The standard treatment of resectable pancreatic cancer is surgery followed by adjuvant chemotherapy. Due to the complication rate of pancreatic surgery and the high rate of primary irresectability, neoadjuvant concepts are increasingly used for pancreatic cancer. Neoadjuvant therapy is better tolerated than adjuvant and might decrease the surgical complication rate from pancreatic surgery. In contrast to neoadjuvant chemoradiation, the nutritional status improves during neoadjuvant chemotherapy. Also, the survival of patients who develop postoperative complications after neoadjuvant therapy is comparable to those without complications whereas the survival of patients who underwent upfront surgery and then develop surgical complications is impaired. Moreover, large data base analyses suggest a down-sizing effect and improvement of overall survival by neoadjuvant therapy. Neoadjuvant chemotherapy appears to be equally efficient in converting irresectable in resectable disease and more efficient with regard to systemic tumor progression and overall survival compared to neoadjuvant chemoradiation therapy. Despite these convincing findings from mostly small phase II trials, neoadjuvant therapy has not yet proven superiority over upfront surgery in randomized trials. The standard treatment for patients with resectable pancreatic cancer is surgery followed by adjuvant chemotherapy (CTx): after the ESPAC-1 trial had suggested a survival benefit for an adjuvant CTx, Oettle et al. have demonstrated a significant improvement of the recurrence-free and overall survivals for patients with pancreatic cancer after a curative resection. Further trials have confirmed these results and have shown survival improvements for combination CTx ,3.Theoretical advantages of neoadjuvant over adjuvant treatments are the treatment of circulating tumor cells and micrometastases prior to surgery, higher probability of R0 resection rates and a better patient selection due to exclusion of patients from surgery with progressive disease under neoadjuvant treatment. On the other hand, toxic neoadjuvant regimen may be harmful since these would hamper the surgical outcome. On the other hand, some (surgeons) may consider the risk of disease progression under therapy as a major disadvantage for the patient.Neoadjuvant therapy has become the standard of care for most gastrointestinal cancers: randomized trials have demonstrated beneficial effects for esophageal, gastric and rectal cancer ,5,6. In Considering these findings from other gastrointestinal cancers, the introduction of neoadjuvant therapy for pancreatic cancer appears reasonable and has gained massive popularity over the past 20 years . While iAlthough several analyses suggest beneficial effects of neoadjuvant therapy for pancreatic cancer, no randomized controlled trials have yet proven superiority of neoadjuvant therapy over upfront surgery.The basis of this review is an extensive PubMed literature research on the latest publications on the respective fields. Whenever available, review articles were accepted if the methodology was felt appropriate for the question of this review.Due to the close contact to the superior mesenteric vessels and the celiac arteries, resectability of localized pancreatic cancer is often hampered by the infiltration of these structures. Since vascular resections are considered critical or even impossible by many surgeons, the resectability of such advanced tumors is highly depending on the individual expertise of the surgeon.In contrast to single-center series, recent meta-analyses have demonstrated that venous and arterial resections are associated with a higher perioperative mortality and impaired long-term survival . Thus, tConsidering these continuous changes in the surgical attitude, it is no wonder that resectability criteria varied among surgeons and associations. The current concept of local resectability is based on the extent of vascular involvement: tumors without contact to the major mesenteric or celiac vessels are considered resectable. In contrast, a tumor contact to the superior mesenteric or hepatic arteries of more than 180° is currently considered unresectable as well as an infiltration of the superior mesenteric vein (SMV) without the possibility of reconstruction . The curThose tumors which are neither classified as resectable nor unresectable are classified as borderline resectable . While sOf note, the concept of borderline resectability also introduces major bias in several ways: the spectrum of “borderline” includes a wide range of clinical situations: a short-segment infiltration of the SMV close to the confluence has a higher chance of R0 resection than a tumor contact to the superior mesenteric artery (SMA) up to 180°—both are classified as borderline resectable. Moreover, secondary resectability is a difficult primary study endpoint of clinical studies on neoadjuvant therapy for borderline resectable pancreatic cancer considering the different surgical attitudes to the treatment of this subgroup. Therefore, the definitions of primary and secondary (ir-)resectability must be analyzed with caution when comparing these end-points of different studies after neoadjuvant therapy.Further terms used in the literature are “potentially resectable” and “locally advanced” Both terms do not have a clear definition. While potentially resectable summarizes resectable and borderline resectable disease, locally advanced is generally considered unresectable disease without distant metastases at this moment  14]. Un. Un14]. The aim of an adjuvant therapy is the reduction of tumor recurrence after an apparently curative resection, and the aim of a palliative therapy is the relief of symptoms in incurable disease. Both are usually associated with a survival benefit, which, however, might be minimal or absent for palliative therapy. While adjuvant and palliative therapies are well defined, a clear definition of “neoadjuvant” therapy is currently lacking. Any preoperative therapy in resectable cancer as well as therapies which might result in surgery in case of tumor response, are considered “neoadjuvant” in the literature. Furthermore, a pretreatment of patients with synchronously metastatic disease  is also considered “neoadjuvant”, if a resection of the pancreatic primary and the liver metastases may be considered in a subset of patients without tumor progression. Consequently, the current literature varies widely regarding the patient populations for neoadjuvant therapy.Neoadjuvant therapy may be based on different treatment modalities. Due to the lack of an efficient chemotherapy for pancreatic cancer, neoadjuvant therapy was historically based on CRTx  GemcitabIt appears reasonable to treat patients with locally advanced tumors with the aim of achieving secondary resectability (downsizing), since surgery is considered the only curative treatment option for these patients. Consequently, most of the literature on neoadjuvant treatment is about patients with borderline or locally advanced (unresectable) pancreatic cancer. Due to the high local effect, CRTx has historically been preferred in this situation. However, a recent analysis of 575 patients with locally advanced pancreatic cancer revealed higher secondary resectability rates after Folfirinox chemotherapy compared to gemcitabine-based CRTx .Another, oncologically much more interesting concept is the preoperative treatment of patients with primarily resectable pancreatic cancer to decrease recurrence rate and to improve long-term survival .Perioperative mortality has dramatically improved during the past 30 years, and large database analyses have demonstrated lower perioperative mortality and better long-term survival in large-volume centers ,20. HoweDue to the substantial complication rate and negative impact on the quality of life related to pancreatic surgery, about 40% of patients deny further treatments or are not considered eligible for adjuvant therapy by their treating physicians . Consequn = 115) received neoadjuvant therapy, while only 50 patients were operated upfront with potential adjuvant therapy. In this analysis, the survival of patients who completed a multimodality treatment was significantly superior to those who did not. These patients with complications from surgery had the same survival as patients who underwent neoadjuvant therapy without surgery. In contrast, survival did not differ between patients with or without major surgical complications, if they had received neoadjuvant therapy. Moreover, the long-term survival of patients who completed adjuvant therapy without surgical complications was not different from patients after neoadjuvant therapy ..15].When this score was adopted to assess the histological response to neoadjuvant CTx, 13 patients (54%) had II, but higher degrees of histological response were not observed. Moreover, more than 80% of the tumors revealed cytopathic effects in response to the neoadjuvant treatment . In the In an analysis of nearly 15,000 patients of the US National Cancer Database from 2006 to 2012, tumors after neoadjuvant therapy had lower T- and N-stages. Moreover more patients had R0 resections after neoadjuvant therapy . AssuminA recent meta-analysis of the current literature confirms these findings of lower T- and N-stages, lower rates of perineural and lymphovascular infiltration rates as well as higher R0-resection rates after neoadjuvant therapy .The standard (re-)staging examination for pancreatic cancer is computed tomography (CT), which depicts vascular invasion and distant metastases. After neoadjuvant therapy, new metastases may be detected by CT-scanning. However, the exact local tumor regression in response to neoadjuvant therapy is not exactly described by CT . TherefoMost cancers of the pancreatico-biliary tract express the tumor marker CA 19.9, and this serum marker is therefore considered the standard tumor marker for pancreatic cancer. In the palliative treatment of patients with pancreatic cancer CA 19.9 is a well-established response marker . Also, CIn the setting of neoadjuvant therapy, CA 19.9 serum levels have also been investigated in several phase II trials. In most studies, CA 19.9 serum levels decreased during the neoadjuvant therapy. Although low CA 19.9 levels after neoadjuvant therapy were associated with higher resectability rates, a decrease in CA 19.9 serum levels in response to therapy was not associated with the radiological , histoloThe lack of significance of the CA 19.9 response in the setting of neoadjuvant therapy in the majority of studies and the controversies between these studies is mainly due to various time points of the pre-therapeutic CA 19.9 measurements and the heterogeneity of the treatments. Mellon et al. only used CA 19.9 levels after biliary stenting, and Katz et al. and Tzeng only included CA 19.9 levels in the absence of hyperbilirubinemia ,49,51. INone of the published studies evaluated CA 19.9 serum levels during the treatment. Considering this limitation and the controversial results of the literature, serum CA 19.9 levels are not suitable for early prediction of the histological response to neoadjuvant therapy.18Fluoro-Desoxy-Glucose (FDG) has been used for several tumor entities to predict tumor response to neoadjuvant therapy [PET based on  therapy ,42. For  therapy .For pancreatic cancer, PET-scanning has also been used pre- and post-therapeutically, and FDG-uptake decreased during neoadjuvant therapy in all studies ,45,54,55Since the assessment of borderline resectable and locally advanced disease varies between centers depending on the surgical expertise, a potential tumor progression under neoadjuvant therapy, which precludes surgery, can only be evaluated in primarily resectable pancreatic cancer.The resectability rate after neoadjuvant CTx for resectable pancreatic cancer was 77% in the O´Reilly trial and 89% in the study reported by Heinrich et al. ,27. In bDespite the higher local effect of neoadjuvant CRT as measured by the histological response, the resectability rates after neoadjuvant CRTx appear lower with 58–74% compared to neoadjuvant CTx ,34,35. TDue to the lack of adequately powered randomized trials, the current evidence for an effect of neoadjuvant therapy on long-term outcome arises from phase II trials and retrospective database analyses. An extensive review of the literature regarding the survival estimates of neoadjuvant treatments has been performed by Gillen et al. . AccordiNeoadjuvant chemotherapy for primarily resectable disease has only been reported from a few centers. These phase II trials report a median survival of 26.5 months and 27.2 months after two-months of neoadjuvant CTx with or without adjuvant CTx [Very recent large database analyses also suggest a survival benefit for patients who received neoadjuvant therapy . Interes1mm vs. R1direct), both subgroups of patients should be candidates for neoadjuvant therapy [The current standard treatment for patients with pancreatic cancer is surgery followed by adjuvant CTx. However, a significant proportion of patients cannot undergo surgery or receive adjuvant therapy . Many pa therapy . Moreove therapy . ConsequCurrently, neoadjuvant therapy is mainly used for borderline and unresectable pancreatic cancer, when surgery is considered inefficient or impossible. Due to its stronger local efficacy, CRTx has been preferred over chemotherapy to down-size the tumor and enable secondary resectability for a long time .Since neoadjuvant is generally better tolerated than adjuvant therapy, patients are most likely to receive a sufficient treatment dosage and have the highest probability to receive a multimodality treatment at all, if the treatment starts prior to surgery—also for primarily resectable cancer. Several phase II trials and large database analyses suggest beneficial effects of neoadjuvant therapy including low surgical complication rate, high treatment exposure and local treatment effect with convincing overall survival. Although one might argue that patients with resectable pancreatic cancer should not be put at risk of treatment related complications or disease progression due to an ineffective treatment, this effect is extremely rare. In contrast, a large proportion of patients who undergo upfront surgery will not benefit the effects of an adjuvant therapy due to surgical complications . TherefoInterestingly, the use of neoadjuvant therapy has expanded over the past years without a clear scientific background . HoweverIn theory, the rationale for neoadjuvant therapy in patients with resectable pancreatic cancer would be the treatment of micrometastases prior to surgery, while the rationale in patients with unresectable cancer would be the secondary resectability of the tumor. These rationales are merging in patients with borderline resectable disease. Although the definitions of local resectability appear clear in the literature, the different groups of resectability are largely overlapping in the clinical routine. Consequently, the data on borderline resectable and irresectable cancer cannot be separately analyzed. Also, most studies on resectable tumors include borderline resectable tumors since they were judged technically resectable.The current literature suggests a stronger local effect of neoadjuvant CRTx considering the histological response to the treatment ,34,35. MMoreover, the estimated probability of micrometastases at the moment of surgery for most patients with pancreatic cancer is very high . Since sAlternatively to neoadjuvant CTx and CRTx for locally advanced pancreatic cancer, local ablative treatments such as irreversible electroporation (IRE) and high intensity focused ultrasound (HIFU) have been introduced into the management of locally unresectable tumors ,64,65. TThe interpretation of the current literature is still difficult due to differences in definitions and inclusion criteria. First, different definitions for “neoadjuvant” are used in the literature. Also, the definition of local resectability varies between the studies and is highly dependent on the individual expertise of the center. Also, the concept of borderline resectability is not followed in the recent trials on neoadjuvant therapy . MoreoveUnfortunately, solid randomized trials are lacking in order to confirm the theoretical advantages of neoadjuvant therapy. Major problems of trials on neoadjuvant therapy are the different attitudes of surgeons and oncologists and the preoperative cytological/histological confirmation of adenocarcinoma. Thus, randomized trials are highly anticipated to clarify which patient benefits from neoadjuvant therapy, and to define the ideal multimodality treatment for resectable, borderline and unresectable pancreatic cancer. Although all centers agree that patients with locally advanced disease and those with a high risk of an R1-resection should receive neoadjuvant therapy in order to increase the chance of a complete resection, patients with primarily resectable cancer potentially benefit the most from neoadjuvant therapy. Before new treatment combinations are tested for the different scenarios, such randomized trials should prove the concept of neoadjuvant therapy for the different indications. Following the dramatic improvements in surgical outcome of pancreatic surgery many studies are currently performed in order to test new treatment concepts and to evaluate the true role of neoadjuvant therapy. These studies should be actively supported in order to further improve long-term outcome of patients with resectable, borderline resectable and primarily unresectable pancreatic cancer (f.e. ,67,68,69


Cristina Mendes de Oliveira's name appeared incorrectly as “Cristine Mendes de Oliveira.”In the article, “Genotypic distribution of HHV-8 in AIDS individuals without and with Kaposi sarcoma: Is genotype B associated with better prognosis of AIDS-KS?”,


The MET receptor tyrosine kinase represents a promising target in cancer. PIK3CA activating mutations are common in several tumor types and can potentially confer resistance to anti-receptor tyrosine kinase therapy.E545K and PIK3CAH1047R, as well as in MET-expressing head and neck cancer cells with endogenous PIK3CA mutations. Endpoints included PI3K pathway activation, cell proliferation, colony-forming ability, cell death, wound-healing, and an in vivo model.MET and/or PI3K pathway inhibition was assessed in NIH3T3 cells harboring MET-activating point mutation with or without ectopic expression of PIK3CAE545K and PIK3CAH1047R confer resistance to MET inhibition in MET-driven models. PIK3CAH1047R was more potent than PIK3CAE545K at inducing resistance in PI3K pathway activation, cell proliferation, colony-forming ability, induction of cell death and wound-healing upon MET inhibition. Resistance to MET inhibition could be synergistically overcome by co-targeting PI3K. Furthermore, combined MET/PI3K inhibition led to enhanced anti-tumor activity in vivo in tumors harboring PIK3CAH1047R. In head and neck cancer cells the combination of MET/PI3K inhibitors led to more-than-additive effects.PIK3CAPIK3CA mutations can lead to resistance to MET inhibition, supporting future clinical evaluation of combinations of PI3K and MET inhibitors in common scenarios of malignant neoplasms featuring aberrant MET expression and PIK3CA mutations.The online version of this article (doi:10.1186/s12943-017-0660-5) contains supplementary material, which is available to authorized users. MET is the receptor tyrosine kinase (RTK) for scatter factor/hepatocyte growth factor (SF/HGF). Aberrant activation of the SF/HGF-MET axis is common in a wide range of human malignancies and promotes cell growth, invasiveness, metastatic potential, angiogenesis, and resistance to cell death following chemotherapy and radiotherapy –7. The eKRAS in cetuximab-treated patients with colorectal cancer. The authors found that 42.3% of the tumors harbored at least one KRAS mutation. Cetuximab was beneficial in terms of overall survival only in patients with wild-type KRAS. In order to optimize patient selection in this subgroup of patients, assessment of KRAS mutational status is currently performed prior to cetuximab therapy [KRAS mutations (exon 3 and 4) equally had an impact on responses to panitumumab in patients with colorectal cancer [Paralleling the preclinical and clinical developments in the field of RTK-targeted therapy, awareness on potential mechanisms of therapeutic resistance is an issue of major relevance. One such resistance mechanism is the presence of activating mutations affecting different members of signaling pathways downstream of RTKs, such as the MAPK and the PI3K pathways. The actual clinical relevance of this phenomenon was clearly outlined by Karapetis et al. , who ass therapy . In a lal cancer .PIK3CA-encoded p110α catalytic subunit of PI3K (PIK3CA) and tend to cluster at hotspots E542 and E545 within the helical domain, as well as H1047 in the kinase domain. Previous in vitro studies have shown that PIK3CA hotspot mutations lead to increased kinase activity and confer variable oncogenic features [E545K and PIK3CAH1047R were tumorigenic in mouse mammary models [E545K-induced tumors developed more slowly and were histologically less aggressive than PIK3CAH1047R-driven tumors. In contrast to these findings in mammary tumors, Trejo et al. [H1047R alone was not sufficient to promote initiation in lung cancer models. Instead, co-presence of BRAFV600E was required for initiation. In such context both mutations cooperated in tumor progression.Mutations along the MAPK pathway are well-established sources of resistance to RTK inhibitors, particularly against EGFR targeted therapies . Importafeatures , 16. Mory models . Howevero et al.  reportedThe PI3K-AKT-mTOR pathway is a critical regulator of cell metabolism, proliferation, survival, apoptosis and cell motility . PI3K acH1047R are equally more resistant to MET inhibition than their wild-type counterpart. In all cases, PIK3CA-induced resistance to MET inhibitors could be reverted by co-targeting PI3K.The implications of synchronous presence of aberrantly active MET and PIK3CA mutations in cancer has not been previously explored. Therefore, we hypothesized that in preclinical models of MET-driven tumors sensitive to MET inhibition, PIK3CA mutations may confer resistance to MET inhibition. Our data demonstrate that PI3K signaling remains active upon MET inhibition in tumors harboring PIK3CA mutations, rendering such tumors resistant to MET inhibition. In vivo tumor models harboring PIK3CAPIK3CA mutations. In this particular disease, PIK3CA are commonly encountered (over one-third of cases), in contrast with the more seldom MAPK or JAK/STAT mutations [Complementarily, we sought to assess the impact of dual MET/PI3K inhibition in models displaying ligand-induced MET activation and PI3K mutations. For this purpose, we selected cellular models of HNC endogenously harboring utations . Moreoveutations . Howeverutations . In HNC NIH3T3 cells stably expressing MET-activating mutation M1268T were provided by Dr. Laura Schmidt  and maintained as previously described .NIH3T3 MET M1268T cells were transfected using standard reverse transfection protocols with Lipofectamine 2000 (Invitrogen), using the following plasmids: PIK3CA E545K and H1047R . StableHNC cell lines FaDu (PIK3CA wild-type) and Detroit-562 (PIK3CA H1047R) were obtained from the American Type Culture Collection  and cultured in Minimum Essential Medium  supplemented with FCS 10%, antibiotic-antimycotic, and non-essential aminoacids . SCC-61 (PIK3CA E542K) cells were provided by Prof. M. Pruschy  and cultured in DMEM with supplements , 29. No The MET tyrosine kinase inhibitor tepotinib  was kindly provided by Merck KGaA . PI3K was inhibited with pictilisib . Both drugs were dissolved in DMSO and stored at−20 °C. DMSO concentrations were normalized in all experimental conditions.Recombinant human SF/HGF was purchased from R&D Systems and prepared following the manufacturer’s instructions. Treatments were carried out as described in Results.Cells were lysed with Non-ident NP40 buffer as previously described . XenograMembranes were incubated with appropriate secondary antibodies and signals were detected with the ECL kit (Amersham Pharmacia Biotech) or with infrared fluorescence on an Odyssey imager (Li-Cor biosciences).Immunoprecipitation was performed using the Dynabeads® Protein G kit following manufacturer’s instructions .With the exception of samples from in vivo tumors and organotypic slices, immunoblots shown are representative of at least three independent experiments.Cell viability was determined using a resazurin sodium salt reduction assay (Sigma). Briefly, after treatment for 48 h, cells were supplemented with medium containing 44 μM resazurin. Resazurin reduction was colorimetrically measured 1 and 6 h later (570/600 nm) with a Tecan Reader (Tecan Group Ltd.). Results were normalized to vehicle-treated controls and represent the mean of at least three independent experiments.50) value determination, resazurin assays were carried out after 72 h of treatment with increasing doses of tepotinib and pictilisib (0 to 10 μM). EC50 values were determined using the GraphPad software (version 5.03).For half maximal effective concentration . For this assay, cells were treated for 3 days and stained with green-fluorescent calcein-AM (viable cells) and red-fluorescent ethidium homodimer-1 (dead cells). Images from four independent experiments were captured under a fluorescent microscope (Leica DC 300 F) and number of live and dead cells quantified using ImageJ (imagej.nih.gov/ij/).Caspase-3 enzymatic activity was determined via a fluorogenic assay based on the caspase-3 specific substrate Ac-DEVD-AMC . The substrate was added to cell lysates after 72 h of treatment and fluorescence was measured at excitation and emission wavelengths of 380 nm and 460 nm respectively, with an Infinite 200 plate reader (Tecan Group Ltd.). Caspase-3 activity was normalized to protein content and results shown are representative of at least three independent experiments.“Analyze Particles” plug-in of ImageJ (imagej.nih.gov/ij/). Experiments were performed three times.Cells were plated the day before treatment at densities ranging from 500 to 2500 cells/well in 6-well plates and then treated with the indicated drug concentrations. Colonies were allowed to form for 5–7 days and subsequently fixed and stained with 2% crystal violet dissolved in 1:3 methanol and 2:3 acetic acid (v:v). Colonies (>50 cells) were scored and quantified using the OrisTMCell Migration Assembly Kit (AMS Biotechnology). Cells were seeded in 96-well plates containing cell stoppers  and left to attach overnight. Thereupon, cell stoppers were removed and cells were treated as indicated. Pictures were captured at baseline and after 48 h using a Leica DC 300 F microscope. Images obtained from at least three independent experiments were quantified with the ImageJ software (imagej.nih.gov/ij/). Results are presented as number of invading cells for NIH3T3 cells (scattering invasion pattern) or as percentage of wound-closure relative to baseline for HNC cell lines (pushing-front invasion pattern).Cell migration was assessed with the H1047R-transfected NIH3T3 METM1268T cells were injected subcutaneously in both flanks of 7 to 9 weeks-old female nude mice . Tumor growth was estimated by regular caliper measurements using the following formula:All animal experiments were conducted in strict compliance with Swiss Federal guidelines. Vector and PIK3CA3, animals were randomly allocated to one of four treatment groups : vehicle , tepotinib 50 mg/kg alone, pictilisib 50 mg/kg alone, and combination of both drugs. Drugs were administered via oral gavage and daily measurements were recorded. Animals were euthanized 4 h after the last treatment, either at the scheduled endpoint or prematurely when interruption criteria were met (tumor volume superior to 1 cm3 or persistent tumor bleeding). For determination of in vivo proliferation, 5-bromo-2′-deoxyuridine  was injected intra-peritoneally 4 h before euthanasia.where V = tumoral volume, L = larger dimension and W = shorter dimension. Upon tumors reaching an average of 150–200 mmAs a complementary assessment method, we generated organotypic tissue cultures as previously described  from freTumor xenograft tissue arrays of 5 mm were generated using the EZ-TMA Manual Tissue Microarray Kit 4 (IHC World). Sections of 5 μm were then cut, deparaffinized and rehydrated. Antigen retrieval was performed by heating slides for 10 min in a microwave oven (600–700 W) in Tris-EGTA buffer (pH 9.0). Sections were incubated overnight at 4 °C with specific primary antibodies: BrdU (1:300), pS6 (1:800), and p-MET (1:150). BrdU was purchased from Abcam and the remaining antibodies from Cell Signaling Technology.Slides were washed and incubated with secondary antibodies from Life Technologies (goat anti-rabbit 488 and goat anti-rat 555), 1:500, for 1 h at room temperature. Slides were scanned using the Pannoramic Midi digital slide scanner and analyzed by CellQuant software (3DHISTECH Ltd).U-test unless otherwise reported. Tumor sizes were compared by one-way ANOVA. Statistical significance was set at p < 0.05. Definitions for p-values are as follows throughout the manuscript: *p = 0.01–0.05, **p = 0.001–0.01, ***p < 0.001. Absence of labels implies non-significant differences.Statistical analysis and graphic representation of data was performed with GraphPad (version 5.03). Data are presented as relative averages ± SD or fold-induction ± SD, as indicated. Statistical comparisons were performed using the Mann-Whitney M1268T. Ectopic expression of METM1268T leads to constitutive MET kinase activation and results in high sensitivity to MET inhibitors such as tepotinib or SU11274 [We first sought to assess the impact of PIK3CA mutations in models mimicking clinical scenarios featuring MET constitutive activation, such as gastric or lung cancer , 33. For SU11274 , 34.M1268T cells were subsequently transfected with vectors containing the two commonest PIK3CA mutations encountered in human cancer: PIK3CAE545K  and PIK3CAH1047R (kinase domain). Cells transfected with the empty pBabe vector (termed hereafter ‘vector cells’) were used as control  displayed increased proliferation relative to vector cells and PIK3CAH1047R mutant cells (H1047R cells) , while H1047R cells proliferated slower than vector cells , and the findings resulting from immunoblots and cell death assays (suggesting resistance), we performed colony-forming assays over a period of 7 days in order to avoid the differences potentially due to the substantially higher proliferation rate of E545K with respect to vector and H1047R cells. These assays confirmed significant resistance of both E545K and H1047R cells upon MET inhibition when compared to vector cells. Resistance was successfully reversed by co-targeting PI3K , the number of cells injected was 500,000 for vector cells and 100,000 for H1047R cells. Tumors in the H1047R group reached an average volume of 150 mm3 17 days after injection, versus 24 days in the vector group , as well as higher efficacy of pictilisib in the same group . H1047R tumors treated with pictilisib alone displayed a significantly higher percentage of BrdU positive nuclei  and SCC-61 (E542K) , 29. FaD7R and SC50 concentrations of tepotinib  and pictilisib . Combination treatment led to more effective abrogation of AKT phosphorylation than either tepotinib or pictilisib alone, but p-S6 levels were generally unaltered  than either FaDu or SCC-61 cells  datasets available at the cBio Portal for Cancer Genomics  revThese figures are globally rendered more complex when considering different subgroups of patients with different subtypes of a same cancer entity. With respect with breast cancer for instance, KRAS mutation rate in metastatic breast cancer was estimated at approximately 12% . Along tAnother important aspect is that different tumor entities have variable degrees of dependence on signaling inputs from given RTKs. For this reason, in the present study we sought to assess differences between models featuring MET constitutive activation and models with ligand-induced activation. Our findings indicate that ectopically-expressed PIK3CA hotspot mutations confer resistance to MET inhibition in initially-sensitive MET-driven models, both in vitro and in vivo. In ligand-dependent HNC cellular models, dual MET and PI3K blockade was superior to single inhibition on downstream signaling and wound-healing ability. Importantly however, stronger synergism between MET and PI3K inhibition was observed only in HNC cells harboring PIK3CA mutations. Importantly, the effect of MET inhibition was not only restored but also enhanced by co-targeting PI3K in both sets of models.MET-gene amplification and were extremely sensitive to MET inhibition. Further in the same direction, small cohorts of patients who experience drastic and prolonged disease remission after MET inhibition indicate that at least subgroups of gastric cancers are indeed strongly dependent on MET signaling [oncogene addiction [The differences seen between MET-driven models and non-MET-driven models endogenously harboring PIK3CA mutations are most relevant also in terms of pre-therapeutic stratification. It is increasingly evident that human cancers and their derived preclinical models display differential degrees of dependence on signaling by specific RTKs as well as other kinases. Therefore, while some malignancies quite uniquely rely on one given RTK, the same is not true for some other cancer types. For instance, with respect to MET, McDermott et al.  assessedignaling , 46. Sucddiction . Classicddiction . Most ilddiction .In contrast, entities like HNC do not feature oncogene addiction for any given RTK, and tend to rely more on ligand-receptor oncogenic loops, as well as on RTK cooperation and redundant signaling , 24, 49.H1047R is a stronger effector of resistance to MET inhibitors, but further translational studies should address this issue given the variable prevalence of helical or catalytic domain mutations seen in different human malignancies .Specifically regarding the potential relevance of MET/PI3K combinations, in the context of a retrospective review of over 2000 patients, Maryam et al.  reportedOur current study provides evidence for the first time supporting the notion of co-targeting MET and PI3K as a potential therapeutic strategy in common clinical scenarios, featuring both constitutive and ligand-induced MET activation along with PIK3CA mutations.While the combinatorial strategy we evaluate here could be therapeutically exploited, it is crucial that upcoming clinical trials consider the characteristics described above, namely mutational status of downstream signaling pathways and activation status of selected RTKs, in order to identify the subgroups of patients most likely to benefit from treatment.MET, the receptor tyrosine kinase (RTK) for scatter factor/hepatocyte growth factor, is involved in acquisition and maintenance of critical oncogenic and invasive features in several types of human cancer and represents a promising molecular therapeutic target. In parallel, accumulating evidence supports the notion that intracellular signaling pathways downstream of RTKs, such as the PI3K pathway, constitute potential sources of resistance to RTK-targeted cancer therapy. Along the same lines, PIK3CA activating mutations are widespread in cancer. In this study we have examined the impact of common PIK3CA helical and catalytic domains hotspot mutations upon MET inhibition in preclinical MET-driven and head and neck cancer models, providing therefore a first evaluation of MET targeting in the context of common PI3K mutations. The results suggest a potential added benefit of this combinatorial approach in common clinical scenarios featuring aberrant MET activation and PIK3CA mutations, warranting evaluation in upcoming clinical trials.Additional file 1: Figure S1.M1268T PIK3CA-mutated cell lines. A, transfection and expression of mutated variants E545K and H1047R was confirmed by detection of the HA tag after immunoprecipitation of HA and detection of p110α. B, comparison of cell proliferation in cells transfected with control vector, pBabe-E545K, and pBabe-H1047R. C, dose-response plots upon exposure to tepotinib and pictilisib and EC50 values with 95% confidence intervals (CI) for both drugs. (PNG 108 kb)Generation of NIH3T3 METAdditional file 2: Figure S2.Resistance to MET inhibition in H1047R tumors in vivo is accompanied by sustained PI3K pathway activation. A, representative images of p-MET and pS6 stained tumors. MET inhibition (tepotinib 50 mg/kg) effectively abrogated MET phosphorylation both in vector and H1047R, but decrease in S6 phosphorylation was only seen in vector tumors. B, immunoblots of AKT in tumoral lysates (two per condition). Right: determination of densitometric values. C, organotypic tissue cultures (left) were used to evaluate AKT phosphorylation levels (center) upon single and dual MET/PI3K inhibition after 3 days of ex vivo culture. Right: determination of densitometric values. (PNG 4913 kb)Additional file 3: Figure S3.50 value determination in FaDu (PIK3CA wild-type), Detroit-562 (PIK3CAH1047R), and SCC-61 (PIK3CAE542K). Bottom: EC50 values and 95% confidence intervals for MET inhibitor tepotinib and PI3K inhibitor pictilisib. (PNG 46 kb)Sensitivity to MET and PI3K inhibitors in HNC cell lines. Top: Dose-response plots for EC


Regarding phase II metabolism, UGT2B7, 1A8 and 1A3 showed highest activity in glucuronidation of tamoxifen bisphenol and metabolite E. Anti-estrogenic metabolites (Z)-4-hydroxytamoxifen, (Z)-endoxifen and (Z)-norendoxifen inhibited the activity of CYP2C enzymes while tamoxifen bisphenol consistently induced CYPs similar to rifampicin and phenobarbital. On the transcript level, highest induction up to 5.6-fold was observed for CYP3A4 by tamoxifen, (Z)-4-hydroxytamoxifen, tamoxifen bisphenol and (E)-metabolite E. Estrogen-like tamoxifen metabolites are formed in CYP-dependent reactions and are further metabolized by glucuronidation. The induction of CYP activity by tamoxifen bisphenol and the inhibition of CYP2C enzymes by anti-estrogenic metabolites may lead to drug–drug-interactions.Tamoxifen, a standard therapy for breast cancer, is metabolized to compounds with anti-estrogenic as well as estrogen-like action at the estrogen receptor. Little is known about the formation of estrogen-like metabolites and their biological impact. Thus, we characterized the estrogen-like metabolites tamoxifen bisphenol and metabolite E for their metabolic pathway and their influence on cytochrome P450 activity and ADME gene expression. The formation of tamoxifen bisphenol and metabolite E was studied in human liver microsomes and Supersomes™. Cellular metabolism and impact on CYP enzymes was analyzed in upcyte® hepatocytes. The influence of 5 µM of tamoxifen, anti-estrogenic and estrogen-like metabolites on CYP activity was measured by HPLC MS/MS and on ADME gene expression using RT-PCR analyses. Metabolite E was formed from tamoxifen by CYP2C19, 3A and 1A2 and from desmethyltamoxifen by CYP2D6, 1A2 and 3A. Tamoxifen bisphenol was mainly formed from The online version of this article (10.1007/s00204-017-2147-y) contains supplementary material, which is available to authorized users. In addition, there are many more metabolites with unknown or even estrogen-like properties. Of the latter, tamoxifen bisphenol (4-[1-(4-hydroxyphenyl)-2-phenylbut-1-en-1-yl]phenol, Tam-Bis), (Z)- and (E)-metabolite E  were found to be full agonists regarding estrogen-regulated mRNA expression in vitro, leading to cell proliferation in estrogen-dependent breast cancer cell lines - and (E)-Met E may hamper Tam therapy success.Tamoxifen (Tam) is the standard therapy for estrogen receptor (ER)-positive breast cancer in premenopausal women and in men and an alternative to aromatase inhibitors in postmenopausal women. As a selective estrogen receptor modulator (SERM) Tam acts by competitive binding to the ER, which leads to tissue-dependent receptor inhibition or activation, e.g. in breast or bone, respectively (Shang and Brown N-desmethyltamoxifen (DM-Tam) and the anti-estrogenic metabolites Endox and 4OH-Tam are known to depend on the activity of certain CYP enzymes, namely 2D6, 2B6, 3A4 and 2C9  inhibiting various members of the CYP family  were prepared as described  without calcium or magnesium were purchased from Life Technologies GmbH, Darmstadt. Trypsin/EDTA was obtained from Fisher scientific GmbH, Nidderau. Fetal calf serum (FCS) was purchased from Sigma–Aldrich, Steinheim. Plastic lab ware and collagen type I from rat tail were purchased from BD Biosciences, Heidelberg.Supersomes™ were obtained from Corning Incorporated, Wiesbaden, Germany, and were specific for CYP isoenzymes CYP1A1, 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 3A4, 3A5 and 19A1. All Supersomes™ contained oxidoreductase. Cytochrome b5 was present in the Supersomes™ of CYP2A6, 2B6, 2C8, 2C9, 3A4 and 3A5. UDP-glucuronosyltransferases (UGT)containing Supersomes™ were specific for the following isoenzymes: 1A1, 1A2, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B10, 2B15 and 2B17. Upcyte® Hepatocyte growth medium containing supplement A, B and 2 mM S-mephenytoin and atorvastatin ; propafenone and 5-OH-propafenone ; [2H7]-5-hydroxypropafenone hydrochloride, bupropion hydrochloride, hydroxybupropion hydrochloride, [2H3]-hydroxybupropion hydrochloride, 4′-hydroxymephenytoin and [2H3]-4′-hydroxymephenytoin were synthesized in house. For metabolism studies of Tam metabolites, tamoxifen and (Z)-4-hydroxytamoxifen were purchased from Sigma-Aldrich, Steinheim and [2H5]-N-desmethyl tamoxifen from Toronto Research Chemicals, Toronto. N-desmethyl tamoxifen was a gift from Klinge Pharma GmbH, Munich. (Z)-endoxifen (> 98% (Z)), tamoxifen bisphenol, (E)- and (Z)-metabolite E, glucuronides of 4OH-Tam, Endox, Tam-Bis and both isomers of Met E, [2H5]-Bisphenol, (E/Z)-[2H5]-metabolite E, [2H3]-tamoxifen, (E)- and (Z)-[2H5]-4-OH-tamoxifen, (E)- and (Z)-[2H5]-Tamoxifen-4-O-glucuronide were synthesized in house ; 2 in a humidified incubator. Before starting experiments, cells were cultivated in hepatocyte culture medium containing 0.5% (v/v) DMSO (Preculture medium) for 5 days (density of 0.8 × 104 cells/cm2) and were subcultivated in high-performance medium containing 0.1% (v/v) DMSO (Metabolism conditioning medium) for 3–5 days . The medium was replaced every 2–3 days. All plastic ware used for cell cultivation was coated with collagen type I. For validation of both cell lines, the base line CYP activity and the induction of CYP enzymes upon treatment with the prototypical inducers rifampicin (Rifa) and phenobarbital (PB) on the level of protein expression and enzymatic activity, as well as the mRNA expression of ADME related genes, were determined .upcyte® hepatocytes 653-03 and 653-03 #138 (653-03 2D6) were obtained from upcyte technologies GmbH, Hamburg. The CYP2D6-expressing subclone #138 was generated by lentiviral transduction as a derivative of the CYP2D6 lacking 653-03 cell line by upcyte technologies GmbH. Cells were cultivated in hepatocyte culture medium at 37 °C and 5 °% CO(E)- and (Z)-Met E) in 100 mM sodium phosphate buffer (pH 7.4) for 10 min on ice. The reaction was started by adding a NADPH regenerating system  to the samples and immediately put in a 37 °C water bath for 30 min. The reaction was stopped with the same volume of acetonitrile (ACN) containing 1% of acetic acid together with the corresponding deuterated internal standards. Regarding incubations with Supersomes™ containing UGTs, 5 µg of protein was incubated with 250 mM MgCl2 and 2.5 ng of alamethicin for 30 min on ice, followed by 10 min at 37 °C in a water bath. Subsequently, the Supersomes™ were incubated with 10 µM of substrates - and (Z)-Met E) and the glucuronidation was started by addition of 10 mM of uridine 5′-diphospho-glucuronic acid to the samples in a water bath at 37 °C for 30 min. The reaction was stopped as described above. Metabolites were analyzed by HPLC-ESI-MS/MS.Supersomes™ (20 pmol CYP enzyme) or pooled human liver microsomes  were pre-incubated in duplicates with 50 µM of substrates - and (Z)-Metabolite E, HLM were incubated with increasing substrate concentrations ranging from 1 to 50 µM as described above. As control for each concentration a sample without NADPH-regenerating system was prepared.For the assessment of formation kinetics of Tam-Bis, For metabolism studies in ucyte® hepatocytes, the conditioned cells were incubated as triplicates with 1 µM of Tam (control 0.01% DMSO) in high-performance medium for up to 144 h without medium change. The culture medium was collected after 24, 48, 72, 96 and 144 h followed by metabolite measurements using HPLC-ESI-MS/MS.n ≥ 5) and the expression of ADME genes  were assessed by incubating cells with 5 µM of the test substances -Met E, (Z)-Met E, control 0.05% DMSO) in high-performance medium for 72 h with daily renewal of treatment medium. The induction potency of CYP activity by Tam-Bis was evaluated by incubating cells with increasing concentrations of Tam-Bis ranging from 50 pM to 5 µM.The influence of Tam metabolites on the activity of CYP enzymes , 5 µM amodiaquine (CYP2C8), 100 µM tolbutamide (CYP2C9), 100 µM S-mephenytoin (CYP2C19), 25 µM bupropion (CYP2B6), 5 µM propafenone (CYP2D6) and 35 µM atorvastatin (CYP3A4). Metabolites formed were quantified via HPLC-MS/MS as described previously . cDNA was synthesized using TaqMan Reverse Transcription Reagents  and the expression of 25 genes representative of phase I and phase II drug metabolism and transport was quantified by Fluidigm’s BioMark HD high-throughput quantitative chip platform . For relative gene expression analysis, the delta-delta-Ct (ΔΔCt) method was used: the Ct values of the target mRNA were first normalized to those of the endogenous control glyceraldehyde-3-phosphate dehydrogenase (GAPDH) followed by dividing values of experimental samples by their corresponding DMSO-treated controls to calculate the x-fold changes of mRNA expression.(E)- and (Z)-Tam-Bis-Gluc, the AUC of the peaks of both isomers were combined for the calculation of the calibration curves and of concentration in the cell media samples. In the case of supersome™ incubations, the isomers of (E)- and (Z)-Tam-Bis-Gluc were quantified separately.For HPLC-MS/MS measurements, calculation of linear calibration curves and quantification were performed using MassHunter Quantitative Analysis . Regarding −1 min−1 and fmol pmol−1 min−1, respectively, and was calculated as the mean ± SD. For formation kinetics Vmax and Km was determined via Michealis-Menten kinetic using GraphPad Prism (version 5.04) after background substraction of the corresponding control sample. The intrinsic Clearance (Clint) was given as Vmax/Km ratio. Regarding Tam-Bis formation from hydroxylated Tam metabolites, due to substrate inhibition at higher concentrations Clint was determined from the initial slope of the curve. Concentrations of Tam and its metabolites in hepatocyte incubations are given as mean values ± SD. Activity of CYP enzymes in induction assays is given as the fold-change (mean ± SD) compared to the corresponding DMSO-treated control. Differences between induced and control samples were compared using Welch’s t-test , when the fold-change was considered biologically relevant (1.5-fold increase or decrease in fold-changes) and corrected for multiple testing according to Bonferroni. Gene expression patterns of treated cells are given as fold-change compared to the corresponding DMSO-treated control samples, as described above. An unpaired t-test assuming unequal variances was used for testing of differences in the expression patterns, when the fold-change was considered biologically relevant (1.5-fold increase or decrease in fold-changes) and corrected using Bonferroni adjustments. In induction and inhibition experiments corresponding EC50 and IC50 values were calculated by nonlinear regression. In all experiments a level of probability (p) ≤ 0.05 for statistical testing was considered significant. The data and statistical analysis comply with the recommendations on experimental design and analysis in pharmacology  background substraction of these levels was done in all incubations. In HLM and supersome™ incubations the formation rate is given as nmol µg(Z)- and (E)-Met E from Tam and relevant precursors. Both isomers of Met E were formed from Tam, DM-Tam and DDM-Tam with 8-273 times higher formation rates compared to the control, indicating a NADPH-dependent, microsomal reaction. Formation rates for the (Z)-isomer were highest for Tam with up to 9.4 pmol mg−1 min−1, followed by DDM-Tam and DM-Tam with 4 pmol mg−1 min−1 -isomer was metabolized mainly from Tam, followed by DDM-Tam and DM-Tam -isomer were only approximately 10% compared to (Z)-Met E. Tam-Bis was also formed in a NADPH-dependent, microsomal reaction from the anti-estrogenic Tam metabolites 4OH-Tam, Endox and Norendox with up to 18 times higher formation rates compared to the control. Here, highest formation rates were observed from 4OH-Tam with 3.3 pmol mg−1 min−1, followed by Endox and Norendox -isomer and 2–6 times for the (E)-isomer, respectively  were incubated with and without a NADPH-regenerating system to characterize the formation of the estrogen-like Tam metabolites Tam-Bis, (Z)-Met E highest formation rates were found from Tam by CYP2C19, CYP3A4/5 and 1A2 with up to 8.9 fmol pmol−1 min−1. CYP2D6, 2B6 and 1A1 showed additional but less pronounced activities in the side chain cleavage of Tam to (Z)-Met E -Met E were obtained by CYP2D6 with 5.6 fmol pmol−1 min−1, followed by CYP1A2 and CYP3A4/5 with 5.1 and 4.6 fmol∙pmol−1 min−1, respectively. For the metabolism from DDM-Tam to (Z)-Met E, CYP2D6 showed highest activity with 7.9 fmol pmol−1 min−1, followed by CYP3A4 with 3.2 fmol pmol−1 min−1 -Met E was comparable to that of the (Z)-isomer but with approximately 10-times lower formation rates. Accordingly, highest formation rates catalyzed by CYP2C19 were observed using Tam as a substrate. The metabolic rate from DM-Tam and DDM-Tam to (E)-Met E did not differ between the main contributors CYP2D6, 3A4 and 1A2 -Met E to Tam-Bis by CYP2B6 and of (Z)-Met E to Tam-Bis by CYP2C19 was observed - and the (E)-isomer, respectively. For the glucuronidation of (Z)-Met E highest formation rates were found for UGT2B7 with 143 pmol mg−1 min−1, followed by 1A9 and 1A3, whereas the (E)-isomer was mainly glucuronidated by UGT1A8, 2B7 and 1A3 with 4–14 times higher formation rates as its (Z)-isomer /(Z)-ratio 0.6), UGT1A8 mainly produced the (E)-isomer and UGT2B17 the (Z)-isomer of Tam-Bis-Gluc - and (E)-Met E from metabolic precursors HLM were incubated with increasing substrate concentrations. Tam showed highest Vmax rates of 9.2 pmol mg−1 min−1 for (Z)-Met E, whereas DDM-Tam showed lowest Km concentrations for the formation to this isomer. Regarding (E)-Met E the Km values did not differ between Tam and DM-Tam. The intrinsic Clearance (Clint) for Tam, DM-Tam and DDM-Tam to both isomers of Met E showed highest values for the precursor Tam of 0.112 and 0.049 µl mg−1 min−1, respectively, followed by DDM-Tam and DM-Tam -Met E were in general 2-times lower than for the (Z)-isomer. Regarding Tam-Bis formation, the hydroxylation of (Z)-Met E was two times faster than from the (E)-isomer (17.2 pmol mg−1 min−1 compared to 8.1 pmol mg−1 min−1), whereas Km values were comparable (8.2 µM from (Z)- and 10.4 µM from (E)-Met E). Therefore, highest Clint was observed from (Z)-Met E with 2.103 µl mg−1 min−1, exceeding the Clint from anti-estrogenic Tam metabolites to Tam-Bis up to 40-fold and up to 16-fold from the corresponding (E)-isomer  were used as models for the absence or presence of functional CYP2D6 enzyme activity, resembling opposite phenotypes that are strongly associated with Tam metabolism -Met E that decreased over time -isomer were, like in previous in vitro findings, at about 10% of those of (Z)-Met E at all time points -Met E and 2.0 nM for (E)-Met E, thus exceeding the concentrations of their aglycones up to 45-fold in the cell media  followed by CYP3A4 (178 nM) and CYP2B6 (187 nM). For the remaining CYP enzymes CYP2C8, 2C9 and 1A2 EC50 concentrations above 400 nM were observed (Table E) and CYP3A4 (Z) with similar EC50 potency for CYP3A4 of 308 and 277 nM for (Z)- and (E)-Met E, respectively -Met E. Although some additional ADME genes were upregulated following Met E isomer or differently regulated upon anti-estrogenic treatment, data were not consistent among both cell lines and therefore remain elusive.On the level of gene expression, a number of ADME genes were differently expressed upon exposure to Tam or 4-hydroxylated anti-estrogenic metabolites; however, only those genes that were significantly regulated in both cell strains were considered for evaluation: In accordance with the induction of enzyme activity by Tam and 4OH-Tam, CYP3A4 gene expression was most strongly upregulated up to 5.6-fold upon 4OH-Tam treatment . Furthermore, the expression of CYP1A1 was induced up to 2.3-fold by Endox. In contrast, downregulation was observed for CYP2A6 and transporter gene SLC10A1 by 4OH-Tam up to twofold . With respect to estrogen-like Tam metabolites, the most frequent expression alterations of ADME genes in both cell strains were observed upon Tam-Bis treatment: CYP3A4, CYP1A1 and efflux transporter ABCG2 were induced up to 4.5-fold, whereas expression levels of solute carrier family transporters SLC10A1 and SLC22A7 were up to 2.5-fold lower. For Met E, CYP1A1 expression was upregulated 3.3-fold by (Z)-Endox and, therefore, impacts clinical outcome - and (E)-Met E show full estrogen-like properties by activating ER in breast cells  strongly depend on the CYP2D6 phenotype which may also impact the formation of estrogen-like metabolites, hepatocyte cells representative of a poor metabolizer phenotype and their CYP2D6 expressing derivatives (extensive metabolizer) were used. As expected, DM-Tam was observed as the primary metabolite which was subsequently hydroxylated. Moreover, a strong CYP2D6 dependency of Endox could be confirmed as this metabolite was only detected in the supernatant of the 653-03 2D6 cells. Noteworthy, Endox-glucuronide was found in small concentrations in the presence of 653-03 cells confirming the relevance of a minor contribution of Endox formation via CYP2D6-independent pathways from 4OH-Tam - to (Z)-isomer were independent of the CYP2D6 phenotype, which is also in line with our in vitro data. With respect to phase II metabolism, the concentrations of the glucuronides of Tam-Bis and Met E were more than 10-times higher compared to the unconjugated compounds. Thus, glucuronidation appears to be an important clearance path for estrogen-like metabolites confirming previous data -Met E to its glucuronide was smaller compared to the respective ratio of the (Z)-isomers indicating a faster glucuronidation of the (E)-isomer, a finding that resembles data for the (E)-isomers of 4OH-Tam and Endox -Met E stimulation suggests that these Tam metabolites act through PXR-activated induction of gene regulation  activity - and (E)-Met E were identified for the first time and integrated into the complex metabolism of Tam Supplementary material 2 (PNG 3687 KB)Supplementary material 3 (PNG 42 KB)Supplementary material 4 (PNG 119 KB)


IgG4-related disease (IgG4-RD) is a rare fibroinflammatory condition that can affect almost any organ, characterized by swollen lesions and often by eosinophilia and elevated serum IgG4 concentrations. The diagnosis of IgG4-RD is a challenging task: in fact, single or multiple organs can be affected and clinical, serological, and histological findings can be heterogeneous. In IgG4-RD, the involvement of salivary glands is observed in 27% to 53% of patients. Several organ-specific conditions, now recognized as different manifestations of IgG4-related sialadenitis (IgG4-RS), were viewed in the past as individual disease entities. The study of salivary glands may sometimes be complex, because of the number of pathological conditions that may affect them, often with overlapping clinical pictures. Integration of different imaging techniques is often required in the case of swelling of salivary glands, even though biopsy remains the gold standard for a definite diagnosis of IgG4-RS. Thus, in this review, we discuss new insights in the pathogenesis of IgG4-RD, focusing on its clinical aspects and the tools that are currently available for a correct differential diagnosis when the salivary glands are involved. Presentfibrosis –8. These+ T cells are the most abundant cells in IgG4-RD lesions; given the eosinophil infiltrate and the levels of IgG4 and IgE, a prominent role of T helper cells type 2 (Th2) cells has been proposed. However, Mattoo et al. [+ CTLs in the blood of patients with IgG4-RD [+ CTLs infiltrated tissue lesions and were the dominant CD4+ T cells at disease sites, while CD4+GATA3+ Th2 cells were sparse [+ CTLs, detected in both peripheral blood and fibrotic lesions of IgG4-RD patients, seems to actively contribute to the disease process, particularly to tissue injury and fibrosis. CD4+ CTLs, bearing SLAMF7 on their surface, might be actively involved in the fibrotic processes in IgG4-RD, releasing profibrotic mediators such as IL-1β, TGF-β, and INF-γ [+SLAMF7+ CTLs slowly declines following B cell depletion in IgG4-RD patients, suggesting a direct link between B cells lineage and CD4+SLAMF7+ CTLs in the pathogenetic mechanisms of IgG4-RD. However, these cells do not express CD20 on their surface and thus are not a target of anti-CD20 treatment, suggesting that CD4+ CTLs are sustained by B cells and plasmablasts.In the last decade, several pathophysiological mechanisms, potentially responsible for the development of IgG4-RD, have been described. B cells and plasmablasts seem to play an important role in IgG4-RD, secreting autoantibodies or acting as antigen presenting cells in the expansion of pathogenic T cells , 9. Oligo et al.  demonstr IgG4-RD . By usine sparse . This clnd INF-γ . To a cend INF-γ . In addihigh Tfh cells have been detected in peripheral blood of patients, functionally effective in driving IgG4 production from autologous B cells and responsive to steroid treatment [Recently, a role of T follicular helper (Tfh) cells in IgG4-RD pathogenesis has also been proposed –17. Highreatment , 16. A dMoreover, these new data on T cell subpopulations suggest novel pathogenetic mechanisms and innovative therapeutic approaches to IgG4-RD.IgG4-RD mainly involves middle-aged to elderly males, unlike classic autoimmune diseases such as systemic lupus erythematosus and Sjogren's syndrome (SSj) that mostly affect females. IgG4-RD occurs in a subacute form in most patients, without the rapid onset of general symptoms such as fever. A minority of patients have weight loss, dramatic elevations of acute phase markers, and other manifestations of systemic inflammation. IgG4-RD typically comes to medical attention because of single-organ involvement, but more widespread disease is often observed following an accurate work-up . Involve+ plasma cells at histology. When serological criteria  are not fulfilled, the disease is considered “probable” and “possible” when only the clinical and serological criteria are met. Less stringent criteria are used in the case of type I autoimmune pancreatitis (AIP) and Mikulicz's disease (MD): in these localizations, histological data are not essential [+ plasma cells in tissues are a more specific finding, but not exclusive of IgG4-RD. It is in fact observed in ANCA-associated vasculitis and urticarial vasculitis, in hematological malignancies, in pancreatic or lung cancer and sarcoma, in tumor of salivary glands and lymphoma, in infections, in inflammatory bowel diseases and diverticulitis, and in rheumatoid arthritis and histiocytosis. However, the other typical hystopathological findings of the disease are not present in any of these disorders. Therefore, histology remains, up to now, mandatory for differential diagnosis; clinicopathological correlation is also essential.The diagnosis of IgG4-RD is a challenging task: in fact, single or multiple organs can be affected and clinical, serological, and histological findings can be heterogeneous. IgG4-RD can be suspected in the presence of swollen lesions or diffuse/localized swelling in one or more organs. The diagnostic work-up involves laboratory investigations and imaging such as ultrasonography, computerized tomography (CT) scan, magnetic resonance imaging (MRI), and positron emission tomography (PET). Histopathology is mandatory for diagnostic purposes and also to exclude neoplastic or other inflammatory disorders . CriteriIn a few cases, spontaneous remission of the disease has been reported and “watchful waiting” represents an option in case of involvement limited to submandibular glands and lymph nodes. When vital organs are affected, or the disease has an aggressive course, treatment is necessary to prevent organ dysfunction. IgG4-RD has a significant response to treatment with immunosuppressants. Steroids represent the cornerstone of treatment. It has been shown that steroid treatment improves the function of affected organs, prevents organ damage, and decreases the rate of recurrence . ResponsSteroid-sparing agents are often employed, as in other autoimmune disorders, to maintain disease control with lower steroid dosage or without steroids. Azathioprine, methotrexate, and mycophenolate mofetil have all been used, and less often 6-mercaptopurine and cyclophosphamide .+ B cells and serum IgG4 levels suggests that IgG4 are mainly produced by short-lived plasmablasts and plasma cells, rapidly depleted by a treatment affecting mature B cells. Rituximab therapy reduces inflammatory infiltrate and also, albeit partially, fibrosis [In patients with recurrent or refractory disease, the monoclonal anti-CD20 Rituximab has been proven to be effective, inducing a decrease in serum IgG4 levels and a rapid clinical response. The parallel decrease in CD20fibrosis .In IgG4-RD, the involvement of salivary glands is observed in 27% to 53% of the patients . SeveralIn IgG4-RS, the swelling of lacrimal and salivary glands is mostly, but not exclusively, bilateral and painless and persists generally for more than 3 months . Submand+ plasmablasts) led to the diagnosis.In our cohort of 20 IgG4-RD patients , salivary gland involvement was observed in 3 cases. Diagnosis of IgG4-RD, according to the above described criteria [The other two patients were affected by both MD and Kuttner's tumor, with AIP and retroperitoneal fibrosis. Their clinical picture was characterized by longer disease duration , higher serum IgG4 titer (1870 mg/dL and 304 mg/dL at the diagnosis), and more frequent relapses of the disease. In one patient, an enlargement of the parotid was detected by PET analysis ; in the + plasma cells infiltration, and the response to steroids in IgG4-RD. However, in a few cases, the two disorders can coexist. Nakashima et al. [+ plasma cells in the minor salivary glands of 3 patients. On the other hand, Yamamoto et al. [In the presence of a mass lesion of salivary glands, the key point is to exclude a tumor. Differential diagnosis involves salivary gland neoplasms and other malignancies such as lymphoma or metastatic tumors. As an IgG4-producing marginal zone B cell lymphoma was described, to exclude a lymphoma may not always be easy . Other ea et al.  describea et al.  reporteda et al.  describeo et al.  describeThe study of salivary glands may sometimes be complex, because of the number of pathological conditions that may affect them, often with overlapping clinical pictures. Integration of different imaging techniques is often required in the case of swelling of salivary glands, even though biopsy remains the gold standard for a definite diagnosis of IgG4-RS. Among the different methods of imaging useful for the study of salivary glands, ultrasonography represents the most widely used technique. This is mainly due to its noninvasiveness and high tolerability for the patient, as well as for the low cost. Even if some ultrasonography features can be shared by IgG4-RS and SSj , the recPET is an emerging diagnostic option in the context of IgG4-RD, since organ lesions accumulate 18F-fludeoxyglucose at high concentration. Although this technology does not allow a specific distinction between inflammatory and cancerous lesions, it can be very useful in the identification of organ involvement beyond the salivary glands, such as the pancreas, retroperitoneum, and periaortic tissue, that can be affected by IgG4-RD, even if clinically silent. Moreover, this technique could be a tool for targeting specific biopsy sites, monitoring disease activity, and also evaluating response to treatment . HoweverEven if CT scan and MRI are useful techniques in the diagnosis of salivary glands swelling, they have some limitations in IgG4-RS diagnosis. Recently, Shimizu et al.  have con+ plasmacytes per high-power field and a ratio of IgG4+ to IgG+ cells greater than 40%. However, a tissue-specific cut-off for tissue IgG4+ plasma cells in IgG4-RD has not yet been validated. Ectopic germinative centers and occasional eosinophilic infiltration in the affected tissue are common findings. Given the recent description of a case of marginal zone B cell lymphoma that produced IgG4, Takano et al. [Biopsy remains mandatory for establishing a diagnosis and should be analyzed as described above. However, slight differences in the histological pattern can be seen in tissue sample from labial salivary glands, parotid glands, or submandibular glands. For example, an intense tissue fibrosis seems to be a common feature of biopsies obtained from submandibular glands , 40, ando et al.  recommeno et al. . In lineIgG4-RD is a multisystem disease that can sometimes involve single organs such as the lacrimal and salivary glands. When the disease is suspected, the two key points are as follows: (1) the differential diagnosis from solid tumors and lymphomas  and (2) the study of multiple organ localizations (by means of imaging techniques). Biopsy of affected organs remains so far the gold standard for diagnosis. Therapy is presently based on the use of steroids and immunosuppressants, but recent insights into pathogenic mechanisms forecast new more disease-tailored therapeutic approaches.


Acute and repetitive pancreatic injury was induced in MET-/- and control mice with cerulein, and chronic injury by feeding mice Lieber-DeCarli diets containing alcohol with or without enhancement of repetitive pancreatic injury. We examined the exocrine pancreas of these mice histologically for acinar death, edema, inflammation and collagen deposition and changes in the transcriptional program. We show that MET expression is relatively low in normal adult pancreas. However, MET levels were elevated in ductal and acinar cells in human pancreatitis specimens, consistent with a role for MET in an adaptive repair mechanism. We report that genetic deletion of MET in adult murine acinar cells was linked to increased acinar cell death, chronic inflammation and delayed recovery (regeneration) of pancreatic exocrine tissue. Notably, increased pancreatic collagen deposition was detected in MET knockout mice following repetitive injury as well alcohol-associated injury. Finally, we identified specific alterations of the pancreatic transcriptome associated with MET signaling during injury, involved in tissue repair, inflammation and endoplasmic reticulum stress. Together, these data demonstrate the importance of MET signaling for acinar repair and regeneration, a novel finding that could attenuate the symptomology of pancreatic injury.Acinar cells represent the primary target in necroinflammatory diseases of the pancreas, including pancreatitis. The signaling pathways guiding acinar cell repair and regeneration following injury remain poorly understood. The purpose of this study was to determine the importance of Hepatocyte Growth Factor Receptor/MET signaling as an intrinsic repair mechanism for acinar cells following acute damage and chronic alcohol-associated injury. Here, we generated mice with targeted deletion of MET in adult acinar cells (MET Pancreatitis is an excruciating and debilitating disease with no available treatments. The primary cause of pancreatitis involves acinar cell endoplasmic reticulum stress and/or the premature activation of pancreatic digestive enzymes in acinar cells, causing the loss of pancreatic acinar cells. Recent findings, however, challenge the latter paradigm . The disTissue injury and inflammation are critical processes for tissue remodeling. However, failure to resolve these responses can lead to the destructive complications of chronic inflammation. Mouse models of pancreatic injury revealed the remarkable capacity of the exocrine pancreas for regeneration ,4. AcuteThe Hepatocyte Growth Factor Receptor (MET) is a tyrosine kinase that is known to participate in inflammatory responses and is critical for the self-renewing capability of stem cells in several cancers ,9. MET i-/- mice following two models of cerulein-induced injury–acute and repetitive acute injury. Furthermore, several inflammatory, macrophage and endoplasmic reticulum stress markers were markedly upregulated in MET-/- pancreas under these conditions. The requirement for MET was not exclusive to acinar regeneration following cerulein-induced injury, as we found increased acinar cell death, chronic inflammation with collagen deposition in MET-/- mice following chronic alcohol intake. Finally, we identified several novel targets downstream of acinar MET important for endoplasmic reticulum stress response, tissue inflammation and collagen deposition whose expression is altered in response to recurrent injury and chronic alcohol-associated injury, particularly in a mouse model of alcohol dependent pancreatic fibrosis. Our data support an important role for MET signaling for acinar regeneration and repair following acute and chronic injury.In the present study, we investigated the role of MET for regeneration of the exocrine pancreas following cerulein-induced acute and recurrent (repetitive) acute injury, alcohol-associated chronic injury and alcohol-associated chronic injury in combination with repeated episodes of acute injury. In the normal adult pancreas, MET expression is low. We show elevated MET levels in ductal and acinar cells in human chronic pancreatitis (CP) specimens as well as transient upregulation of MET in the exocrine pancreas of cerulein-treated mice, consistent with a role for MET as an adaptive repair mechanism. We generated a novel, conditional MET knockout model in which MET is specifically deleted in mature acinar cells. Using this model we show increased acinar cell death, inflammatory infiltrates with edema in the pancreata of MET-/- animals were generated by crossing METfl/fl mice [fl/fl, Bac-Ela-CreErT and MET-/- strains were maintained on the C57BL/6J background. Littermate METfl/fl or wild-type C57BL/6J mice were used as controls. Acinar-specific knockout of MET was induced by tamoxifen treatment of 5–9 week old male mice (75 mg/kg) by oral gavage for three consecutive days and used one week following tamoxifen treatment. Cre-mediated MET knockout was confirmed by PCR using the following forward and reverse primers sets for MET  and CRE recombinase .All animals were cared for in accordance with the Office for Protection from Research Risks (OPRR) and Animal Welfare Act guidelines under an animal protocol approved by the University of Texas Medical Branch Animal Care and Use Committee (IACUC). This study was approved by the University of Texas Medical Branch IACUC. Experimental MET/fl mice  with Bac/fl mice . METfl/fFor mild acute cerulein-induced damage, age-matched male mice (N = 5/group) received seven hourly spaced intraperitoneal injections of cerulein (Bachem) at (50 μg/kg) dissolved in Dulbecco's Phosphate-Buffered Saline (DPBS) with 0.1% BSA or saline (control). Mice were euthanized at the indicated times following the last cerulein injection. For recurrent cerulein-induced damage age-matched male mice (N = 5/group) received six hourly spaced intraperitoneal injections of cerulein or saline, on alternate days three times. Mice were euthanized 72 h following the last cerulein injection. For alcohol-feeding studies, male mice (N = 3-4/group) were housed individually and sensitized to a Liber-DeCarli ethanol diet  ad libitum for two weeks, and sustained on a diet containing 3.5% ethanol for up an additional 14 weeks. During weeks 7, 9 and 11, two cohorts of mice were injected with 7 hourly spaced cerulein injections (50 μg/kg), three times per week. Individual animal weights and food intake were recorded daily. Animals were intraperitoneally injected with 100 μg/kg BrdU (Sigma) in phosphate buffered saline (PBS) 24 h prior to the euthanasia.3 piece of pancreas was immediately stored in RNAlater (Qiagen) solution at 4°C for RNA isolation. The remaining pancreatic tissues were halved and frozen in liquid nitrogen for protein analysis or fixed in 10% neutral buffered formalin for the histological analysis. Human pancreas samples were obtained from discarded pancreatic tissue following surgical resection, in accordance with the policies and practices of the Institutional Review Board of the University of Texas Medical Branch.Mice were sacrificed by cardiac puncture with cervical dislocation, blood was clotted for 1 h at room temperature and centrifuged  to obtain serum. Pancreas and other tissues were removed, dissected and portions stored or processed for RNA or protein analysis, and immunohistochemistry. For RNA analysis a ~2x2 mm5’CCTCATGCCATCATGCGTC3’, 5’CAATCTCACGCTCGGCAGTA3’; COL1A1 (NM_007742) 5’CCTCCTGACGCATGGCC3’, 5’ATACAGATCAAGCATACCTCGGG3’; COL1A2 (NM_007743) 5’TCTGTCCTAGTCGATGGCTGC3’, 5’CAGAGGTGCAATGTCAAGGAAC3’; COL3A1 (NM_009930) 5’AAGGATGGAGAGTCAGGAAGACC3’, 5’CCATTGCGTCCATCAAAGC3’; EMR1 (NM_010130) 5’CCTCATTCACTGTCTGCTCAACC3’, 5’GAAGTCTGGGAATGGGAGCTAAG3’; HGF (NM_010427) 5TGATAAAGGAAAGTTGGGTTCTTACTG3’, 5’TCGCCTCTCTCATGAACATCG3’;IL1β (NM_008361) 5’TATGAGCTGAAAGCTCTCCACCTC3’, 5’GTCGTTGCTTGGTTCTCCTTG3’; RelA (NM_009045) 5’CTGTGCCTACCCGAAACTCAAC3’, 5’GGTTTGAGATCTGCCCTGATG3’; sXBP1  5’CCTTCCTGCTCCTCATGGC3’, 5’CGCACACAGCAGTTCTTCTCTG3’; TNFα (NM_013693) 5’CTCATCAGTTCTATGGCCCAGAC3’, 5’TCCTCCACTTGGTGGTTTGC3’. Primers for CHOP were designed according to Oslowski and Urano (2011) [5’ CGGCTACCACATCCAAGGAA3’, 5’GCTGGAATTACCGCGGT3’), and data are expressed as fold change relative to the controls.Total pancreatic RNA was isolated using the guanidinium thiocyanate-cesium chloride . Total Ro (2011) . All qRTAntibodies against the following were used for IHC: BrdU , insulin , Ki67, a cellular marker for proliferation , Ly6G, a marker for neutrophils , isolated macrophages of mouse origin , mouse MET , human MET . The following dilutions were used for western analysis: α-amylase , β-actin  and mouse MET .2O2 for 10 min to block endogenous peroxidase activity, washed with PBS and blocked in 10% horse serum for one hr. Samples from normal (N = 13) or CP (N = 16) patients were stained with human MET antibody  overnight and counterstained with Harris Hematoxylin (Statlab). Stained sections were graded blind by two individuals for MET staining intensity (MSI) in acinar and ductal cells as: 0 = baseline, 1 = low, 2 = medium or 3 = high.Paraffin embedded human pancreatic sections were deparaffinized, rehydrated and subjected to antigen retrieval in modified citrate buffer, pH 6.1 (DAKO) in a steamer for 20 min. Slides were treated with 3% HFormalin-fixed mouse pancreatic sections were processed as described above and analyzed using MetaMorph software (Molecular Devices)  as folloPancreatic tissue samples were homogenized and analyzed by SDS-PAGE and Western blot analysis and the resulting digitized blots were quantified using MetaMorph software as previously described ,24.Blood ethanol measurements were performed using 50 μl of whole heparinized blood in GC auto sampler vials as previously described . A standStatistical significance was determined by Student’s t-test, 1-way ANOVA with a Dunnett’s post hoc analysis or a multiple t-test with Holm-Sidak correction for multiple analysis as indicated, using GraphPad Prism 6 (GraphPad Software). P<0.05 was considered statistically significant. Data are presented as a mean value ± SEM.Serum HGF levels are known to be elevated in CP patients and are positively correlated with disease severity . Accordi).Acinar cells constitute the primary site of injury in CP ,3,4. TheS1 Figfl/fl mice with a pancreatic acinar specific CreErT line (BAC-Ela-CreErT) under the control of the elastase I gene promoter. The Cre recombinase was activated in differentiated and adult acinar cells in response to tamoxifen  or saline and sacrificed either 1 h or 48 h following the last injection  stress induces the unfolded protein response (UPR), a signaling cascade critical for re-establishing acinar cell homeostasis [-/- mice relative to METfl/fl mice is envisioned to be an adaptive response to injury in the absence of MET activity .Quantitative RT-PCR (qRT-PCR) detected pronounced upregulation of the proinflammatory mediators TNFα, and RelA in METeatments . Of noteeostasis . Splicedeostasis –31. Howeeostasis . The sigactivity . As expe-/- and METfl/fl mice with repetitive cerulein injections. In this model, mice were injected with six, hourly-spaced intraperitoneal injections of cerulein, on alternate days for three days to perpetuate a microenvironment of severe inflammation and collagen deposition [-/- mice relative to wild type animals  infiltrates were detected (data not shown) and serum lipase levels were directly comparable and tested within normal range between METfl/fl and MET-/- mice -/- and M animals . These crol mice . qRT-PCRn of HGF . qRT-PCRn of HGF . Of notecollagen . Consistpression . As expe-/- mice .-/- mice following high alcohol intake, in the absence or presence of the exacerbating effects of cerulein. We reasoned that if MET signaling is critical for acinar renewal, we would detect increased acinar death, and chronic inflammation with collagen deposition in MET-/- mice following high alcohol intake and cerulein treatment. To test this, METfl/fl and MET-/- mice were fed Lieber-DeCarli diets containing 3.5% (v/v) alcohol for 8 weeks. Two cohorts of mice were subjected to repeated episodes of recurrent injury elicited by 3 weeks of alternate, daily i.p. injections of cerulein or saline as a control. Pancreatic morphology and MET expression was examined 3 weeks following the last injection, a time at which fibrosis has been shown to develop in the alcohol-cerulein model [fl/fl and MET-/- mice and tested within normal range (-/- acini consistent with a targeted acinar knockout (data not shown). Morphometric analyses detected a significant preponderance of macrophage (+) and B cell (CD79B+) infiltration (data not shown) in the pancreata of MET-/- fed alcohol, particularly in MET-/- mice fed alcohol and co-treated with cerulein, relative to METfl/fl mice. No neutrophil (Ly6G+) infiltrates were detected in MET-/- and METfl/fl mice consistent with a chronic inflammatory response (data not shown). Moreover, qRT-PCR confirmed significant increases in the expression of the ER stress marker sXBP1 and to a greater extent CHOP-10, in mice deficient in MET, consistent with the chronic nature of the injury , increased expression of fibrotic collagens  and increased collagen deposition. Interestingly, serum lipase levels, a measure of immediate pancreatic damage, were directly comparable between the two MET genotypes following acute and repetitive cerulein-induced injury, suggesting that MET signaling does not directly affect the magnitude of acinar cell injury. Rather, acinar MET signaling contributes to the resolution of inflammation and the promotion of tissue repair following acute and repetitive cerulein induced injury.Acinar cells that survive cerulein-induced injury are capable of regenerating the pancreatic exocrine tissue. In METhase see . Followi-/- mice, which may represent a compensatory mechanism for acinar injury in the absence of MET signaling. Alternatively, the increase in HGF expression may be a consequence of the pronounced macrophage infiltrate noted in mice deficient in MET following acute and repetitive injury, since macrophage derived HGF is critical for wound repair.Emerging evidence suggests that an early and sustained activation of inflammatory signaling in acinar cells is responsible for the intense local and systemic inflammatory responses detected during pancreatic injury ,38. TNFα-/- mice relative to METfl/fl animals. One possible explanation for this observation is that the increased expression of TNFα in the MET-/- mice may result in stellate cell activation and proliferation [-/- mice, may promote stromal changes that result in collagen deposition [-/- mice relative to METfl/fl animals, and animals fed alcohol alone. Consistent with our earlier data indicating that acinar repair is initiated following injury in MET-/- mice, serum lipase levels were directly comparable between experimental cohorts. Our data are consistent with the hypothesis that when the self-renewing capacity of the exocrine pancreas is overwhelmed, inflammation ensues resulting in irreversible tissue damage. It is tempting to speculate that the loss of MET in acinar cells appears to lower the threshold at which irreparable damage occurs.The association between alcohol and CP has been recognized for well over a century. Recent population studies reported that alcohol increased the risk of CP 2–4 fold, depending on the dose and duration of alcohol exposure. By itself, heavy alcohol consumption in humans and rodent models does not cause substantial pancreatic damage. Rather alcohol is proposed to function as an etiologic sensitizer, rendering acinar cells more susceptible to injury from external stressors or genetic factors ,35. Our feration . Alternaposition ,37. When-/- mice relative to METfl/fl controls may indicate a compensatory mechanism for driving acinar regeneration in the absence of MET. Consistent with this, CHOP10 remained directly comparable between MET-/- and METfl/fl mice under these conditions. Thus, in the absence of MET, reparative ER stress responses predominate over pro-apoptotic pathways in an effort to elicit the resolution of inflammation and promote the tissue repair. Conversely, CHOP10 levels were ~15 fold higher in MET-/- mice relative to METfl/fl animals following recurrent cerulein-induced injury, correlating closely with acinar cell loss, sustained inflammation and collagen deposition. sXBP1 levels remained directly comparable between both experimental groups under these conditions. Of note, we show increased sXBP1 and CHOP10 expression following alcohol-associated injury in the absence or presence of cerulein in MET-/- mice relative to the wild-type controls. Additional studies will be required to fully understand how MET signaling may affect ER stress responses to favor the regeneration of injured acinar cells.The ER is a multifunctional organ responsible for the synthesis and correct folding of proteins destined for secretion. Pancreatic acinar cells maintain a high level of protein secretion, and as such the maintenance acinar ER homeostasis is critical. Pancreatic ER stress or the unfolded protein response (UPR) results in the activation of the early protective factor sXBP1 as well as pro-apoptotic signals such as CHOP10 ,45. StudIn summary, pancreatic acinar cells possess a remarkable capacity for regeneration. Here we present a novel role for MET signaling in pancreatic plasticity following acute and chronic models of acinar injury. Our findings are timely and may form the basis for targeted therapeutics designed to promote repair of the exocrine pancreas.S1 Fig(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S4 Fig-/- and METfl/fl mice following recurrent cerulein injury. B, Representative pancreatic sections analyzed for histological changes display increased tissue injury (cell death), repair (cell proliferation) and macrophage invasion in MET-/- mice relative to METfl/fl controls following recurrent cerulein induced injury .A, Comparable levels of serum lipase were noted in MET(TIF)Click here for additional data file.S5 FigAll experimental groups of mice exhibited equivalent serum lipase levels (A) and weight gain (B). Note the comparable drop in weight in MET-/- and METfl/fl mice treated with cerulein (Cer).(TIF)Click here for additional data file.


Knowledge of key drivers for antibiotic prescribing in pediatric lower respiratory tract infection (LRTI) could support rational antibiotic use. Thus, we aimed to determine the impact of clinical and laboratory factors on antibiotic prescribing in children and adolescents with febrile LRTI.Pediatric patients from the standard care control group of a randomized controlled trial (ProPAED) investigating procalcitonin guided antibiotic treatment in febrile LRTI were included in a multivariate logistic regression analysis to evaluate the impact of laboratory and clinical factors on antibiotic prescribing.The standard care control group of the ProPAED study comprised 165 LRTI patients , out of which 88 (55%) received antibiotic treatment. Factors significantly associated with antibiotic prescribing in patients with complete clinical and laboratory documentation (n = 158) were C-reactive protein , white blood count beyond age-dependent reference range , body temperature , and pleuritic pain . Dyspnea  and wheezing  were inversely associated with antibiotic prescribing.Laboratory markers were strong drivers of antibiotic prescribing in children with febrile lower respiratory tract infections, in spite of their known poor prediction of antibiotic need. Building on current guidelines for antibiotic treatment in children with febrile LRTI, a reliable decision algorithm for safe antibiotic withholding considering the laboratory and clinical factors evaluated in this study has the potential to further reduce antibiotic prescribing. Lower respiratory tract infections (LRTI) bear high mortality and morbidity in children and adolescents with community acquired pneumonia (CAP) being the leading cause of death worldwide with 1.3 million fatal pediatric cases each year , 3. HoweThis is a hypothesis generating post-hoc analysis from a randomized controlled trial (ProPAED) evaluating PCT as a biomarker to reduce antibiotic treatment of LRTI in children and adolescents . BrieflyHaemophilus influenzae type b vaccination status . Example codes of the main analyses can be found in the supplemental data  The interaction between pleuritic pain and age (stratified by 2 years of age), was assessed, since pain is difficult to be assessed children < 2 years. (2) Elastic net regression was used to verify robustness of selected covariates. (3) Remaining missing variables (<3%) were multiply imputed by fully conditional specification using the R package mice and re-tested in the logistic regression analysis . All statal data .Both the ethics committee of the University of Basel and Kanton Aargau approved the trial protocol. Written informed consent was obtained from all patients or their care takers. The trial was registered with the International Standard Randomized Controlled Trial Number Register (ISRCTN 17057980).Out of 169 patients in the standard care group, a total of 165 patients with complete data sets for the current analyses were included. Of these, 93 (56%) received antibiotic treatment . The majEleven of 19 assessed clinical and laboratory variables showed a significant association with antibiotic prescribing . Of thesFor multivariate logistic regression analysis: 158 patients with complete clinical and laboratory findings of all variables were included . Of thosThe multivariate model explained the antibiotic prescribing in 126 patients , with a correct prediction of antibiotic prescription of 83%  and a correct prediction of withholding antibiotic therapy of 76% . Characteristics of falsely predicted patients are summarized in Subgroup analysis indicated that the association of pleuritic pain with antibiotic prescribing was reduced in younger children  compared to the OR in the full analysis , and older children only ; including a formal interaction term for this age-dependent association reached however not statistical significance of pleuritic pain in neither group. Elastic net regression suggested lowest misclassification error  when 9 covariates are included in a multivariate model . A simpler/more regulized model with only 4 covariates  could be favored, when accepting a larger error, within one standard error of the minimum. Using a higher penalty  for covariable selection, the same 4 covariates would be selected  to obtain lowest misclassification error.Results of univariate regression analysis did not significantly change when estimating coefficients from multiply imputed datasets . The multivariate regression results changed slightly, with pleuritic pain removed in backward deletion due to a p-value of 0.056 ; all other 5 variables  were retained with a p-value < 0.05.This hypothesis generating study investigated associations of laboratory and clinical findings with antibiotic prescription in children and adolescents with LRTI presenting to the pediatric emergency department. The main finding of this study was the strong association of elevated routine laboratory parameters (CRP and WBC) with increased antibiotic prescribing. Four clinical factors  were also associated with antibiotic prescribing and explained, together with the laboratory parameters, 80% of the antibiotic prescription behavior.Mycoplasma pneumoniae, influenza virus, or adenovirus is associated with elevated WBC as well, blurring its diagnostic performance in LRTI [Although the diagnostic utility of the laboratory values is known to be limited, in this study they have been found to play a major role for antibiotic prescribing. The strongest association with prescription of antimicrobial agents was found in the multivariate analysis for higher than normal CRP. CRP has been shown in the past to have low predictive values as a single and combined biomarker for LRTI in need of antimicrobial treatment –25. In a in LRTI –33.In the ProPAED study, physicians were encouraged to order WBC and CRP at study enrolment in all age groups. This might have introduced bias, as laboratory evaluation is not routinely necessary in young children. However, including only patients > = 2 years of age in a subanalysis did not change the result: CRP and WBC remained significantly associated with antibiotic prescribing ).Clinical parameters predictive for antibiotic prescribing were pleuritic pain and body temperature. Pleuritic pain may be associated with more severe forms of pneumonia particularly with exudative pleural effusions or empyema, for which hospitalization and intravenous antibiotic treatment is required. However, previous studies have shown that sensitivity of pleuritic pain for severe pneumonic infection in young children is limited as children with any LRTI or non-complicated CAP can present with chest or abdominal pain without evidence of effusions or empyema , 35. FurBody temperature has been shown to be a predictor of antibiotic prescribing in a large Italian study including all kinds of respiratory tract infections in pediatric primary care setting. Fever >38°C resulted in a two-fold chance of being treated with antibiotics  , 37. HowTachypnea is included in WHO, Infectious Diseases Society of America (IDSA) and British Thoracic Society (BTS) guidelines as a clinical sign for severity of LRTI disease , 4, 39. Incomplete pneumococcal vaccination status was associated with increased antibiotic prescription in univariate regression analyses, but not in the multivariate model. However, incomplete vaccination might drive physicians to suspect pneumococcal infection in those not vaccinated, and this may influence their prescription behavior. It may be well possible, that incomplete vaccination status might be associated with antibiotic use in future studies.Wheezing and dyspnea were independently associated with lower rates of antibiotic prescribing: Particularly wheezing is associated mostly with viral infections, which is also reflected by international guidelines where antibiotic treatment is not recommended for children with mild fever, wheeze, and signs of upper respiratory tract infections , 4.In multivariate analysis, the combination of six factors including CRP, dyspnea, WBC, body temperature, pleuritic pain and wheezing explained 80% of antibiotic prescribing and resulted in more than half of the patients presenting with LRTI to the emergency departments being treated with antibiotics. Importantly the present study demonstrates that there may be an opportunity for further reduction of antibiotic prescribing for febrile LRTI based on the availability of improved biomarkers and clinical parameters. Although some clinical symptoms did not show a statistically significant association with antibiotic prescription in our study , their consideration in future larger studies could still be of advantage: with our sample size we calculate a power of only < 60% to detect such a significant univariate OR ≤2.One limitation of the study is that the impact of chest radiographs on antibiotic prescribing could not be assessed due to potential selection bias. Chest radiographs were not mandatory for the initial evaluation during the ProPAED study resulting in chest radiographs being performed in only 87% of all ProPAED patients. However, a background re-assessment of the chest radiographs based on the WHO standard  showed aA limitation of the original ProPAED trial with a potential impact on this analysis is the treatment of the intervention group and the standard care control group by the same emergency department team. First, a Hawthorne effect for being under study observation and second, a spill-over effect for learning from the rather restrictive PCT guidance intervention group cannot not be excluded. Especially, as the antibiotic prescription rate was indeed lower in the ProPAED control group compared to historical controls from the same center . The facA further limitation may be that interactions between patients, parents and physicians were not included as endpoints in the original ProPAED study. Thus, these variables could not be included in the regression analysis. So far, large-scale studies have provided valuable insights into non-clinical factors influencing physicians when making decisions on LRTI treatment. Ahmed et al. showed that pediatric patients are at lowest risk for antibiotic exposure when visiting pediatricians in contrast to emergency department doctors or family practitioners, as the antibiotic prescribing rate in pediatricians is one-third of the rate in emergency department staff . Moro etOur model did explain 80% of decisions to prescribe antibiotics but leaves 20% unexplained. Several large studies as well as the latest meta-analysis of Lucas et al. demonstrated that a main factor driving antibiotic prescribing in pediatric LRTI is diagnostic uncertainty , 45, 46.CRP and WBC were strong drivers of antibiotic prescribing in children with febrile LRTI, in spite of their known poor predictive value for antibiotic requirement. In contrast, antibiotics were used prudently in patients with dyspnea and wheezing. Building on current guidelines for antibiotic treatment in children with febrile LRTI, a reliable decision algorithm for safe antibiotic withholding considering the laboratory and clinical factors evaluated in this study may have the potential to further reduce antibiotic prescribing.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S4 Table(PDF)Click here for additional data file.S5 Table(PDF)Click here for additional data file.S6 Table(PDF)Click here for additional data file.S7 Table(PDF)Click here for additional data file.S8 Table(PDF)Click here for additional data file.S9 Table(PDF)Click here for additional data file.S1 Fig(PDF)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S3 Fig(PDF)Click here for additional data file.S4 Fig(PDF)Click here for additional data file.S5 Fig(PDF)Click here for additional data file.S1 Text(PDF)Click here for additional data file.