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supplementary Table S1 to S3; Supplementary Figure S1 to S9

The ectonucleotidases CD39 and CD73 catalyze extracellular ATP to immunosuppressive adenosine, and as such, represent potential cancer targets. We investigated biological impacts of CD39 and CD73 in pancreatic ductal adenocarcinoma (PDAC) by studying clinical samples and experimental mouse tumors. Stromal CD39 and tumoral CD73 expression significantly associated with worse survival in human PDAC samples and abolished the favorable prognostic impact associated with the presence of tumor-infiltrating CD8+ T cells. In mouse transplanted KPC tumors, both CD39 and CD73 on myeloid cells, as well as CD73 on tumor cells, promoted polarization of infiltrating myeloid cells towards an M2-like phenotype, which enhanced tumor growth. CD39 on tumor-specific CD8+ T cells and pancreatic stellate cells also suppressed IFNγ production by T cells. Although therapeutic inhibition of CD39 or CD73 alone significantly delayed tumor growth in vivo, targeting of both ectonucleotidases exhibited markedly superior antitumor activity. CD73 expression on human and mouse PDAC tumor cells also protected against DNA damage induced by gemcitabine and irradiation. Accordingly, large-scale pharmacogenomic analyses of human PDAC cell lines revealed significant associations between CD73 expression and gemcitabine chemoresistance. Strikingly, increased DNA damage in CD73-deficient tumor cells associated with activation of the cGAS–STING pathway. Moreover, cGAS expression in mouse KPC tumor cells was required for antitumor activity of the CD73 inhibitor AB680 in vivo. Our study, thus, illuminates molecular mechanisms whereby CD73 and CD39 seemingly cooperate to promote PDAC progression.

BACKGROUND: After resection, colorectal cancer liver metastases (CRLM) surrounded by a desmoplastic rim carry a better prognosis than the metastases replacing the adjacent liver. However, these histopathological growth patterns (HGPs) are insufficient to guide clinical decision-making. We explored whether the adaptive immune features of HGPs could refine prognostication. METHODS: From 276 metastases resected in 176 patients classified by HGPs, tissue microarrays were used to assess intratumoral T cells (CD3), antigen presentation capacity (MHC class I) and CD73 expression producing immunosuppressive adenosine. We tested correlations between these variables and patient outcomes. RESULTS: The 101 (57.4%) patients with dominant desmoplastic HGP had a median recurrence-free survival (RFS) of 17.1 months compared to 13.3 months in the 75 patients (42.6%) with dominant replacement HGP (p = 0.037). In desmoplastic CRLM, high vs. low CD73 was the only prognostically informative immune parameter and was associated with a median RFS of 12.3 months compared to 26.3, respectively (p = 0.010). Only in dominant replacement CRLM, we found a subgroup (n = 23) with high intratumoral MHC-I expression but poor CD3(+) T cell infiltration, a phenotype associated with a short median RFS of 7.9 months. CONCLUSIONS: Combining the assessments of HGP and adaptive immune features in resected CRLM could help identify patients at risk of early recurrence.

The ectonucleotidases CD39 and CD73 catalyze extracellular ATP to immunosuppressive adenosine, and as such, represent potential cancer targets. We investigated biological impacts of CD39 and CD73 in pancreatic ductal adenocarcinoma (PDAC) by studying clinical samples and experimental mouse tumors. Stromal CD39 and tumoral CD73 expression significantly associated with worse survival in human PDAC samples and abolished the favorable prognostic impact associated with the presence of tumor-infiltrating CD8+ T cells. In mouse transplanted KPC tumors, both CD39 and CD73 on myeloid cells, as well as CD73 on tumor cells, promoted polarization of infiltrating myeloid cells towards an M2-like phenotype, which enhanced tumor growth. CD39 on tumor-specific CD8+ T cells and pancreatic stellate cells also suppressed IFNγ production by T cells. Although therapeutic inhibition of CD39 or CD73 alone significantly delayed tumor growth in vivo, targeting of both ectonucleotidases exhibited markedly superior antitumor activity. CD73 expression on human and mouse PDAC tumor cells also protected against DNA damage induced by gemcitabine and irradiation. Accordingly, large-scale pharmacogenomic analyses of human PDAC cell lines revealed significant associations between CD73 expression and gemcitabine chemoresistance. Strikingly, increased DNA damage in CD73-deficient tumor cells associated with activation of the cGAS–STING pathway. Moreover, cGAS expression in mouse KPC tumor cells was required for antitumor activity of the CD73 inhibitor AB680 in vivo. Our study, thus, illuminates molecular mechanisms whereby CD73 and CD39 seemingly cooperate to promote PDAC progression.

Background: Helicobacter pylori ( H. pylori ) prevalence in Canada has been estimated to be around 20% to 30%. However, H. pylori prevalence is declining in industrialized countries. We conducted a retrospective study on a population of patients referred for esophagogastroduodenoscopy (EGD) in a Canadian quaternary hospital to see the current prevalence of H. pylori infection and identify its main risk factors. Methods: We performed a retrospective cross-sectional study from the Electronic Medical Records of 500 patients who visited our endoscopy clinic and who had biopsies to search for H. pylori infection. In addition to the outcome of the biopsies, we collected demographic characteristics of patients, EGD indication and endoscopic findings. Results: The overall prevalence of H. pylori was 13.0% (65/500) among our population. We found no association with age, sex, tobacco or alcohol consumption. However, we noticed a significantly higher prevalence of H. pylori among African (25.0%; 8/32), Asian (30.8%; 4/13) and South American (34.9%; 15/43) born subjects when compared to the Caucasian group (8.0%; 28/350) (all P < 0.05). Conclusions: The prevalence of H. pylori in Canada is declining, particularly among its Caucasian population. The race seems to be the strongest risk factor associated with this infection. J Clin Med Res. 2020;12(11):687-692 doi: https://doi.org/10.14740/jocmr4348

Most patients with hepatocellular carcinoma (HCC) are diagnosed at a late stage and have few therapeutic options and a poor prognosis. This is due to the lack of clearly defined underlying mechanisms or a dominant oncogene that can be targeted pharmacologically, unlike in other cancer types. Here, we report the identification of a previously uncharacterized oncogenic signaling pathway in HCC that is mediated by the tyrosine kinase Yes. Using genetic and pharmacological interventions in cellular and mouse models of HCC, we showed that Yes activity was necessary for HCC cell proliferation. Transgenic expression of activated Yes in mouse hepatocytes was sufficient to induce liver tumorigenesis. Yes phosphorylated the transcriptional coactivators YAP and TAZ (YAP/TAZ), promoting their nuclear accumulation and transcriptional activity in HCC cells and liver tumors. We also showed that YAP/TAZ were effectors of the Yes-dependent oncogenic transformation of hepatocytes. Src family kinase activation correlated with the tyrosine phosphorylation and nuclear localization of YAP in human HCC and was associated with increased tumor burden in mice. Specifically, high Yes activity predicted shorter overall survival in patients with HCC. Thus, our findings identify Yes as a potential therapeutic target in HCC.

86 Background: Patients with left-sided metastatic colorectal cancer (mCRC) without mutations in RAS or BRAF V600E are eligible to a combination treatment of an anti-EGFR monoclonal antibody in association with chemotherapy. This combination is associated with a survival benefit and is recommended by oncology guidelines as first line therapy for RAS/BRAF wild type left-sided mCRC patients. The CHUM (University of Montreal Hospital Center) has implemented reflex testing of RAS/BRAF mutations for mCRC since October 22nd 2019, and was one of the only hospitals in Canada to do so. With this study, we want to demonstrate that reflex testing of these mutations improves the quality of care received by patients. Methods: In this retrospective study, we reviewed pathological and clinical data of 101 patients with newly diagnosed mCRC that had their pathological diagnosis done at the CHUM. Patients diagnosed between July 1st 2017 and October 21st 2019, before implementation of reflex testing, (“Before reflex testing” group, n = 62 patients) were compared to patients diagnosed between October 22nd 2019 and December 31st 2021 (“After reflex testing” group, n = 39 patients). Results: The average delay between the mCRC biopsy and the result of the RAS/BRAF mutations was reduced to 25 days (CI95% 22.6 – 28.1) in the “After reflex testing” group, compared to 154 days (CI95% 111.2 – 202.5) in the “Before reflex testing” group. Respectively 66% (41/62) of patients in the “Before reflex testing” group and 69% (27/39) of patients in the “After reflex testing” received a systemic treatment. At initiation of systemic therapy, results of RAS/ BRAF mutations were available for 85% (35/41) of patients in the “After reflex testing” group, versus only 17% (7/41) of patients in the “Before reflex testing” group. Among patients eligible for an anti-EGFR monoclonal antibody and who received it, 100% of them (6/6) received it in the first line setting in the “After reflex testing” group, compared to 33% (3/9) in the “Before reflex testing” group. Conclusions: Our study shows that with reflex testing of RAS/ BRAF mutations, oncologists have quicker access to all relevant information to make the best treatment decision for their patients. It allows eligible patients to receive an anti-EGFR therapy in the first line setting, as recommended by oncology guidelines.

Nodular regenerative hyperplasia (NRH) is one of the most frequent causes of noncirrhotic intrahepatic hypertension, but is a difficult histologic diagnosis. The expression of glutamine synthetase (GS) and cytokeratin 7 (CK7) has been reported to be increased in other regenerative/vascular conditions, while CK7 and BerEP4 are also markers of hepatic progenitor cells. The aims of this study were to investigate the use of GS, CK7, and BerEP4 as the potential markers for NRH. This is a retrospective case series of NRH at Centre Hospitalier de l'Universite de Montreal between 1993 and 2013. Normal liver from partial hepatectomies for tumors were used as controls. GS, CK7, CK19, and BerEP4 immunohistochemical stains were performed on all specimens. Immunohistochemical staining patterns were scored from 0 to 3+. NRH was identified in 46 samples obtained from 26 patients. Liver chemistry profile was cholestatic in 45% of the patients. In 93% of the NRH cases, there was abnormal zone 2 expression of GS. Weak panacinar GS staining was seen in all the NRH cases. Aberrant CK7 expression was present in all cases of NRH, but were not associated with cholestasis. BerEP4 was overexpressed in 47% of the NRH cases (P

Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD), 2 incurable neurodegenerative disorders, share the same pathological hallmark named TDP43 (TAR DNA binding protein 43) proteinopathy. This event is characterized by a consistent cytoplasmic mislocalization and aggregation of the protein TDP43, which loses its physiological properties, leading neurons to death. Antibody-based approaches are now emerging interventions in the field of neurodegenerative disorders. Here, we tested the target specificity, in vivo distribution, and therapeutic efficacy of a monoclonal full-length antibody, named E6, in TDP43-related conditions. We observed that the antibody recognizes specifically the cytoplasmic fraction of TDP43. We demonstrated its ability in targeting large neurons in the spinal cord of mice and in reducing TDP43 mislocalization and NF-κB activation. We also recognized the proteasome as well as the lysosome machineries as possible mechanisms used by the antibody to reduce TDP43 proteinopathy. To our knowledge, this is the first report showing the therapeutic efficacy and feasibility of a full-length antibody against TDP43 in reducing TDP43 proteinopathy in spinal neurons of an ALS/FTLD mouse model., A full-length antibody against TDP43 reduces TDP43 proteinopathy in spinal neurons of an amyotrophic lateral sclerosis/Frontotemporal lobar degeneration mouse model.

Herbal and dietary supplements are frequently used as weight loss supplements. However, they account for 20% of drug-induced liver injury. Garcinia cambogia's (GC) active compound, hydroxycitric acid, can be found among those supplements. We report a 26-year-old woman who had been taking GC for 7 months when she presented with subacute liver failure and ultimately required a liver transplantation. This report highlights the risk of liver injury after long-term use of GC and demonstrates the importance of considering a close and prolonged monitoring of patients in a tertiary liver transplant center.

To test the hypothesis that the cerebrospinal fluid (CSF) could provide a spreading route for pathogenesis of amyotrophic lateral sclerosis (ALS), we have examined the effects of intraventricular infusion during 2 weeks of pooled CSF samples from sporadic ALS patients or control CSF samples into transgenic mice expressing human TDP43WT which do not develop pathological phenotypes. Infusion of ALS-CSF, but not of control CSF, triggered motor and cognitive dysfunction, as well as ALS-like pathological changes including TDP43 proteinopathy, neurofilament disorganization and neuroinflammation. In addition, the neuron-specific translational profiles from peptide analyses of immunoprecipitated ribosomes revealed dysregulation of multiple protein networks in response to ALS-CSF altering cytoskeletal organization, vesicle trafficking, mitochondrial function, and cell metabolism. With normal mice, similar ALS-CSF infusion induced mild motor dysfunction but without significant TDP43 pathology in spinal neurons. We conclude that the CSF from sporadic ALS contains factors that can transmit and disseminate disease including TDP43 proteinopathy into appropriate recipient animal model expressing human TDP43. These findings open new research avenues for the discovery of etiogenic factors for sporadic ALS and for the testing of drugs aiming to neutralize the ALS-CSF toxicity.

The immune landscape of the tumor microenvironment (TME) is a determining factor in cancer progression and response to therapy. Specifically, the density and the location of immune cells in the TME have important diagnostic and prognostic values. Multiomic profiling of the TME has exponentially increased our understanding of the numerous cellular and molecular networks regulating tumor initiation and progression. However, these techniques do not provide information about the spatial organization of cells or cell-cell interactions. Affordable, accessible, and easy to execute multiplexing techniques that allow spatial resolution of immune cells in tissue sections are needed to complement single cell-based high-throughput technologies. Here, we describe a strategy that integrates serial imaging, sequential labeling, and image alignment to generate virtual multiparameter slides of whole tissue sections. Virtual slides are subsequently analyzed in an automated fashion using user-defined protocols that enable identification, quantification, and mapping of cell populations of interest. The image analysis is done, in this case using the analysis modules Tissuealign, Author, and HISTOmap. We present an example where we applied this strategy successfully to one clinical specimen, maximizing the information that can be obtained from limited tissue samples and providing an unbiased view of the TME in the entire tissue section.

In developed countries, colorectal cancer is the second cause of cancer-related mortality. Chemotherapy is considered a standard treatment for colorectal liver metastases (CLM). Among patients who develop CLM, the assessment of patient response to chemotherapy is often required to determine the need for second-line chemotherapy and eligibility for surgery. However, while FOLFOX-based regimens are typically used for CLM treatment, the identification of responsive patients remains elusive. Computer-aided diagnosis systems may provide insight in the classification of liver metastases identified on diagnostic images. In this paper, we propose a fully automated framework based on deep convolutional neural networks (DCNN) which first differentiates treated and untreated lesions to identify new lesions appearing on CT scans, followed by a fully connected neural networks to predict from untreated lesions in pre-treatment computed tomography (CT) for patients with CLM undergoing chemotherapy, their response to a FOLFOX with Bevacizumab regimen as first-line of treatment. The ground truth for assessment of treatment response was histopathology-determined tumor regression grade. Our DCNN approach trained on 444 lesions from 202 patients achieved accuracies of 91% for differentiating treated and untreated lesions, and 78% for predicting the response to FOLFOX-based chemotherapy regimen. Experimental results showed that our method outperformed traditional machine learning algorithms and may allow for the early detection of non-responsive patients.

Immune checkpoint blockade has not yet been effective in patients with mismatch repair proficient metastatic colorectal cancer. Targeting immunosuppressive metabolic pathways is being explored as a new immunotherapeutic approach. We assessed whether CD73, the rate limiting enzyme that catalyzes the degradation of extracellular AMP into immunosuppressive adenosine, could be an immunological determinant of colorectal liver metastases (CRLMs). By immunofluorescence on tissue microarrays, intratumoral CD73 expression (tCD73) was analyzed in 391 CRLMs resected in 215 patients, and soluble CD73 (sCD73) was measured by ELISA in the pre-operative serum of 193 patients. High tCD73 was associated with worse pathological features, such as multiple and larger CRLMs, and poorer pathologic response to pre-operative chemotherapy. The median time to recurrence and disease-specific survival after CRLM resection was significantly shorter in patients with high tCD73 (11.0 and 46.4 months, respectively) compared with low tCD73 (19.0 and 61.5 months, respectively). tCD73 was strongly associated with patient outcomes independently of clinicopathological variables. sCD73 did not correlate with tCD73. Patients with high levels of sCD73 also had shorter disease-specific survival. Our results suggested that CD73 in CRLMs may be prognostically informative and may help select patients more likely to respond to adenosine pathway blocking agents.

Human epidermal growth factor receptor 2 (HER2) dysregulation is associated with tumorigenesis in gastric/gastroesophageal junction cancer; however, the number of patients with HER2-positive disease is unclear, possibly due to differing scoring criteria/assays. Data are also lacking for early disease. We aimed to assess the HER2-positivity rate using approved testing criteria in a large, real-life multinational population. HER2-positivity was defined as an immunohistochemistry staining score of 3+, or immunohistochemistry 2+ and HER2 amplification detected by in situ hybridization. A total of 4949 patients were enrolled and results showed that 14.2% of 4920 samples with immunohistochemistry results were HER2-positive. HER2-positivity was significantly higher in males (16.1% vs. 9.6% in females), in gastroesophageal versus stomach tumors (22.1% vs. 12.9%), in biopsy versus surgical samples (18.3% vs. 13.0%), in intestinal tumor subtypes versus diffuse (21.5% vs. 4.8%) and mixed types (21.5% vs. 8.5%) (P

Introduction Malignant rhabdoid tumor (MRT) is defined as a high-grade sarcoma derived from an uncertain cell of origin. Its diagnosis is associated with poor prognosis and patient’s life expectancy is greatly reduced. Material and method Here, we describe a unique case of 9-month-old boy who presented with a large MRT arising from the soft tissue of the neck. Following intensive multimodal treatment, the patient benefited from a 25 years’ remission until the discovery of multiple liver metastases. Conclusion MRT of soft tissue needs to be distinguished from other soft tissue neoplasms, as MRT is highly aggressive and is usually associated with a poor outcome. In addition, this is the longest remission time reported in a patient with soft tissue MRT and this might be related to the use of early intensive multimodal treatments.

Background and Aims CD14+ mononuclear phagocytes [MNPs] and T cells infiltrate colon in ulcerative colitis [UC]. Here we investigated how CD14+ MNPs and the cytokines they produce shape the colonic effector T cell profile. Methods Colonic or mesenteric lymph node [mLNs] CD4+ T cells isolated from UC or Crohn’s disease [CD] patients were stimulated with cytokines or autologous CD14+ MNPs. Cytokine expression was assessed by intracytoplasmic staining and multiplex ELISA. Unsupervised phenotypic multicolour analysis of colonic CD14+ MNPs was performed using the FlowSOM algorithm. Results Among CD14+CD64+HLA-DR+SIRPα + MNPs, only the pro-inflammatory cytokine-producing CD163− subpopulation accumulated in inflamed UC colon and promoted mucosal IL-1β-dependent Th17, Th17/Th1, Th17/Th22 but not Th1 responses. Unsupervised phenotypic analysis of CD14+CD64+ MNPs segregated CD163− monocyte-like cells and CD163+ macrophages. Unexpectedly, IL-12, IL-1β and CD163−, but not CD163+, cells induced IL-8 expression in colonic CD4+ T cells, which co-expressed IFN-γ and/or IL-17 in UC and not CD. The CD163− monocyte-like cells increased the frequency of IL-8+IL-17+/−IFN-γ +/− T cells through IL-1β and IL-12. Finally, colonic IL-8+ T cells co-expressing GM-CSF, TNF-α and IL-6 were detected ex vivo and, promoted by IL-12 in the mucosa and mLNs in UC only. Conclusions Our findings established a link between monocyte-like CD163− MNPs, IL-12, IL-1β and the detection of colonic memory IL-8-producing CD4+ T cells, which might all contribute to the pathogenesis of UC.

The drug targets IL23 and IL12 regulate pathogenicity and plasticity of intestinal Th17 cells in Crohn's disease (CD) and ulcerative colitis (UC), the two most common inflammatory bowel diseases (IBD). However, studies examining Th17 dysregulation in mesenteric lymph nodes (mLNs) of these patients are rare. We showed that in mLNs, CD could be distinguished from UC by increased frequencies of CCR6+CXCR3-RORγ+Tbet-CD4+ (Th17) memory T cells enriched in CD62Llow effector memory T cells (TEM), and their differentially expressed molecular profile. Th17 TEM cells (expressing IL17A, IL17F, RORC, and STAT3) displayed a higher pathogenic/cytotoxic (IL23R, IL18RAP, and GZMB, CD160, PRF1) gene signature in CD relative to UC, while non-pathogenic/regulatory genes (IL9, FOXP3, CTLA4) were more elevated in UC. In both CD and UC, IL12 but not IL23, augmented IFNγ expression in Th17 TEM and switched their molecular profile toward an ex-Th17 (Th1*)-biased transcriptomic signature (increased IFNG, and decreased TCF7, IL17A), suggesting that Th17 plasticity occurs in mLNs before their recruitment to inflamed colon. We propose that differences observed between Th17 cell frequencies and their molecular profile in CD and UC might have implications in understanding disease pathogenesis, and thus, therapeutic management of patients with IBD.

e18762 Background: Febrile neutropenia is a serious complication of chemotherapy leading to hospitalization in cancer patients. According to a practice guidelines published by ASCO (American Society of clinical Oncology) and IDSA (Infectious Diseases Society of American) in 2018, patients meeting the criteria for low-risk neutropenia according to the MASCC score (Multinational Association for Supportive Care in Cancer Score) could be treated as outpatient and thus avoid hospitalization. The objective of the study was to assess the number and proportion of patients who were hospitalized for febrile neutropenia in university hospital that would have met the low risk criteria of febrile neutropenia. We also wanted to know if these patients had experienced a favorable outcome during hospitalization. Methods: We performed a retrospective study including all patients admitted for febrile neutropenia in 3 hospitals in Quebec City during the period from January 1, 2018 to December 31, 2019. We excluded patients with leukemia, as well as stem cell transplant patients. The chart review retrospectively established the MASCC score for each patient. We also established according to predefined criteria whether the clinical course was favorable or unfavorable. Results: A total of 177 hospitalizations met our inclusion criteria. We found that 101/177 (57.1%) of hospitalized patients met the criteria for low-risk neutropenia according to the MASCC score (score of 21 and above). Of this number 74/177 (41.8%) presented all the criteria suggested for receiving outpatient treatment. In these patients 70/177 (39.5%) presented a favorable evolution during hospitalization and thus 4/177 (2.3%) presented an unfavorable evolution. Among these, 2 patients presented with infections considered major (2 bacteremia), 1 patient developed acute renal failure, and 1 other patient developed delirium. There was no death or admission to the intensive care unit in these 4 patients. Conclusions: According to this retrospective study, about 40% of patients admitted for febrile neutropenia filled the criteria of low risk febrile neutropenia and could be treated as outpatient. Given this represents a significant proportion of patients, a protocol for systematic follow-up of outpatient treatment with low-risk febrile neutropenia should be put in place.

BACKGROUND Pelvic endoscopic ultrasound-guided fine-needle aspiration (PEUS-FNA) of rectal or perirectal lesions is safe, minimally invasive, and well tolerated. It provides valuable information, which can greatly influence patient management. Herein, the authors present what to their knowledge is the largest series to date of PEUS-FNA. METHODS PEUS-FNA specimens were retrieved from the archives of the study institution from January 2001 to March 2015. Only patients with solid pelvic lesions were examined. The cytopathology findings, immunohistochemistry, corresponding histology, and clinical data were collected. For analysis of accuracy, atypical or suspicious results were classified as “negative.” The sensitivity and specificity of PEUS-FNA were calculated in a subset of patients with available surgical pathology. RESULTS A total of 127 cases meeting the current study criteria were obtained from patients who underwent PEUS-FNA at the study institution between January 2001 and March 2015. The mean age of the patients was 60 years, and 53% were female. Pelvic lesions were comprised of 72% masses and 28% lymph nodes, with a mean mass diameter of 27.38 mm (range, 5-100 mm). PEUS-FNA was positive for malignancy in 45% of cases, atypical/suspicious in 4.7% of cases, and negative for malignancy in 50.3% of cases. Surgical pathology was available for 44 patients. PEUS-FNA demonstrated 89.3% sensitivity, 100% specificity, a diagnostic accuracy of 93.2%, a positive predictive value of 100%, and a negative predictive value of 84.2%. No complications were noted. CONCLUSIONS PEUS-FNA is safe and effective for the investigation of pelvic lesions. Cancer Cytopathol 2016;124:836-41. © 2016 American Cancer Society.

While reactive microgliosis is a hallmark of advanced stages of amyotrophic lateral sclerosis (ALS), the role of microglial cells in events initiating and/or precipitating disease onset is largely unknown. Here we provide novelin vivoevidence of a distinct adaptive shift in functional microglial phenotypes in preclinical stages of superoxide dismutase 1 (SOD1)-mutant-mediated disease. Using a mouse model for live imaging of microglial activation crossed withSOD1G93AandSOD1G37Rmouse models, we discovered that the preonset phase of SOD1-mediated disease is characterized by development of distinct anti-inflammatory profile and attenuated innate immune/TLR2 responses to lipopolysaccharide (LPS) challenge. This microglial phenotype was associated with a 16-fold overexpression of anti-inflammatory cytokine IL-10 in baseline conditions followed by a 4.5-fold increase following LPS challenge. While infusion of IL-10R blocking antibody, initiated at day 60, caused a significant increase in markers of microglial activation and precipitated clinical onset of disease, a targeted overexpression of IL-10 in microglial cells, delivered via viral vectors expressed under CD11b promoter, significantly delayed disease onset and increased survival ofSOD1G93Amice. We propose that the high IL-10 levels in resident microglia in early ALS represent a homeostatic and compensatory “adaptive immune escape” mechanism acting as a nonneuronal determinant of clinical onset of disease.SIGNIFICANCE STATEMENTWe report here for the first time that changing the immune profile of brain microglia may significantly affect clinical onset and duration of disease in ALS models. We discovered that in presymptomatic disease microglial cells overexpress anti-inflammatory cytokine IL-10. Given that IL-10 is major homeostatic cytokine and its production becomes deregulated with aging, this may suggest that the capacity of microglia to adequately produce IL-10 may be compromised in ALS. We show that blocking IL-10 increased inflammation and precipitated clinical disease onset, whereas overexpression of IL-10 in microglia using a gene therapy approach significantly delayed disease onset and increased survival of ALS mice. Based on our results, we propose that targeted overexpression of IL-10 in microglia may have therapeutic potential in ALS.

Inflammatory immune cells can modulate activation of hepatic stellate cells (HSCs) and progression of liver fibrosis. Type 3 inflammation characterized by production of interleukin-17A (IL-17) and IL-22 by innate and adaptive immune cells is implicated in many inflammatory conditions of the gut and can be counteracted by regulatory T cells (Tregs), but its contribution to liver fibrosis is still poorly understood. Here, we evaluated the contribution of type 3 inflammation in liver fibrosis using clinical liver biopsies, in vitro stimulation of primary HSCs, and in vivo mouse models. We report dysregulated type 3 responses in fibrotic lesions with increased IL-17+CD4+/FOXP3hiCD4+ ratio and increased IL-17 and IL-22 production in advanced liver fibrosis. Neutrophils and mast cells were the main sources of IL-17 in situ in humans. In addition, we demonstrate a new profibrotic function of IL-22 through enhancement of transforming growth factor-β signaling in HSCs in a p38 mitogen-activated protein kinase-dependent manner. In vivo, IL-22RA1 knockout mice exhibited reduced fibrosis in response to thioacetamide and carbon tetrachloride. Blocking either IL-22 or IL-17 production using aryl hydrocarbon receptor or RAR-related orphan receptor gamma-t antagonists resulted in reduced fibrosis. Together, these data have identified a pathogenic role for type 3 immune response mediated by IL-22 in driving liver fibrosis during chronic liver injury.

Summary Neutrophils are detected in inflamed colon in Crohn’s disease (CD). However, whether the frequency and/or activation of circulating or gut tissue neutrophils correlate with endoscopic severity remains to be investigated. A cohort of 73 CD patients was prospectively enrolled according to endoscopic severity and treatment history. Individuals with active disease were stratified using the Montreal classification. Harvey–Bradshaw Index (HBI) and Simple Endoscopic Score for Crohn’s Disease (SES-CD) were performed at the time of ileocolonoscopy. Frequency of neutrophils and their expression of CD66b and CD64 were assessed in paired blood and colonic biopsies using flow cytometry. The percentage of neutrophils increased in inflamed colon and correlated with SES-CD in the entire cohort of patients examined, as well as in the subgroup with inflammatory (B1) active disease. SES-CD further correlated with neutrophil CD66b expression in mucosa but not blood and, conversely, with neutrophil CD64 expression in blood but not mucosa. However, the evaluation of neutrophil activation in mucosa when compared to blood reflected disease activity more clearly. Finally, a neutrophil activation power index (CD66b in mucosa X CD64 in blood) that correlated with SES-CD discriminated between patients with mild and severe disease. In conclusion, the frequency and activation of colonic neutrophils correlated with SES-CD, highlighting that mucosal neutrophils are associated with disease severity in CD.

The cytoplasmic aggregation of TAR DNA-binding protein-43 (TDP-43) is a hallmark of degenerating neurons in amyotrophic lateral sclerosis (ALS) and subsets of frontotemporal dementia (FTD). In order to reduce TDP-43 pathology, we generated single-chain (scFv) antibodies against the RNA recognition motif 1 (RRM1) of TDP-43, which is involved in abnormal protein self-aggregation and interaction with p65 NF-κB. Virus-mediated delivery into the nervous system of a scFv antibody, named VH7Vk9, reduced microgliosis in a mouse model of acute neuroinflammation and mitigated cognitive impairment, motor defects, TDP-43 proteinopathy, and neuroinflammation in transgenic mice expressing ALS-linked TDP-43 mutations. These results suggest that antibodies targeting the RRM1 domain of TDP-43 might provide new therapeutic avenues for the treatment of ALS and FTD.

3584Background: As approximately 80% of patients recur and die of their cancer after undergoing curative-intent resection of colorectal cancer liver metastasis (CRLM) combined with systemic chemoth...

Hematoxylin and eosin (H&E) staining is a well-established technique in histopathology. However, immunohistochemistry (IHC) interpretation is done exclusively with hematoxylin counterstaining. Our goal was to investigate the potential of H&E as counterstaining (H&E-IHC) to allow for visualization of a marker while confirming the diagnosis on the same slide. The quality of immunostaining and the fast-technical performance were the main criteria to select the final protocol. We stained multiple diagnostic tissues with class I IHC tests with different subcellular localization markers (anti-CK7, CK20, synaptophysin, CD20, HMB45, and Ki-67) and with double-staining on prostate tissues with anti-high molecular weight keratins/p63 (DAB detection) and p504s (alkaline phosphatase detection). To validate the efficacy of the counterstaining, we stained tissue microarrays from the Canadian Immunohistochemistry Quality Control (cIQc) with class II IHC tests (ER, PR, HER2, and p53 markers). Interobserver and intraobserver concordance was assessed by κ statistics. Excellent agreement of H&E-IHC interpretation was observed in comparison with standard IHC from our laboratory (κ, 0.87 to 1.00), and with the cIQc reference values (κ, 0.81 to 1.00). Interobserver and intraobserver agreement was excellent (κ, 0.89 to 1.00 and 0.87 to 1.00, respectively). We therefore show for the first time the potential of using H&E counterstaining for IHC interpretation. We recommend the H&E-IHC protocol to enhance diagnostic precision for the clinical workflow and research studies.

Nonalcoholic fatty liver disease (NAFLD) is now the most common progressive liver disease in developed countries and is the second leading indication for liver transplantation due to the extensive fibrosis it causes. NAFLD progression is thought to be tied to chronic low-level type 1 inflammation originating in the adipose tissue during obesity; however, the specific immunological mechanisms regulating the progression of NAFLD-associated fibrosis in the liver are unclear. To investigate the immunopathogenesis of NAFLD more completely, we investigated adipose dysfunction, nonalcoholic steatohepatitis (NASH), and fibrosis in mice that develop polarized type 1 or type 2 immune responses. Unexpectedly, obese interleukin-10 (IL-10)/IL-4-deficient mice (type 1-polarized) were highly resistant to NASH. This protection was associated with an increased hepatic interferon-γ (IFN-γ) signature. Conversely, IFN-γ-deficient mice progressed rapidly to NASH with evidence of fibrosis dependent on transforming growth factor-β (TGF-β) and IL-13 signaling. Unlike increasing type 1 inflammation and the marked loss of eosinophils seen in expanding adipose tissue, progression of NASH was associated with increasing eosinophilic type 2 liver inflammation in mice and human patient biopsies. Finally, simultaneous inhibition of TGF-β and IL-13 signaling attenuated the fibrotic machinery more completely than TGF-β alone in NAFLD-associated fibrosis. Thus, although type 2 immunity maintains healthy metabolic signaling in adipose tissues, it exacerbates the progression of NAFLD collaboratively with TGF-β in the liver.

Introduction Malignant rhabdoid tumor (MRT) is defined as a high-grade sarcoma derived from an uncertain cell of origin. Its diagnosis is associated with poor prognosis and patient's life expectancy is greatly reduced. Material and method Here, we describe a unique case of 9-month-old boy who presented with a large MRT arising from the soft tissue of the neck. Following intensive multimodal treatment, the patient benefited from a 25 years' remission until the discovery of multiple liver metastases. Conclusion MRT of soft tissue needs to be distinguished from other soft tissue neoplasms, as MRT is highly aggressive and is usually associated with a poor outcome. In addition, this is the longest remission time reported in a patient with soft tissue MRT and this might be related to the use of early intensive multimodal treatments.

There is emerging evidence that the misfolding of superoxide dismutase 1 (SOD1) may represent a common pathogenic event in both familial and sporadic amyotrophic lateral sclerosis (ALS). To reduce the burden of misfolded SOD1 species in the nervous system, we have tested a novel therapeutic approach based on adeno-associated virus (AAV)-mediated tonic expression of a DNA construct encoding a secretable single-chain fragment variable (scFv) antibody composed of the variable heavy and light chain regions of a monoclonal antibody (D3H5) binding specifically to misfolded SOD1. A single intrathecal injection of the AAV encoding the single-chain antibody in SOD1(G93A) mice at 45 days of age resulted in sustained expression of single-chain antibodies in the spinal cord, and it delayed disease onset and extension of life span by up to 28%, in direct correlation with scFv titers in the spinal cord. The treatment caused attenuation of neuronal stress signals and reduction in levels of misfolded SOD1 in the spinal cord of SOD1(G93A) mice. From these results, we propose that an immunotherapy based on intrathecal inoculation of AAV encoding a secretable scFv against misfolded SOD1 should be considered as potential treatment for ALS, especially for individuals carrying SOD1 mutations.

BACKGROUND: Activation of hepatic stellate cells (HSCs) is a key event in the initiation of liver fibrosis. CD4 T cells can modulate positively or negatively this process. Briefly, Th1 cells despite their pro-inflammatory properties have anti-fibrogenic properties in contrast to Th2 cells. We and others have demonstrated that IL-17A produced by Th17 cells has pro-fibrogenic properties as it promotes activation of HSCs via different mechanisms. Th17 cells also produce IL-22, an enigmatic cytokine with proinflammatory and hepatoprotective properties. In addition, IL-10 produced by regulatory T cells (Treg) negatively modulates activation of HSCs. AIMS: We hypothesized that liver fibrosis progression results from an alteration in the Th17/Treg ratio leading to an imbalance in the pro-fibrotic cytokine profile within the liver. METHODS: We examined ex vivo the frequency of Th17 and Treg populations and the cytokine profile of intrahepatic lymphocytes isolated from liver biopsy samples (n=32). We validated these cytokines profiles using in vivo model of fibrosis in transgenic mice and primary human HSCs. RESULTS: We observed increased Th17/Treg ratio in advanced (F4, Metavir) as compared to moderate or non-fibrosis (F0-F2). Furthermore, we observed a bias towards Th17/Th9 cytokine profile in fibrotic livers with viral-hepatitis, whereas the cytokine profile was Th17/Th2 in non-viral hepatitis. All biopsies exhibited a 5-fold increase in IL-22 in fibrotic livers (p=0.0082) irrespective of aetiology. In vivo, lack of IL-22 signaling protects against thioacetamide-induced fibrosis. IL-22RA1 Knockout mice have reduced collagen deposition measured by picro-sirius red staining (p=0.0009) and pro-fibrotic genes expression (ACTA2, LOXL2, TIMP-I, TGFb1, COL1A1) in comparison to wild-type littermates. In vitro stimulation of primary human HSCs with IL-22 sensitized them to suboptimal doses of TGF-b. RNA-seq analysis demonstrated activation of p38 in HSCs in response to IL-22 and chemical inhibition of p38 suppressed the pro-fibrogenic effect of IL-22. CONCLUSIONS: Our results suggest a dysregulated Th17/Treg ratio in advanced fibrosis coupled with distinct cytokine profile dependant on the aetiology of liver disease. Finally, we have identified IL-22 as a common factor in advanced liver fibrosis acting through sensitization of HSCs to TGF-b in a p38-dependent manner. FUNDING AGENCIES: CIHR

Vancomycin-resistant enterococci (VRE) are an important cause of health care-acquired infections (HAIs). Studies have shown that active surveillance of high-risk patients for VRE colonization can aid in reducing HAIs; however, these screens generate a significant cost to the laboratory and health care system. Digital imaging capable of differentiating negative and “nonnegative” chromogenic agar can reduce the labor cost of these screens and potentially improve patient care. In this study, we evaluated the performance of the WASPLab Chromogenic Detection Module (CDM) (Copan, Brescia, Italy) software to analyze VRE chromogenic agar and compared the results to technologist plate reading. Specimens collected at 3 laboratories were cultured using the WASPLab CDM and plated to each site's standard-of-care chromogenic media, which included Colorex VRE (BioMed Diagnostics, White City, OR) or Oxoid VRE (Oxoid, Basingstoke, United Kingdom). Digital images were scored using the CDM software after 24 or 40 h of growth, and all manual reading was performed using digital images on a high-definition (HD) monitor. In total, 104,730 specimens were enrolled and automation agreed with manual analysis for 90.1% of all specimens tested, with sensitivity and specificity of 100% and 89.5%, respectively. Automation results were discordant for 10,348 specimens, and all discordant images were reviewed by a laboratory supervisor or director. After a second review, 499 specimens were identified as representing missed positive cultures falsely called negative by the technologist, 1,616 were identified as containing borderline color results (negative result but with no package insert color visible), and 8,234 specimens were identified as containing colorimetric pigmentation due to residual matrix from the specimen or yeast ( Candida ). Overall, the CDM was accurate at identifying negative VRE plates, which comprised 84% (87,973) of the specimens in this study.

Cytoplasmic TDP-43 aggregation is a pathological hallmark of amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Here we investigated the role of exosomes in the secretion and propagation of TDP-43 aggregates. TDP-43 was detected in secreted exosomes from Neuro2a cells and primary neurons but not from astrocytes or microglia. Evidence is presented that protein aggregation and autophagy inhibition are factors that promote exosomal secretion of TDP-43. We also report that levels of exosomal TDP-43 full length and C-terminal fragment species are upregulated in human amyotrophic lateral sclerosis brains. Exposure of Neuro2a cells to exosomes from amyotrophic lateral sclerosis brain, but not from control brain, caused cytoplasmic redistribution of TDP-43, suggesting that secreted exosomes might contribute to propagation of TDP-43 proteinopathy. Yet, inhibition of exosome secretion by inactivation of neutral sphingomyelinase 2 with GW4869 or by silencing RAB27A provoked formation of TDP-43 aggregates in Neuro2a cells. Moreover, administration of GW4869 exacerbated the disease phenotypes of transgenic mice expressing human TDP-43A315T mutant. Thus, even though results suggest that exosomes containing pathological TDP-43 may play a key role in the propagation of TDP-43 proteinopathy, a therapeutic strategy for amyotrophic lateral sclerosis based on inhibition of exosome production would seem inappropriate, as in vivo data suggest that exosome secretion plays an overall beneficial role in neuronal clearance of pathological TDP-43.

Pelvic endoscopic ultrasound-guided fine-needle aspiration (PEUS-FNA) of rectal or perirectal lesions is safe, minimally invasive, and well tolerated. It provides valuable information, which can greatly influence patient management. Herein, the authors present what to their knowledge is the largest series to date of PEUS-FNA.PEUS-FNA specimens were retrieved from the archives of the study institution from January 2001 to March 2015. Only patients with solid pelvic lesions were examined. The cytopathology findings, immunohistochemistry, corresponding histology, and clinical data were collected. For analysis of accuracy, atypical or suspicious results were classified as "negative." The sensitivity and specificity of PEUS-FNA were calculated in a subset of patients with available surgical pathology.A total of 127 cases meeting the current study criteria were obtained from patients who underwent PEUS-FNA at the study institution between January 2001 and March 2015. The mean age of the patients was 60 years, and 53% were female. Pelvic lesions were comprised of 72% masses and 28% lymph nodes, with a mean mass diameter of 27.38 mm (range, 5-100 mm). PEUS-FNA was positive for malignancy in 45% of cases, atypical/suspicious in 4.7% of cases, and negative for malignancy in 50.3% of cases. Surgical pathology was available for 44 patients. PEUS-FNA demonstrated 89.3% sensitivity, 100% specificity, a diagnostic accuracy of 93.2%, a positive predictive value of 100%, and a negative predictive value of 84.2%. No complications were noted.PEUS-FNA is safe and effective for the investigation of pelvic lesions. Cancer Cytopathol 2016;124:836-41. © 2016 American Cancer Society.

Recently, systems have been developed to create total laboratory automation for clinical microbiology. These systems allow for the automation of specimen processing, specimen incubation, and imaging of bacterial growth. In this study, we used the WASPLab to validate software that discriminates and segregates positive and negative chromogenic methicillin-resistant Staphylococcus aureus (MRSA) plates by recognition of pigmented colonies. A total of 57,690 swabs submitted for MRSA screening were enrolled in the study. Four sites enrolled specimens following their standard of care. Chromogenic agar used at these sites included MRSASelect (Bio-Rad Laboratories, Redmond, WA), chromID MRSA (bioMérieux, Marcy l'Etoile, France), and CHROMagar MRSA (BD Diagnostics, Sparks, MD). Specimens were plated and incubated using the WASPLab. The digital camera took images at 0 and 16 to 24 h and the WASPLab software determined the presence of positive colonies based on a hue, saturation, and value (HSV) score. If the HSV score fell within a defined threshold, the plate was called positive. The performance of the digital analysis was compared to manual reading. Overall, the digital software had a sensitivity of 100% and a specificity of 90.7% with the specificity ranging between 90.0 and 96.0 across all sites. The results were similar using the three different agars with a sensitivity of 100% and specificity ranging between 90.7 and 92.4%. These data demonstrate that automated digital analysis can be used to accurately sort positive from negative chromogenic agar cultures regardless of the pigmentation produced.

Approximately 20% cases of familial amyotrophic lateral sclerosis (ALS) are caused by mutations in the gene encoding Cu/Zn superoxide dismutase (SOD1). Recent studies have shown that methylene blue (MB) was efficient in conferring protection in several neurological disorders. MB was found to improve mitochondrial function, to reduce reactive oxygen species, to clear aggregates of toxic proteins, and to act as a nitric oxide synthase inhibitor. These pleiotropic effects of relevance to ALS pathogenesis led us to test MB in two models of ALS, SOD1(G93A) mice and TDP-43(G348C) transgenic mice. Intraperitoneal administration of MB at two different doses was initiated at the beginning of disease onset, at 90 days of age in SOD1(G93A) and at 6 months of age in TDP-43(G348C) mice. Despite its established neuroprotective properties, MB failed to confer protection in both mouse models of ALS. The lifespan of SOD1(G93A) mice was not affected by MB treatment. The declines in motor function, reflex score, and body weight of SOD1(G93A) mice remained unchanged. MB treatment had no effect on motor neuron loss and aggregation or misfolding of SOD1. A combination of MB with lithium also failed to provide benefits in SOD1(G93A) mice. In TDP-43(G348C) mice, MB failed to improve motor function. Cytosolic translocation of TDP-43, ubiquitination and inflammation remained also unchanged after MB treatment of TDP-43(G348C) mice.

3586Background: Approximately 80% of patients recur after curative-intent resection of colorectal cancer liver metastasis (CRLM) and systemic chemotherapy. Immune profiling may help prognostication...

Peroxisome proliferator-activated receptor-γ (PPARγ) agonism potently reduces circulating triglycerides (TG) in rodents and more modestly so in humans. This study aimed to quantify in vivo the relative contribution of hepatic VLDL-TG secretion and tissue-specific TG clearance to such action. Rats were fed an obesogenic diet, treated with the PPARγ full agonist COOH (30 mg·kg−1·day−1) for 3 wk, and studied in both the fasted and refed (fat-free) states. Hepatic VLDL-TG secretion rate was not affected by chronic COOH in the fasted state and was only modestly decreased (−30%) in refed rats. In contrast, postprandial VLDL-TG clearance was increased 2.6-fold by COOH, which concomitantly stimulated adipose tissue TG-derived lipid uptake and one of its major determinants, lipoprotein lipase (LPL) activity, in a highly depot-specific manner. TG-derived lipid uptake and LPL were indeed strongly increased in subcutaneous inguinal white adipose tissue and in brown adipose tissue, independently of the nutritional state, whereas of the three visceral fat depots examined (epididymal, retroperitoneal, mesenteric) only the latter responded consistently to COOH. Robust correlations (0.5 < r < 0.9) were observed between TG-derived lipid uptake and LPL in adipose tissues. The agonist did not increase LPL in muscle, and its enhancing action on postprandial muscle lipid uptake appeared to be mediated by post-LPL processes involving increased expression of fatty acid binding/transport proteins (aP2, likely in infiltrated adipocytes, FAT/CD36, and FATP-1). The study establishes in a diet-induced obesity model the major contribution of lipid uptake by specific, metabolically safe adipose depots to the postprandial hypotriglyceridemic action of PPARγ agonism, and suggests a key role for LPL therein.