.35 MenB-FHbp has been approved for the prevention of IMD in several countries and regions, including the United States, Canada, Europe, and Australia.38 Another MenB vaccine, MenB-4C , also has a recombinant FHbp component (nonlipidated variant 1.1 from subfamily B) as well as 2 other recombinant protein antigens and an OMV.39 Thus, MenB-4C is different from MenB-FHbp, which contains two variants of a single antigen to afford broad coverage.40MenB-FHbp  is a bivalent, recombinant protein MenB vaccine composed of equal amounts of 2 recombinant lipidated FHbp antigens, one from subfamily A (variant A05) and the other from subfamily B (variant B01).41 an hSBA titer ≥1:4 is the accepted correlate of protection against meningococcal disease,43 and hSBA response rates based on this correlate have been used as surrogates for meningococcal vaccine efficacy.43 The SBA response rate has been specifically correlated with natural protection for the serogroup C and A polysaccharide vaccines.41 Because serogroup-specific polysaccharides are not variable, a single strain from each serogroup was sufficient to infer broad vaccine coverage. MenB OMV vaccines are also efficacious and vaccine-elicited hSBA titers correlated with protection against the target strain causing the epidemic.46 Accurately predicting strain coverage of protein-based vaccines is more complex using hSBA than for vaccines targeting capsular polysaccharides, given that protein sequence diversity and variability in expression levels differ among the different meningococcal disease strains.47 For example, PorA is the predominant target for serum bactericidal antibodies conferring protection after OMV vaccine immunization.49 PorA is a cell surface porin whose small cell surface exposed region has a high degree of sequence diversity. It has been estimated that protective immunity would need to be demonstrated with strains expressing 20 different PorA serosubtypes to protect against approximately 80% of sporadic MenB disease-causing strains in the United States.50 Historically, OMV vaccines have contained 1 PorA and have not demonstrated protection against strains with PorA sequences that are heterologous in amino acid sequence compared with the vaccine antigen.51 Therefore, selection of representative test strains to demonstrate that vaccine-elicited antibodies can be effective against a meningococcal disease strain is of paramount importance for protein-based vaccines.The serum bactericidal assay using human complement (hSBA) measures complement-dependent, antibody-mediated lysis of meningococcal bacteria. An hSBA titer is defined as the highest serum dilution killing ≥50% of assay bacteria;53 In an early assessment of the potential breadth of MenB-FHbp coverage, 100 MenB isolates with diverse FHbp variants, geographic origins, and genetic backgrounds were tested in hSBAs using MenB-FHbp immune rabbit serum.35 Of the 100 strains tested, 87 were killed in these hSBAs.35 Analysis of the 13 strains that were not killed suggested that the FHbp surface expression level on a given MenB strain affected the hSBA response. A threshold FHbp surface expression level was subsequently determined, above which isolates were predictably killed in hSBA.54 Additional investigations of potential factors determining strain susceptibility found that killing was largely independent of FHbp sequence variant, multilocus sequence type, or PorA subtype.35Immune sera elicited by MenB-FHbp in preclinical and early clinical studies demonstrated broad bactericidal antibodies that could kill diverse MenB strains containing FHbp subfamily A and B variants heterologous to the vaccine FHbp variants A05 and B01.33 An unbiased approach was used to select four antigenically and epidemiologically diverse representative test strains for use in MenB-FHbp immunogenicity studies.27 Selection criteria included expression of FHbp variants heterologous to the vaccine antigens and adequately reflecting the diversity of FHbp in MenB disease isolates, low to medium FHbp surface expression levels, and low baseline hSBA seropositivity rates. These four primary MenB test strains express FHbp variants from both FHbp subfamilies  describe the strategy and criteria used to select the 10 additional test strains, and (ii) present data demonstrating that the immune responses measured by hSBA using the four primary MenB strains are predictive of the responses obtained using 10 additional test strains, which further demonstrate and support the broad coverage of the immune response elicited by MenB-FHbp.33 For the MenB isolate collection, US strains were from the Active Bacterial Core Surveillance sites (2000–2005), covering ~13% of the population. European isolates (2001–2006) were from the public health laboratories of Norway, France, Czech Republic, and the Health Protection Agency in Manchester  and were collected systematically (every seventh or eighth isolate was included by order received at the country’s reference laboratory) and represented ~13% of invasive MenB isolates during the period.55 The strains expressing FHbp variant A07 were obtained from an extension of the MenB isolate collection that included an additional 551 disease-causing MenB strains from Spain and Germany (n = 1814). The extended MenB isolate collection was used as A07-expressing strains in the MenB isolate collection were not suitable because of the low surface expression of FHbp on these strains, high baseline seropositivity, and lack of readily available source of complement.Nine of the 10 additional MenB test strains were obtained from a collection of 1263 invasive disease-causing MenB strains (the MenB isolate collection).52The criteria used to select the additional MenB test strains were (i) FHbp variant prevalence among MenB disease-causing strains in the United States and/or Europe, (ii) the FHbp variant needed to be different from those expressed by MenB primary test strains, (iii) in vitro FHbp expression levels at or below median levels for the respective FHbp variant group to ensure that the strain was representative of the variant group it belonged to, (iv) technical compatibility in the hSBA, and (v) being considered a predominant clonal complex for the variant group (if a predominant complex existed). Strains meeting these criteria also needed to be technically compatible in the hSBA, including adequate availability of suitable human complement lots Fig. . StrainsThe 10 additional selected MenB test strains express FHbp variants A06, A07, A12, A15, A19, A29, B03, B09, B15, and B16 which differ from the ones in the four primary test strains  and have different sequences compared to the vaccine antigens Table .Table 1C32 Serological responses to the 10 additional hSBA strains were assessed in a subgroup of the study subjects. The majority of subjects had hSBAs ≥ lower limit of quantitation  1 month after dose 2 and 1 month after dose 3 for each of the primary  and the 10 additional MenB test strains  .58The MenB-FHbp–elicited responses measured by hSBA to the four primary and 10 additional MenB test strains were evaluated using sera from individual vaccine recipients. By determining the proportion of vaccinated subjects with functional bactericidal antibodies, assessment of the breadth of MenB-FHbp coverage at the individual level was determined, which is not possible using pooled sera. The four primary MenB test strains were selected to represent the diversity of MenB disease-causing IMD and thus support the potential breadth of coverage for MenB-FHbp using hSBA.60 However, MATS only predicts coverage of antigens specific to MenB-4C and is not useful for assessing coverage of other vaccines with different antigen compositions.61 Specifically, MATS measures antigen expression rather than bactericidal activity and is reported as a relative potency compared with a reference strain for each antigen.61 If the relative potency for any one of the component antigens is commensurate with bactericidal activity for MenB-4C immune sera , the strain is considered susceptible to killing.62 However, because sera from vaccinated individuals are not used in MATS,61 the assay is unable to predict the proportion of a population achieving hSBA titers ≥1:4  in response to immunization.Another methodology, the enzyme-linked immunosorbent assay–based Meningococcal Antigen Typing System (MATS), has been used to predict vaccine coverage of MenB-4C.47 For example, hSBAs are labor intensive and can require large quantities of sera and assay-compatible complement, particularly when larger numbers of strains and/or sera are to be assessed. In addition, interlaboratory differences in the performance of the assay reagents and strains used in hSBAs limit comparison of responses and assessments of breadth of coverage between vaccines. A known limitation of PPV analysis is the dependence of the magnitude of the response on prevalence .63 However, it is notable in this analysis that although there was a range of postvaccination responses to the additional strains (at 1 month postdose 2 and postdose 3), PPVs were uniformly high.Of note, limitations in performing hSBAs exist.Taken together, the immunogenicity data obtained from the 10 additional MenB hSBA test strains support the response data obtained from the four primary MenB hSBA test strains and confirm the broad coverage of MenB isolates conferred by MenB-FHbp. This work demonstrates the application of a rigorous assessment of a MenB vaccine’s elicited immune response using the epidemiology of MenB strains with regard to the vaccine antigen sequence and expression, in conjunction with the recognized surrogate of protection (hSBA), and, using this knowledge, led to vaccine licensure.35 Details of the MEASURE assay have been described previously.54 Briefly, 50 µL of bacteria were plated per well of 96-well round-bottom polystyrene plates, then centrifuged and washed with 1% (weight/volume) bovine serum albumin (BSA) in 1X phosphate buffered saline (PBS). Monoclonal antibodies  were added to the bacterial pellets to a final concentration of 6.7 µg/mL (335 ng/well), resuspended, and incubated for 30 min on ice. Cells were washed twice in 1% BSA in PBS followed by addition of biotinylated goat anti-mouse IgG , resuspension, and incubation for 30 min on ice. After two washes, cells were resuspended in streptavidin-phycoerythrin . Cells were washed twice in 1% BSA in PBS and then resuspended in 1% paraformaldehyde. For each well, 20,000 events were acquired on an Accuri C6 flow cytometer and analyzed using Accuri CFlow software (BD Biosciences). The gating strategy of the MEASURE assay has been previously published.54 For each sample, the mean fluorescence intensity (MFI) of the PE channel was determined after gating in the logarithmic forward scatter versus side scatter dot plot. FHbp expression was considered above the level of detection if MFIs were above an arbitrary threshold of at least 100 and three times that of the negative control (mouse IgG) MFI used in that assay. The cutoff level adopted for each FHbp variant group was the observed median mean fluorescence intensity plus 1 standard deviation, using the precision estimate of 25.2% relative standard deviation.For all strains, FHbp surface expression was quantified by the validated MEASURE assay, a flow cytometric assay using monoclonal antibody (MN86–994–11) recognition of a conserved FHbp epitope common to both FHbp subfamilies.32 A total of 900 subjects from each study were to be divided into three subsets (n = 300 each); the 10 additional test strains were allocated across these subsets so that two subsets each included 3 test strains and 1 subset included four test strains. The subsets included samples from 300 subjects to ensure that ≥150 evaluable hSBA results from each study would be obtained. Immune responses measured by hSBA using phase 3 clinical study sera were based on the assay LLOQ, which was an hSBA titer equal to 1:8 or 1:16 depending on the strain.Each of the 10 additional MenB test strains were used in hSBAs to test sera from subjects participating in 2 pivotal phase 3 studies of MenB-FHbp.The PPV for each primary/additional strain pair within an FHbp subfamily was defined as the proportion of subjects responding to the additional strain  among the total number of primary strain responders (hSBA titer ≥LLOQ for the primary strain). PPV analyses assessed whether observed hSBA responses to the four primary strains predicted immune responses to additional strains expressing FHbps from the same subfamily.Further information on research design is available in the Reporting Summary


Dear Editor,Nivolumab, an inhibitor of programmed cell death 1 (PD-1), is an immune checkpoint inhibitor (ICI) that enhances T cell functions by preventing negative regulation of cancer immunity, and it has shown clinically significant efficacy and tolerability in various types of cancer. Based on the results of randomized phase III trials comparing nivolumab with docetaxel , nivolumThis retrospective observational study was performed to obtain real-world data on the prognosis of patients who discontinued nivolumab , treatment but showed no progression. All patients with advanced NSCLC who had received nivolumab monotherapy and discontinued it by 31 March 2016 were initially selected from each institution. Of these 124 patients, 17 who had discontinued nivolumab due to reasons other than disease progression were included in the analysis (Table EGFR mutations or anaplastic lymphoma kinase (ALK) fusion genes; the age at treatment initiation ranged from 61 to 79 years, and the Eastern Cooperative Oncology Group (ECOG) performance status (PS) was good  was defined as the duration from the initiation of nivolumab treatment to disease progression or death. The PFS of the patients ranged from 13 to 580 days, with a median PFS of 163 days  evaluated the survival benefits of nivolumab versus docetaxel in previously treated patients with advanced NSCLC [The CheckMate 153 trial compared patients who discontinued nivolumab within 1 year with those who continued treatment until disease progression or severe AEs, and the major conclusion was that survival was significantly longer in the continuation group than in the discontinuation group . HoweverA key finding of the present study was that some patients had a long survival after nivolumab discontinuation despite no further anti-cancer treatment. We speculate that patients who discontinue treatment due to AEs, especially irAEs, will have long-lasting responses to nivolumab. Haratani et al.  evaluateOur results should be confirmed by a larger-scale study. The present study was retrospective, and the results were influenced by the decisions of physicians, such as the time points of nivolumab discontinuation and initiation of subsequent treatments. In other words, we obtained data reflecting actual clinical settings. In a phase III trial that compared pembrolizumab, a PD-1 inhibitor, with chemotherapy in patients with untreated advanced NSCLC expressing programmed death-1 ligand-1 (PD-L1) in at least 50% of tumor cells, pembrolizumab resulted in longer PFS and overall survival than did chemotherapy . Since tIn conclusion, some patients with previously treated, advanced NSCLC who discontinued nivolumab treatment for reasons other than tumor progression may have potential for a long-lasting treatment response, and irAE onset and a good ECOG PS at nivolumab initiation may be predictive markers of a long-lasting treatment response.Additional file 1: Figure S1. Kaplan–Meier progression-free survival curves of the 17 patients stratified by irAE and PS. (A) Survival curves of patients with or without irAE. (B) Survival curves of patients with good or poor PS before nivolumab treatment.


Nonalcoholic fatty liver (NAFLD) may progress to nonalcoholic steatohepatitis (NASH) and ultimately to cirrhosis and hepatocellular carcinoma (HCC). Prognostic markers for these conditions are poorly defined. The aim of this study was to identify predictive gene markers for the transition from NAFL to NASH and then to poorer conditions. Gene expression omnibus datasets associated with a prediction analysis algorithm were used to create a matrix composed of control subject (n = 52), healthy obese (n = 51), obese with NAFL (n = 42) and NASH patients (n = 37) and 19,085 genes in order to identify specific genes predictive of the transition from steatosis to NASH and from NASH to cirrhosis and HCC and thus patients at high risk of complications. A validation cohort was used to validate these results. We identified two genes, fatty acid binding protein-4 (FABP4) and matrix metalloproteinase-9 (MMP9), which respectively allowed distinguishing patients at risk of progression from NAFL to NASH and from NASH to cirrhosis and HCC. Thus, NAFL patients expressing high hepatic levels of FABP4 and NASH patients expressing high hepatic levels of MMP9 are likely to experience disease progression. Therefore, using FABP4 and MMP9 as blood markers could help to predict poor outcomes and/or progression of NAFL during clinical trial follow-up. Indeed, 25–30% of NAFL patients will develop liver inflammation and progress to NASH and more than 30% of NASH patients will develop severe fibrosis and/or cirrhosis leading to HCC7. NASH is a liver disorder associated with obesity, insulin resistance, type 2 diabetes mellitus (T2D) and metabolic syndrome9. The incidence of NASH has dramatically increased and it is now the leading cause of chronic liver disease and a major public health issue worldwide14. In addition, NASH patients are at higher risk of cardiovascular diseases, which are also the leading cause of mortality in these patients6. Therefore, NAFL and NASH should be diagnosed at an early stage during which they may be reversible using lifestyle changes and/or a pharmacological management16.Nonalcoholic fatty liver disease (NAFLD) includes a wide spectrum of conditions from nonalcoholic fatty liver (NAFL) or simple steatosis to nonalcoholic steatohepatitis (NASH) which may progress to hepatic fibrosis, cirrhosis and ultimately to hepatocellular carcinoma (HCC)18. Non-invasive approaches have been proposed to differentiate NAFL from NASH such as the use of ultrasound or a fatty liver index (FLI) which includes the body mass index (BMI), waist circumference, triglyceride levels and serum gamma-glutamyl transferase (γ-GT) levels and to identify liver fibrosis with numerous non-invasive diagnostic tests19. In parallel, the primary and secondary causes of steatosis such as viral hepatitis C, autoimmune hepatitis, genetic mutations or polymorphisms, alcohol consumption, medications, total parenteral nutrition, congenital or acquired lipodystrophy, have to be ruled out. Based on the poor or contradictory results of these non-invasive approaches, or in the context of clinical trials, liver biopsy is highly recommended18.Guidelines have been recently proposed to determine which patients should be screened for NAFLD such as patients with obesity, T2D and/or metabolic syndrome21. The diagnosis of a patient with a NAS ranging between 3 and 5 being unclear, this score is mainly used to assess NASH progression in clinical trials, but it cannot really be used as a diagnostic tool to identify NASH patients16. Recently, the “Fatty Liver Inhibition of Progression” (FLIP) European Consortium has focused on a diagnostic algorithm for NASH identification  and has proposed a more accurate and reproducible score separating Steatosis, Activity and Fibrosis (SAF)23. However, the histological assessment of NAFLD patients remains strongly observer-dependent and is not fully reproducible25. In addition, it has also been shown that sampling variabilities  may interfere with the diagnosis of NAFLD28. Therefore, the pathologist assessment of NAFLD lesions may be challenging30 and it may be assumed that the prevalence of NASH and its complications is probably underestimated31. Based on these observations, we have previously shown the need to confirm the pathological assessment with an analysis of gene expression levels and that genes involved in inflammatory processes are upregulated in patients with steatosis using high-density oligonucleotide microarrays32.Thus, despite the procedure-related risks of morbidity and mortality, liver biopsy remains the “gold standard” for the diagnosis of NASH. The NASH Clinical Research Network has developed a NAFLD activity score (NAS) based on steatosis grading, the presence of hepatocellular ballooning and lobular inflammation. A NAS below 3 indicates the absence of NASH while a NAS upper 5 supports the diagnosis of NASH. In addition, a fibrosis score may be associated with the NAS30.Despite many years of research to identify non-invasive predictive markers for NASH using sophisticated algorithm approaches, none of the known markers is reliable enough to remove the need to perform liver biopsy due to the large spectrum of disease manifestations ranging from simple steatosis to HCCBecause gene expression is highly specific and very sensitive to environmental changes, exploring comprehensive gene expression levels in liver biopsies may help to identify markers of progression from steatosis to NASH. Thus, the aim of this study was to identify predictive gene markers for the transition from NAFLD to NASH and then to poorer outcomes using hepatic gene expression microarray datasets.http://www.ncbi.nlm.nih.gov/geo/) and were used as learning datasets. Briefly, gene expression levels were measured in human liver biopsies using the same high-density oligonucleotide microarray ([HuGene-1_1-st] Affymetrix® Human Gene 1.1 ST Array [transcript (gene) version]) as previously published. Each array included more than 750,000 unique 25-mer oligonucleotide probes interrogating more than 28,000 genes34. GSE48452 and GSE61260 respectively included a total of 73 and 109 human liver samples grouped into control samples (n = 14 and 38), healthy obese (HO) samples (n = 27 and 24), obese and NAFL samples (n = 14 and 23) and obese and NASH samples (n = 18 and 24). Thus, a total of 52 control samples, 51 HO samples, 37 obese and NAFL samples and 42 obese and NASH samples could be analyzed when both GSE datasets were combined34. The characteristics of the patients who provided these samples are summarized in Table GSE48452 and GSE61260 microarray datasets were selected and downloaded from the genome expression omnibus (GEO) database on NCBI website  with “batchadjust” implemented in “PAMR” for R package at the “pamr.train” function38. Then the threshold was calculated to determine the minimum number of genes allowing distinguishing the four sample groups based on the calculation of the minimum misclassification error for each group leading to a table of true versus predicted values (pamr.confusion), from a nearest shrunken centroid fit (pamr.adaptthresh)38. Thereafter, the genes of interest were sorted based on their best score (pamr.listgenes) and the gene(s) with the best score which survived the thresholding from the nearest shrunken centroid classifier were plotted (pamr.geneplot).Data were analyzed using R 3.5.2 softwareVenn” function in “gplots” for R package, indicating the number of overlapping transcripts in each sample group39.The gene lists generated by the different PAM were compared using the “http://www.ncbi.nlm.nih.gov/geo/) to validate the genes of interest identified using the learning datasets (GSE48452 and GSE61260) after the PAM. Here, gene expression levels were measured using HG-U133_Plus_2.0 array  and CodeLink Human Whole Genome Bioarray , respectively41. The GSE63067 dataset included 7 control samples, 2 NAFL samples and 9 NASH samples41. The GSE17470 dataset included 4 control samples and 7 NASH samples40. The clinical characteristics of the patients who provided these samples were not available. To validate the genes selected in both validation datasets, two independent unsupervised approaches were used, the non-negative matrix factorization (NMF) and the principal component analysis (PCA). The “NMF” (R-package) was used here as an unsupervised clustering method for samples using gene expression microarray data from the validation datasets42. PCAs were performed using “FactoMineR” for R-package. Each sample and the ellipses centered onto the mean representing the 95% confidence interval (CI) were plotted on the PCA. The probabilities associated with the F-test of the variance analysis along the dimension axes (α = 0.05) were calculated43.Two independent human liver biopsy datasets were selected and downloaded  that includes 17 gene-set libraries, an alternative approach to rank enriched terms, and various interactive visualization approaches to display enrichment results45. Among the 17 libraries, we searched WikiPathways 2016 and/or KEGG 2016 (Kyoto Encyclopedia Gene and Genome) libraries. The algorithm calculated the p-value, Z-score and combined score for each signaling pathway identified. Using methods based on the Z-score and combined score has been shown to be the best approach to recover a higher number of correct terms45. Thus, only the signaling pathways with a significant p-value  were ranked based on their combined score and represented as a bar graph and network. Also, to pin-point certain genes associated with a signaling pathway, data were represented as clustergrams for better clarity.The lists of genes identified at each step of the analysis of learning datasets were used as data entries for computing enrichment with existing lists created from prior knowledge organized into gene-set libraries. We used Enrichr, a freely available integrative web-based and mobile software application (http://software.broadinstitute.org/gsea/msigdb/)46. KEGG and Reactome databases were used to identify the genes and signaling pathways significantly related to the NASH group from the learning datasets. Data were represented as enriched set plots.To identify specific molecular signatures, we also used MSigDB database software v5.2 developed by the Broad Institute  approved the study conduct and a written informed consent was obtained from all patients. The study was conducted in accordance with the relevant “Declaration of Helsinki” and “International Conference on Harmonization Good Clinical Practice” guidelines and the French ethical laws.A total of 21 patients who consulted at Paul Brousse hospital between 2004 and 2012 and with a follow-up of at least 4 years were included in this study. They were divided as follows: Controls (n = 7), NAFL patients (n = 6) and NASH patients (n = 8). A histological distinction based on the NAS was used to differentiate NAFL from NASH as previously described®-ND1000 (Thermo Scientific). cDNAs were generated using RivertAid® First Strand cDNA Synthesis (Thermo Scientific), and Syber Green from FastStart Essential DNA Green Master mixes  were used to quantify hepatic fatty acid-binding protein-4 (FABP4) and matrix metallopeptidase-9 (MMP9) mRNA levels using the gene-specific primers described in Supplementary Table Total RNA were extracted from frozen liver biopsies using RNA-STAT 60 reagent (AMS Biotechnology Europe LTD). RNA levels and quality were assessed using NanoDrop® 96 Instrument . Gene expression levels were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels and data were analyzed using LightCycler® 96 SW 1.1 software . For each sample, the gene of interest level to GAPDH level ratio was calculated based on an arbitrary number of copies determined using the standard curve for each gene, as previously described47.Q-RT-PCR was performed using the LightCyclerFABP4 and MMP9 hepatic mRNA levels were assessed using a PCA by plotting each patient and the ellipses centered onto the mean representing the 95% CI. The probabilities associated with the F-test of the analysis of variance along the dimension axes (α = 0.05) were calculated. To determine the individual and combined sensitivity and specificity of both markers, receiver operating characteristic (ROC) curves, the area under the curve (AUC), negative and positive predictive values (PV) and optimal response cut-off points (Ir.eta) were assessed using “pROC” and “Epi” for R package. A one-way ANOVA was used to analyze the distribution between the patient groups using “beeswarm” for R package and was represented by boxplots. Patient clinical data were analyzed to assess the predictive FABP4 and MMP9 mRNA expression levels. NAFL and NASH patients were divided into two subgroups: patients with low FABP4 and MMP9 mRNA levels (NAFL_FABP4_MMP9_L and NASH_FABP4_MMP9_L) and patients with high FABP4 and/or MMP9 mRNA levels (NAFL_FABP4_MMP9_H and NASH_FABP4_MMP9_H). The clinical events, defined as a worsening of the liver disease, were recorded during the follow-up for each patient. In the Data were analyzed using a Fisher’s exact test and represented as circular layouts (“circos plot”) using “circlize” for R package.The institutional review board of the hospital  approved the study and written informed consent was obtained from all patients. Access to this material and all experiments were performed in accordance with the relevant guidelines and regulation of the French ethical laws.34 to create a new learning dataset matrix. The clinical data from this learning dataset were based on the clinical data available in the GEO dataset website and included 52 control subjects, 51 HO patients, 37 obese patients with NAFL and 42 obese patients with NASH classified according to their NAFLD-activity score as previously described21. The clinical characteristics of the different groups of patients are summarized in Table p-value <0.0001, chi-squared test). Importantly, the number of women was higher in control subjects and HO patients than in obese patients with NAFL and NASH , but there was no significant difference in gender distribution between obese patients with NAFL and NASH . As expected, the BMI was significantly higher in the three groups of obese patients compared to control subjects, with significantly higher serum leptin levels and lower serum adiponectin levels. However, no difference in age, BMI, serum leptin and adiponectin levels was observed between the three groups of obese patients.For further biostatistical analyzes Fig. , we combTo confirm the statistical results from the new learning dataset matrix, we used validation datasets (GSE63067 and GSE17470). The clinical characteristics of the patients included in these validation datasets were not available.34. The PCA of the new matrix showed a high variance between the two microarray results of both learning datasets  was built based on the data from human liver biopsies using the same high-density oligonucleotide microarray version (see Methods)Then, a PAM was performed on the standardized and corrected transcriptome arrays including the 182 samples divided into 4 groups. The PAM identified 1,549 genes with a threshold of 0.78, corresponding to an overall low misclassification error rate of 31.5%  were used Fig. .Thus, using a batch-adjust normalization approach associated with a gene prediction algorithm to combine two different Affymetrix® microarray datasets, 1,549 genes were selected to distinguish healthy subjects, HO patients, obese patients with NAFL and obese patients with NASH. This gene signature was also confirmed in two other independent GEO datasets. Interestingly, this specific set of genes allowed identifying a subgroup of 10 patients with NAFL among the 37 patients (27.03%) who were classified as NASH patients. These results strongly suggested that these 10 patients with NAFL shared the same specific gene profile as NASH patients and that this specific gene profile could help to identify the process involved in the progression from steatosis to NASH.i.e. serotonin receptors), nuclear receptors and transcription factors, inflammatory process, and more importantly genes involved in the extracellular matrix remodeling (ECMR)  that were specific to each group of patients were identified. The sets of genes specific to NAFL and NASH patients  were involved in ECMR, apoptosis and carcinogenesis pathways.In addition, the signaling pathways in which the genes identified were involved could be found in each group of patients with an increased expression according to disease the severity, from the inflammatory process to ECMR and oncogenic pathways. Thus, these results also validated the biostatistical workflow approaches used to identify these genes.i.e. the 10 patients who were misclassified) had already a gene signature similar to that of NASH patients.As shown, the confusion matrix identified 12 obese patients with NAFL classified as control subjects (n = 3) or HO patients (n = 9), and 10 obese patients with NAFL classified as patients with NASH Table . These rOAT, GNMT, AASS and CYP2C19) in the NAFL-NASH subgroup  were involved in ECMR, DNA regulation , inflammatory processes and some were involved in insulin resistance and lipid metabolism  and NASH n = 141) were combined to create a new matrix with 150 genes and two groups of patients from the learning datasets . FABP4 was also the top-ranked gene among the 58 genes that allowed distinguishing NAFL-NASH patients from NAFL-Steatosis patients according to the PAM results  also expressed high mRNA expression levels for genes involved in ECMR and inflammatory pathways  as well as Control subjects from NASH patients  while no significant difference was observed between NAFL and NASH patients . As expected, MMP9 mRNA expression levels allowed distinguishing NASH patients from control subjects, and HO patients and NAFL patients  and another with low MMP9 level  pathway, MAPK, JNK, p38 activation and leading to CD4+ and CD8+ T cell activation and interleukin (IL)2, IL6, and IL12 secretion, the nuclear factor activated T-cells (NAFT) calcium-calcineurin pathway and hypoxia pathways and genes related to the AP1 complex (e.g. FRA1 and FRA2 family members). These results showed that, in the one hand, cell signaling pathways, and especially the G2 DNA damage checkpoint, were inhibited, allowing the resumption of cell cycling and entry into mitosis50. On the other hand, inflammatory processes were activated with T-cell and interleukin activation  identified 330 predictive genes (229 were upregulated and 101 were downregulated) for NASH patients with high MMP9 expression levels significantly identified ECMR pathways .To confirm these data, FABP4, MMP9, HELLS and TREM2, were shared between these three groups of patients  and 4 NASH patients had poor outcomes . The other patients did not experience any progression with no change in NAFL score or NAS. The clinical characteristics of these patients are summarized in Table cos Fig. .FABP4 and MMP9 hepatic mRNA expression levels could be used as predictive markers for clinical outcomes in patients with NAFL and/or NASH and we were able to build a prediction model 41. Interestingly, the data showed that 27.03% of NAFL patients (10 out of 37 patients) were re-classified in the group of HO patients. As expected, this result confirmed what was already known: the histological scoring system is limited to classify and predict the outcome of NAFL patients. Indeed, we identified 58 genes allowing differentiating two subgroups of patients with NAFL. Fifty-four out of the 58 genes were upregulated in one third of the NAFL patients (12 out of the 37 patients). These genes were involved in inflammatory and ECMR processes as previously reported in NASH patients63. For the first time, we identified 58 genes among which FABP4 showed the highest expression levels in NAFL patients. FABP4 was co-expressed with genes involved in NASH progression. Then, these 58 genes expressed in patients with steatosis allowed predicting the outcome of these patients, which could help to improve the follow-up and lead to the implementation of an early therapeutic strategy.Using an unbiased machine-learning approach of prediction analysis of microarray data, we combined two independent GEO datasets from two independent cohorts of patients, GSE48452 and GSE61260MMP9 mRNA expression levels. Interestingly, this subgroup of patients had poor outcomes and expressed genes involved in ECMR, inflammation, and carcinogenesis, whereas patients with low MMP9 mRNA expression levels had gene-expression profiling associated with a better outcome.Then, we identified 330 genes specific to a subgroup of NASH patients characterized by high FABP4 and MMP9 hepatic mRNA expression levels in patients who consulted in our institution to confirm if these two genes could be used as prognostic markers. As these prognostic markers were identified in a large cohort of NAFLD patients, this validation cohort was deliberately smaller. Thus, we confirmed retrospectively that FABP4 and MMP9 hepatic mRNA expression levels predicted patient clinical outcome. Indeed, elevated FABP4 hepatic expression levels have been found to positively correlate with NAFLD severity in a human cohort65. Interestingly, high FABP4 serum levels have been reported in NAFLD patients but its use as a prognostic marker in the serum is still controversial70.Finally, we quantified 74. More recently, MMP9 polymorphisms have been associated with the risk of NAFLD and obesity75. Therefore, our data showed that MMP9 hepatic mRNA levels could be used earlier as a prognostic marker to identify NASH patients whose disease could progress to cirrhosis and HCC. To note, a higher MMP-9 serum level has been reported in NAFLD patients compared to control patients but it did not allow distinguishing NAFL patients from NASH patients due to the small sample size and because the authors have not studied patient subgroups76. However, D’Amico and colleagues have previously shown significantly higher MMP-9 plasma levels in NASH patients compared to hepatitis C-infected patients with liver disease77. In addition, MMP-9 levels have been associated with an increase in inflammatory biomarker levels78. Thus, FABP4 and MMP-9 serum levels could be used as non-invasive prognostic markers, especially to identify subgroups of NAFL and NASH patients likely to experience disease progression or not. The next step will be to quantify FABP4 and MMP-9 serum levels in a larger cohort of NAFLD patients to confirm whether these two markers could be used as non-invasive markers.For a decade, MMP9 has been involved in the development and progression of human HCC metastasisFABP4 and MMP9, the proteins of which could be quantified in the serum and used as non-invasive prognostic markers for NAFL and NASH progression.In conclusion, using publicly available GEO datasets and an original machine-learning analysis, we identified gene signatures that could help to determine the outcome of patients with steatosis likely to progress to NASH, and the outcome of patients with NASH likely to progress to cirrhosis and/or HCC. Liver biopsies cannot be avoided, however, the use of predictive markers could strongly reduce the number of biopsies during patient follow-up and enable a better management of the patients. Thus, we identified a predictive gene signature in human liver that could be used in patient clinical follow-up, as well as in clinical trials focused on the development of drugs to treat NASH patients, including two main genes, Supplementary InformationSupplementary Dataset 1Supplementary Dataset 2Supplementary Dataset 3Supplementary Dataset 4


KRAS-mutant as compared to wild-type tumours . Survival analyses revealed the association between mesothelin expression and poor overall survival, whereas lack of PTEN protein expression associated with lower progression-free survival with anti-EGFR-based therapy in the first-line setting for patients with RAS wild-type tumour. Finally, outlier analysis identified putative targetable proteins in 65% of patients lacking a targetable genomic alteration. Our data show that TMP constitutes a promising, novel molecular prescreening tool in mCRC to identify protein expression alterations that may impact on patient outcomes and more precisely guide patient eligibility to clinical trials with novel targeted experimental therapies.Protein biomarkers are widely used in cancer diagnosis, prognosis, and prediction of treatment response. Here we introduce the use of targeted multiplex proteomics (TMP) as a tool to simultaneously measure a panel of 54 proteins involved in oncogenic, tumour suppression, drug metabolism and resistance, in patients with metastatic colorectal cancer (mCRC). TMP provided valuable diagnostic information by unmasking an occult neuroendocrine differentiation and identifying a misclassified case based on abnormal proteins phenotype. No significant differences in protein levels between unpaired primary and metastatic samples were observed. Four proteins were found differentially expressed in  Simultaneous and accurate measurement of many proteins in experimental samples is very important in descriptive and predictive biological research, including comprehensive proteomic surveys, protein network studies, validation of genomic alterations and clinical biomarker development.The accepted ‘gold standard’ for protein measurement is immunoassay, which uses either one (as in immunohistochemistry or IHC) or two (competitive immunoassays) antigen-specific antibodies. Antibody-antigen binding can be measured across multiple matrices such as in fluids, on the surface of cells, within cells or tissues and in organs. The most widely used platform for tissue-based biomarker analyses is IHC, which facilitates the qualitative expression of proteins while preserving tissue architecture. Despite being a relatively easy and inexpensive approach, IHC has its limitations including reproducibility issues and very poor multiplexing capabilities. It is at best a semiquantitative method and is minimally effective for comprehensive analyses.3. However, its application to formalin -fixed paraffin -embedded (FFPE) tissues, which represent the standard preservation method for tumour samples analysed in the clinic, has been hampered by incomplete solubilization of samples5.Targeted proteomics using selected reaction monitoring mass spectrometry (SRM-MS) has emerged as a promising and highly sensitive method for the quantification of multiple proteins within the same tissue sample in an antibody-free setting7. The reliability of this approach for the analysis of proteins in patient tumour tissues has been previously demonstrated13. Our targeted panel of proteins including oncogenic, tumour suppression, drug metabolism, and drug resistance markers, among others were measured in FFPE tissues from 50 patients with mCRC treated at our institution and whose tumours were genomically-profiled as a prescreening strategy for clinical trial recruitment. We performed an exploratory correlative analysis of protein levels with clinicopathological and genomic markers, investigated their association with the patient outcome – including response to standard chemotherapies and combinations with anti-EGFR agents – and assessed their potential as predictive biomarkers for novel targeted experimental therapies currently under investigation.In the present study, we used the Liquid Tissue® proteomic method to reverse formalin-induced crosslinks allowing complete solubilization of all proteins in the sample for the precise quantificationMET amplification status by fluorescence in situ hybridization to identify potentially targetable alterations. FFPE blocks were retrieved from the pathology archive of the Vall d’Hebron University Hospital.Clinicopathological characteristics of patients included in our study are summarized in Table 10. All samples were qualified as evaluable according to the expression levels of the two housekeeping proteins actin and tubulin. We based the selection of the target proteins included in our TMP panel on a literature search for predictive biomarkers of response to targeted/chemo/immunotherapy and also included differentiation proteins with diagnostic value. Among 54 target proteins analysed, 18 (33%) were below the limit of detection of SRM-MS (non-detectable)  with very high levels (10-times higher than those of the lowest detectable case) , markers of stratified epithelia (P63), mesenchymal (Vimentin) and neuroendocrine  differentiation, and primary tumour origin (TTF1). Analysis of differentiation markers showed a lack of expression of P63 and TTF1 in all but 2 cases (4%), consistent with the colorectal origin of our samples. One tumour (ID-87) with P63 expression also showed very high levels of EGFR and KRT5, highly suggestive of squamous cell differentiation. Upon histopathological review, the case turned out to be an anal squamous cell carcinoma and, therefore, excluded from further analyses. The only case in our dataset expressing TTF1 was a liver metastasis (ID-51). Thyroid transcription factor 1 (TTF1) has been considered a highly sensitive and specific marker for primary lung adenocarcinoma. As it is not usually expressed in CRC, TTF1 is used for the differential diagnosis of metastatic adenocarcinomas of colorectal versus lung origin17. Worthy of note, entrapped normal epithelial cells from the lung were the main contributor of KRT7 expression in sample ID-53  and metastatic disease n = 24) were included in our cohort, we investigated differences in protein expression levels according to the tissue of origin. Overall, no significant difference between unpaired primary and metastatic lesions was observed   versus wild-type cases . KRAS and EGFR expression levels were higher in the RAS-mutated subgroup as compared to wild-type (BH adjusted Mann-Whitney p = 0.023 for both comparisons). Median KRAS expression levels were 1192 amol/μg (IQR 1011-1327) and 867 amol/μg (IQR 640-1006) for RAS-mutated and wild-type tumours, respectively (no differences were found according to codon mutated). Median EGFR expression levels were 232 amol/μg (IQR 210-299) in RAS-mutated and 192 amol/μg (IQR 165-206) in wild-type tumours. On the other hand, TOPO1 and TOP2A were significantly underexpressed in RAS-mutated tumours (BH adjusted Mann-Whitney p = 0.023 for both comparisons). Median TOPO1 expression levels were 1408 amol/μg (IQR 1116-1893) and 1033 amol/μg (IQR 953-1131) for RAS wild-type and mutated tumours, respectively. TOP2A levels were 753 amol/μg (IQR 492-936) in RAS wild-type and 441 amol/μg (IQT 354-587) in mutated tumours  in the metastatic setting. In univariate analysis, after multiple testing adjustment, only MSLN levels associated with outcome . No association between other protein expression levels (including EGFR) and response to EGFR targeted therapy was found.A total of 23 22. In our dataset, TS was detectable in a single case (2%) whereas ERCC1 and TOPO1 were expressed in 22% and 100% of mCRC analysed . No association between ERCC1 and benefit from treatment with oxaliplatin-based chemotherapy was found . Similarly, we did not find a significant association between clinical benefit from irinotecan-based therapy and TOPO1 expression when dichotomized at the high-level cut-off .A panel of 19 proteins involved in chemosensitivity or chemoresistance was analysed in our TMP assay. Among those, thymidylate synthase (TS), excision repair cross-complementing group 1 (ERCC1), and Topoisomerase 1 (TOPO1) have been previously studied as putative biomarkers of response to 5-FU, oxaliplatin, and irinotecan, respectivelyPIK3CA exon 9 or 20 mutations  eligible for combination therapy with PI3K inhibitors at our institution.One of the major goals of our study was to explore whether multiplex proteomic analysis could complement current genomic molecular prescreening by identifying abnormal proteins expression in patients without any targetable genomic alteration. Out of 49 patients, 6 (12%) had MET amplification by fluorescence in situ hybridization in our cohort). With regards to other proteins measured in our TMP panel, in the absence of a validated threshold for overexpression by SRM-MS, we conducted Robust regression and Outlier removal (ROUT) analysis23 . A total of 31 outliers (proteins expressed at very high levels) across 12 proteins were identified in 24 individual patients  without PTEN expression (non-detectable), enrichment criteria for PI3K pathway inhibitors. In addition, 1 tumour had very high expression of EGFR (7635 amol/μg) and 4 harboured high expression levels of HER2  remains empirical and does not rely on tumour biomarkers, which could be revealed by a novel multiplatform profiling strategy like ours.Therapies guided by tumour molecular profiling have shown significant clinical benefit in treating patients with advanced cancer. In mCRC, examples of emerging markers include in situ hybridization and chromogenic in situ hybridization to investigate targetable biomarker aberrations28. Authors found major differences in the expression of selected proteins across different metastatic sites, reflecting inter-metastatic tumour heterogeneity, although the limited overlap in term of proteins analyzed  and the different methodology used for protein expression (IHC vs SRM-MS in our study) hampered any comparative analysis.To the best of our knowledge, this is the first study to explore the potential impact of quantitative targeted proteomics in the context of precision oncology in mCRC. A comprehensive molecular profiling of CRC samples has been previously conducted with a multiplatform approach that includes sequencing, IHC, fluorescence In our study, we investigated quantitative protein biomarker profiles of mCRC and integrated the results obtained with available clinical, pathological and genomic data towards advancing insights into predictive and prognostic markers. Our TMP panel included proteins involved in oncogenic, tumour suppression, drug metabolism and resistance as well as tumour differentiation markers that could aid in the standard diagnostic decision-making processes as well as the selection of novel targeted drugs and immunotherapies (antibody-drug conjugates or ADC).30. We expect that proteomics-guided drug development will expand treatment options for patients who are eligible to participate in early phase clinical studies, particularly in view of the increasing array of ADCs and immunotherapeutic approaches, thus repurposing proteomics as an important contender in precision oncology.The major impact of the use of TMP in mCRC may translate in more precisely matching patients to experimental therapies. Recruitment rates in early clinical trials based on genomic markers or targeted IHC in metastatic CRC does not exceed 15%Until validated thresholds for individual biomarkers become available, outlier expression may be a valuable marker for testing novel experimental therapies in the metastatic setting as well as an enrichment strategy for clinical trials. In our present study, we found 29 outliers in 21 patients without any targetable genomic alteration, 12 of which are potentially useful in more precisely guiding the selection of an investigational ADC therapy  or refining a chemotherapy strategy (TOPO1 and TOP2A).31. In mCRC, the value of MSLN expression has not been thoroughly investigated. The prognostic association we found between MSLN and poor prognosis has been previously reported in the literature with both tumour and plasma MSLN levels in early-stage CRC33 thus strengthening the role of this protein in CRC biology. This supports its investigation as a therapeutic target in this yet unexplored tumour indication.With regards to novel prognostic markers and therapeutic targets, our TMP analysis identified MSLN expression in 30% of metastatic CRC. MSLN is a cell-surface glycoprotein whose expression in normal human tissues is restricted to mesothelial cells. Given that it is highly expressed by many solid tumours , it represents an attractive target for ADC developmentRAS wild-type patients. Previous retrospective studies37 showed that this association could well be linked to a prognostic effect of PTEN in this subset of patients39 as opposed to a predictive value40.We also investigated the value of TMP in patient selection for standard-of-care targeted agents. We recognize the difficulties in identifying predictive markers for anti-EGFR and standard therapies in our cohort since these therapies are administered in combination regimens and the small sample size that together do not allow for the proper study of interactions among different markers and therapies. We did, however, identify an association between a lack of PTEN protein expression and shorter time to progression in the first-line setting with chemotherapy plus anti-EGFR in RAS mutation status. KRAS protein levels were significantly higher in RAS mutant vs wild-type CRC, independently of the mutation type. This is not surprising as KRAS mutations occurring in codons 12 and 13 of exon 2 induce stabilization of the protein in a constitutive activation state which, in theory, could also facilitate the detection of higher levels of the total protein in mutant tumours as compared to wild-type41. We also found that TOPO1 and TOP2A expression levels were lower in RAS-mutated tumours, an association not previously reported in the literature, and that reinforces differences in the biology of these tumours. Higher TOPO1 expression in RAS wild-type may favour irinotecan over oxaliplatin in this molecular subgroup, and many trials are assessing irinotecan plus anti-EGFR therapy re-challenge after an initial response in RAS wild-type mCRC. Similarly, high TOP2A levels may suggest an increased sensitivity to topoisomerase II inhibitors43 in RAS wild-type CRC and prompt the exploration of agents not routinely considered as standard-of-care. These results certainly merit further consideration in future clinical trials.Exploratory analysis of proteomics data allowed us to assess the value of complementary data provided by this approach in the context of the molecular diagnostic evaluation of mCRC patients. First, TMP may assist in refining conventional histopathological diagnosis, identifying misdiagnosed cases, and revealing uncommon differentiation that may help clinicians to prioritize alternative treatment regimens. For example, we could confirm an occult neuroendocrine differentiation in one case as well as an atypical proteomics profile which facilitated the identification of a patient sample erroneously submitted as colorectal adenocarcinoma for molecular analyses. Second, primary tumour molecular profiling is generally used for treatment decision even in the metastatic setting where cancer may have acquired new alterations or lost those originally present during carcinogenesis. Our analysis failed to reveal significant differences in protein expression levels between unmatched primary and metastatic samples (continuous protein levels), thus supporting current profiling strategies at least for the targets included in our panel. Finally, comprehensive proteomics analysis may also expose biological differences in clinically or molecularly-defined groups. We examined differences in the biomarker profiles based on In conclusion, this study opens the door to the application of targeted multiplex proteomics in CRC with a potential impact on patient stratification for precision medicine. Our results reinforce the inter-tumour heterogeneity of CRC with unique protein alterations in individual cases that can guide enrolment in early clinical trials and co-development of biomarkers and drugs. We expect that an expanded TMP panel with proteins that represent targets for ADCs and immune checkpoints may bring novel insights on potential combinatorial immunotherapeutic strategies.44 and MET amplification status by fluorescence in situ hybridization. The protocol of this study was approved by the Vall d’Hebron University Hospital Ethical Committee (PR(AG)147-2009) and all methods were performed following relevant guidelines and regulations. Informed consent was obtained from all patients. Clinicopathological characteristics are shown in Table Samples of histologically confirmed invasive CRC diagnosed at Vall d’Hebron University Hospital  were retrospectively identified by a study pathologist. All patients included in the study underwent a molecular prescreening between 2013 and 2014 which included an amplicon sequencing analysis of a panel of 59 genes10. Briefly, tissue sections (10 μM) were cut from FFPE blocks, placed onto Director® microdissection slides, deparaffinized and stained with haematoxylin. Tumour areas were marked by a board-certified pathologist. Microdissection of the marked area was performed using a laser microdissector . A total area of 12 mm2 containing approximately 45,000 malignant cells was microdissected from each tumour and transferred directly into the cap of 0.65 ml tube containing 20 µl of 100% acetonitrile. Acetonitrile was removed by SpeedVac centrifugation at 35 °C for 6 min. The dried dissection pellet was stored at −20 °C. Peptides were extracted from the pellet using the Liquid Tissue® technology as per manufacturer’s instructions . The Liquid Tissue® protocol involves heating the tumour tissue at 95 °C for 90 minutes using the Liquid Tissue® buffer, followed by trypsin digestion for 18 hours at 37 °C. The resulting peptide concentration is measured by the microBCA assay. Heavy labelled internal standards are added along with buffer A (0.1% formic acid). 10 µl of this mixture is injected into the mass spectrometer (Thermo TSQ Quantiva) and a targeted list of peptides . This information was used to identify the peptides from fixed cell lines or FFPE tissue samples. The top two  unique peptides from FFPE or fixed cell lines were chosen for further development. Unlabelled and labelled synthetic peptides of the top two unique peptides were used to assess the analytical performance . Eventually, the peptide that had the best analytical performance was used to quantitate the protein of interest.Assay development and QC was along the lines of our previously published workThe following primary monoclonal antibodies were used: anti-chromogranin A (CHGA), anti-cytokeratin 7 (KRT7), anti-HER2, and anti-mesothelin (MSLN). Before cutting, paraffin blocks were cooled to −10 °C and 3 µm sections were cut with a microtome. To ensure tissue straightening, sections were floated on distilled water at 43 °C. Cut tissues were collected on positively charged Superfrost glass slides and slides were dried overnight at 37 °C. Immunohistochemical stainings of HER2, KRT7 and CHGA were performed using a Benchmark ULTRA autostainer . The slides were heated in the instrument and deparaffinized with EZ prep solution . Heat-induced antigen retrieval was executed using Cell Conditioning 1  for 36 min at 95 °C. Then, the primary antibody was applied as indicated in Supplementary Table Staining of MSLN was carried out using the rabbit Envision-kit . Briefly, the sections were deparaffinized with xylene, dehydrated with decreasing ethanol baths and hydrated with distilled water and then heated in the PTLink for 20 minutes at 95 °C with Dako Target Retrieval Solution pH 9 , diluted 1:10 with distilled water. Endogenous peroxidase activities were inactivated with Dako Envision Flex Peroxidase Blocking Reagent for 15 min at room temperature and then protein block  was applied for 30 minutes at room temperature. Samples were then incubated with rabbit anti-human MSLN monoclonal antibody (clone SP74) at room temperature for 1 hour . Flex HRP was then applied for 30 min at room temperature, followed by incubation with Dako DAB detection solution FLEX-DAB for 5 minutes at room temperature. Slides were counterstained with Merck Haematoxylin Harris (dilution 1:4) for 2 min, dehydrated and mounted with Xylol based mounting medium. IHC stained slides were scanned using NanoZoomer 2.0-HT  and subsequently, the digital images were scored by a pathologist blinded to the MSLN SRM-MS results. For the assessment of MSLN, a semi-quantitative approach was used in which H-scores were generated by multiplying the staining intensity  by the percentage of positive cells (0-100%). MSLN staining was considered positive when at least 15% of the cells showed moderate (2+) to strong (3+) intensity.A descriptive analysis of the variables included in the study was performed. Continuous variables were expressed as median and IQR, and categorical variables were expressed as absolute values and percentages. Comparison of continuous variables was performed with non-parametric Mann-Witney test (two groups) or Kruskal-Wallis test (more than two) with adjustment for multiple testing according to Benjamini and Hochberg (BH) method. Spearman’s rank correlation coefficient was used to study the association between two continuous variables. For the univariate analysis of categorical variables, we used the Chi-squared test or Fisher exact test if cell frequencies were below 5. To assess their association with clinical benefit with standard chemotherapies, we classified patients as responders when the time to progression  in the first-line or second-line treatments exceeded their respective medians .Overall survival (OS) in the metastatic setting as an interval from the diagnosis of metastasis to death. Survival analysis was calculated using the Kaplan–Meier method and the log-rank test was used for statistical comparison. Hazard ratios (HRs) and associated 95% confidence interval were assessed using Cox proportional-hazard. ROUT analysis with a false discovery rate of 1% was conducted to identify outliers. Statistical significance was accepted at the conventional two-sided p < 0.05 threshold. The data analyses were carried out using R version 3.3.3 statistical software package.Supplementary informationSupplementary Table 1


To date, a major research effort on Behçet's syndrome (BS) has been concentrated on immunological aspects. Little is known about the metabolic reprogramming in BS. Citrate is an intermediary metabolite synthesized in mitochondria, and when transported into the cytosol by the mitochondrial citrate carrier—SLC25A1-encoded protein—it is cleaved into acetyl-CoA and oxaloacetate by ATP citrate lyase (ACLY). In induced macrophages, mitochondrial citrate is necessary for the production of inflammatory mediators. The aim of our study was to evaluate SLC25A1 and ACLY expression levels in BS patients. Following a power analysis undertaken on few random samples, the number of enrolled patients was set. Thirty-nine consecutive BS patients fulfilling ISG criteria, and 21 healthy controls suitable for age and sex were recruited. BS patients were divided into two groups according to the presence (active) or absence (inactive) of clinical manifestations. Real-time PCR experiments were performed on PBMCs to quantify SLC25A1 and ACLY mRNA levels. Data processing through the Kruskal-Wallis test and Dunn's multiple comparison test as post hoc showed higher SLC25A1 and ACLY mRNA levels in BS patients compared to those in healthy controls. Therefore, SLC25A1 and ACLY upregulation suggests that metabolic reprogramming in BS involves the citrate pathway dysregulation. Behçet's syndrome (BS) is a multisystemic inflammatory disorder classified among primary vasculitis. The diagnosis is based on clinical signs due to the lack of specific tests. Typical clinical manifestations include oral and genital ulcers, uveitis, and skin lesions. The involvement of the central nervous system, vessel tree, and gastrointestinal tract even if less frequent can be life-threatening. The disease is characterized by a relapsing-remitting course with a period of substantial wellbeing between the phases . BS is mCitrate is a key metabolite for energy production. Citrate is synthesized by citrate synthase from acetyl-CoA and oxaloacetate (OAA) in the mitochondrion where it usually enters the Krebs cycle and promotes oxidative phosphorylation.Immune response of activated macrophages and dendritic cells requires a metabolic reprogramming during which citrate is diverted away from the Krebs cycle toward the cytosol where it can be used to trigger several cellular functions . Part of+ (necessary for fatty acid and sterol synthesis). We refer to CIC plus ACLY as a “citrate pathway” , theOur results shed light on citrate pathway dysregulation occurring in BS. However, further investigations are needed to better understand its role in BS pathogenesis.