CINSARC signature outperforms gold-standard TNM staging and consensus molecular subtypes for clinical outcome in stage II-III colorectal carcinoma.

The outcome of stage II-III colorectal cancer (CRC) is highly variable and therapeutic choice is currently based on TNM staging with a few additional biomarkers. However, studies show that some stage III patients have a better prognosis than some stage II patients. A promising consensus molecular (CMS) classification with prognostic relevance has been developed, but it is not used in daily practice. Our team developed CINSARC, a 67-gene expression prognostic signature, whose prognostic value has been demonstrated in many cancer types. It is applicable to formalin-fixed, paraffin-embedded (FFPE) blocks using NanoString® technology. We investigated whether it could predict outcome in stage II-III CRC. We established the CINSARC classification on the TCGA retrospective cohort comprising 297 stage II-III CRC patients using RNA sequencing and on a second independent cohort comprising 169 cases using NanoString® technology. We compared its recurrence-free and overall survival prognostic value with TNM staging and CMS classification. In the TCGA cohort, we showed that CINSARC significantly splits the population of stage II-III CRC into two groups with different progression-free interval (P = 1.68 × 10-2; HR = 1.87 [1.11-3.16]) and overall survival (P = 3.73 × 10-3; HR = 2.45 [1.31-4.59]) and is a strong prognostic factor in multivariate analysis, outperforming TNM staging and CMS classification. We validated these results in the second cohort by applying CINSARC on FFPE samples with Nanostring® technology. CINSARC is a ready-to-use tool with a robust independent prognostic value in stage II-III CRC.

An unusual phenotype occurs in 15% of mismatch repair-deficient tumors and is associated with non-colorectal cancers and genetic syndromes.

Immunohistochemistry (IHC) and/or MSI-PCR (microsatellite instability-polymerase chain reaction) tests are performed routinely to detect mismatch repair deficiency (MMR-D). Classical MMR-D tumors present a loss of MLH1/PMS2 or MSH2/MSH6 with MSI-High. Other profiles of MMR-D tumors have been described but have been rarely studied. In this study, we established a classification of unusual MMR-D tumors and determined their frequency and clinical impact. All MMR-D tumors identified between 2007 and 2017 were selected. Any profile besides the classical MMR-D phenotype was defined as unusual. For patients with unusual MMR-D tumors, IHC, and PCR data were reviewed, the tumor mutation burden (TMB) was evaluated and clinical and genetic features were collected. Of the 4948 cases of MMR testing, 3800 had both the available IHC and MSI-PCR results and 585 of these had MMR-D. After reviewing the IHC and PCR, 21% of the cases initially identified as unusual MMR-D were reclassified, which resulted in a final identification of 89 unusual MMR-D tumors (15%). Unusual MMR-D tumors were more often associated with non-CRC than classical MMR-D tumors. Unusual MMR-D tumors were classified into four sub-groups: i) isolated loss of PMS2 or MSH6, ii) classical loss of MLH1/PMS2 or MSH2/MSH6 without MSI, iii) four MMR proteins retained with MSI and, iv) complex loss of MMR proteins, with clinical characteristics for each sub-group. TMB-high or -intermediate was shown in 96% of the cancers studied (24/25), which confirmed MMR deficiency. Genetic syndromes were identified in 44.9% (40/89) and 21.4% (106/496) of patients with unusual and classical MMR-D tumors, respectively (P < 0.001). Five patients treated with an immune checkpoint inhibitor (ICI) had a prolonged clinical benefit. Our classification of unusual MMR-D phenotype helps to identify MMR deficiency. Unusual MMR-D phenotype occurs in 15% of MMR-D tumors. A high frequency of genetic syndromes was noted in these patients who could benefit from ICI.

Divergent Roles for Macrophage C-type Lectin Receptors, Dectin-1 and Mannose Receptors, in the Intestinal Inflammatory Response.

Colonic macrophages are considered to be major effectors of inflammatory bowel diseases (IBDs) and the control of gut inflammation through C-type lectin receptors is an emerging concept. We show that during colitis, the loss of dectin-1 on myeloid cells prevents intestinal inflammation, while the lack of mannose receptor (MR) exacerbates it. A marked increase in dectin-1 expression in dextran sulfate sodium (DSS)-exposed MR-deficient mice supports the critical contribution of dectin-1 to colitis outcome. Dectin-1 is crucial for Ly6ChighCCR2high monocyte population enrichment in the blood and their recruitment to inflamed colon as precursors of inflammatory macrophages. Dectin-1 also promotes inflammasome-dependent interleukin-1ß (IL-1ß) secretion through leukotriene B4 production. Interestingly, colonic inflammation is associated with a concomitant overexpression of dectin-1/CCL2/LTA4H and downregulation of MR on macrophages from IBD patients. Thus, MR and dectin-1 on macrophages are important mucosal inflammatory regulators that contribute to the intestinal inflammation.

Outcome of Liver Transplant Patients With Preformed Donor-Specific Anti-Human Leukocyte Antigen Antibodies.

After liver transplantation (LT), the role of preformed donor-specific anti-human leukocyte antigen antibodies (pDSAs) remains incompletely understood. We conducted a retrospective, case-control analysis to determine the impact of pDSAs after LT in 3 French transplant centers (Bordeaux, Lyon, and Toulouse). Among the 1788 LTs performed during the study period, 142 (7.9%) had at least 1 pDSA. The patient survival rate was not different between patients who received an LT with pDSAs and the matched-control group. A liver biopsy was performed 1 year after transplantation in 87 recipients. The metavir fibrosis score did not differ between both groups (1 ± 0.8 versus 0 ± 0.8; P = 0.80). However, undergoing a retransplantation (hazard ratio [HR] = 2.6, 95% confidence interval [CI], 1.02-6.77; P = 0.05) and receiving induction therapy with polyclonal antibodies (HR = 2.5; 95% CI, 1.33-4.74; P = 0.01) were associated with a higher risk of mortality. Nonetheless, high mean fluorescence intensity (MFI) donor-specific antibodies (ie, >10,000 with One Lambda assay or >5000 with Immucor assay) were associated with an increased risk of acute rejection (HR = 2.0; 95% CI, 1.12-3.49; P = 0.02). Acute antibody-mediated rejection was diagnosed in 10 patients: 8 recipients were alive 34 (1-125) months after rejection. The use of polyclonal antibodies or rituximab as an induction therapy did not reduce the risk of acute rejection, but it increased the risk of infectious complications. In conclusion, high MFI pDSAs increase the risk of graft rejection after LT, but they do not reduce medium-term and longterm patient survival. The use of a T or B cell-depleting agent did not reduce the risk of acute rejection.

High tacrolimus intra-patient variability is associated with graft rejection, and de novo donor-specific antibodies occurrence after liver transplantation.

AIM: To investigate the role of tacrolimus intra-patient variability (IPV) in adult liver-transplant recipients. METHODS: We retrospectively assessed tacrolimus variability in a cohort of liver-transplant recipients and analyzed its effect on the occurrence of graft rejection and de novo donor-specific antibodies (dnDSAs), as well as graft survival during the first 2 years posttransplantation. Between 02/08 and 06/2015, 116 patients that received tacrolimus plus mycophenolate mofetil (with or without steroids) were included. RESULTS: Twenty-two patients (18.5%) experienced at least one acute-rejection episode (BPAR). Predictive factors for a BPAR were a tacrolimus IPV of > 35% [OR = 3.07 95%CI (1.14-8.24), P = 0.03] or > 40% [OR = 4.16 (1.38-12.50), P = 0.01), and a tacrolimus trough level of < 5 ng/mL [OR=3.68 (1.3-10.4), P =0.014]. Thirteen patients (11.2%) developed at least one dnDSA during the follow-up. Tacrolimus IPV [coded as a continuous variable: OR = 1.1, 95%CI (1.0-1.12), P = 0.006] of > 35% [OR = 4.83, 95%CI (1.39-16.72), P = 0.01] and > 40% [OR = 9.73, 95%CI (2.65-35.76), P = 0.001] were identified as predictors to detect dnDSAs. IPV did not impact on patient- or graft-survival rates during the follow-up. CONCLUSION: Tacrolimus-IPV could be a useful tool to identify patients with a greater risk of graft rejection and of developing a de novo DSA after liver transplantation.

Long-term liver disease in methylmalonic and propionic acidemias.

BACKGROUND AND OBJECTIVES: Patients affected with methylmalonic acidemia (MMA) and propionic acidemia (PA) exhibit diverse long-term complications and poor outcome. Liver disease is not a reported complication. The aim of this study was to characterize and extensively evaluate long-term liver involvement in MMA and PA patients. PATIENTS AND METHODS: We first describe four patients who had severe liver involvement during the course of their disease. Histology showed fibrosis and/or cirrhosis in 3 patients. Such liver involvement led us to retrospectively collect liver (clinical, laboratory and ultrasound) data of MMA (N = 12) or PA patients (N = 16) from 2003 to 2016. RESULTS: Alpha-fetoprotein (αFP) levels were increased in 8/16 and 3/12 PA and MMA patients, respectively, and tended to increase with age. Moderate and recurrent increase of GGT was observed in 4/16 PA patients and 4/12 MMA patients. Abnormal liver ultrasound with either hepatomegaly and/or hyperechoic liver was observed in 7/9 PA patients and 3/9 MMA patients. CONCLUSIONS: These data demonstrate that approximately half of the patients affected by MMA or PA had signs of liver abnormalities. The increase of αFP with age suggests progressive toxicity, which might be due to the metabolites accumulated in PA and MMA. These metabolites (e.g., methylmalonic acid and propionic acid derivatives) have previously been reported to have mitochondrial toxicity; this toxicity is confirmed by the results of histological and biochemical mitochondrial analyses of the liver in two of our MMA patients. In contrast to the moderate clinical, laboratory or ultrasound expression, severe pathological expression was found for three of the 4 patients who underwent liver biopsy, ranging from fibrosis to cirrhosis. These results emphasize the need for detailed liver function evaluation in organic aciduria patients, including liver biopsy when liver disease is suspected. TAKE HOME MESSAGE: MMA and PA patients exhibit long-term liver abnormalities.

Histological long-term outcomes from acute antibody-mediated rejection following ABO-compatible liver transplantation.

BACKGROUND AND AIM: Acute antibody-mediated rejection (aAMR) is an unusual complication after orthotopic ABO-compatible liver transplantation. To date, the clinical and histological long-term outcomes after aAMR are not well known. METHOD: Herein, we describe nine cases of aAMR that occurred in our liver-transplant center between 2008 and 2016, with an initial and reevaluation liver biopsy available for reexamination. RESULTS: Two patients presented with aAMR at 10.5 (10, 11) days post-transplantation, caused by preformed donor-specific antibodies. Seven other recipients developed de novo donor-specific antibodies and aAMR at 11.2 (3-24) months post-transplantation. Eight of the nine patients received a B-cell targeting agent (rituximab, with or without plasma exchange), associated with polyclonal antibodies (three patients) or intravenous immunoglobulins (three patients). At the last follow up (i.e. 21 [4-90] months post-aAMR), seven patients were alive, including two patients with normal liver tests. Grafts' survival was 66%. A liver biopsy performed at 11.5 (5-48.5) months after the first biopsy showed no significant improvement in aAMR score (from 2 ± 1.3 to 1.6 ± 1.5, P = 0.6), a significant improvement in chronic AMR score (from 37 ± 9 to 25 ± 8, P = 0.003) and an increase in the Metavir score (1.2 ± 0.6 to 2.1 ± 0.9, P = 0.03). CONCLUSION: In this study, a B-cell-depleting agent seemed to improve the prognosis of aAMR in selected cases, but several patients kept active lesions antibody-mediated rejection.

Donor-specific antibodies and liver transplantation.

In contrast to other types of organ transplantation, liver-transplant recipients used to be considered highly resistant to donor-specific antibodies (DSAs). Consequently, most transplant programs did not consider the presence of DSAs at transplantation or during the follow-up. However, since the early 1990s, antibody-mediated pathological lesions have been recognized in ABO-incompatible liver-transplant recipients. Recent data confirm the detrimental effect of preformed and de novo DSAs in ABO-compatible liver transplantation, with inferior clinical outcomes in patients presenting with circulating antibodies. Acute antibody-mediated rejection (AMR), plasma-cell hepatitis, biliary stricture, but also long-term complications, such as chronic rejection, liver ductopenia, and graft fibrosis, are now recognized to be associated with DSAs. Moreover, some non-HLA DSAs are suspected to induce graft dysfunction. Clinical, biological, and histological patterns within AMR need to be clarified. Treatment of these complications has yet to be defined. This article summarizes recent advances concerning the impact of preformed and de novo DSAs in liver transplantation, it defines the complications associated with DSAs, and discusses the potential strategies to manage patients with such complications.

De novo donor-specific anti-HLA antibodies mediated rejection in liver-transplant patients.

The incidence and consequences of de novo donor-specific anti-HLA antibodies (DSAs) after liver transplantation (LT) are not well known. We investigated the incidence, risk factors, and complications associated with de novo DSAs in this setting. A total of 152 de novo liver-transplant patients, without preformed anti-HLA DSAs, were tested for anti-HLA antibodies, with single-antigen bead technology, before, at transplantation, at 1, 3, 6 and 12 months after transplantation, and thereafter annually and at each time they presented with increased liver-enzyme levels until the last follow-up, that is, 34 (1.5-77) months. Twenty-one patients (14%) developed de novo DSAs. Of these, five patients had C1q-binding DSAs (24%). Younger age, low exposure to calcineurin inhibitors, and noncompliance were predictive factors for de novo DSA formation. Nine of the 21 patients (43%) with de novo DSAs experienced an acute antibody-mediated rejection (AMR). Positive C4d staining was more frequently observed in liver biopsies of patients with AMR (9/9 vs. 1/12, P < 0.0001). Eight patients received a B-cell targeting therapy, and one patient received polyclonal antibodies. Only one patient required retransplantation. Patient- and graft-survival rates did not differ between patients with and without DSAs. In conclusion, liver-transplant patients with liver abnormalities should be screened for DSAs and AMR.

Endoscopic ultrasound-guided fine-needle aspiration biopsy coupled with a KRAS mutation assay using allelic discrimination improves the diagnosis of pancreatic cancer.

GOALS AND BACKGROUND: Mutation of the KRAS oncogene is present in 75% to 95% of pancreatic cancer tissues. This study aimed to evaluate whether endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA), combined with analysis of the KRAS mutation, improves the diagnosis of pancreatic cancer in cases of inconclusive or doubtful cytopathologic analysis. PATIENTS AND METHODS: We prospectively included 186 patients with a pancreatic mass (103 men; mean age: 62 y). Cytopathology and KRAS mutations, using TaqMan MGB allelic discrimination, were performed on EUS-FNA material. A final diagnosis was obtained from EUS-FNA analysis and/or a subsequent biopsy if necessary, and/or surgery, and follow-up: these were pancreatic adenocarcinoma (n=104), other malignant pancreatic tumors (n=22), and benign lesions (n=60, including 35 cases of chronic pancreatitis). RESULTS: Inconclusive or doubtful (low-grade dysplasia or atypia) cytopathology was found in 68 cases. Of these, 29 patients who had adenocarcinoma were subsequently diagnosed, including 19 cases with a former KRAS mutation. Sensitivity, specificity, positive and negative predictive values, and overall accuracy of cytopathology alone to diagnose adenocarcinoma were 73%, 100%, 100%, 75%, and 85%, respectively. When KRAS mutation analysis was combined with pathology, these values reached 88%, 99%, 99%, 89%, and 93%, respectively. The performance of EUS-FNA to diagnose malignancy was similarly improved after the KRAS-mutation assay (negative predictive value increased from 67% to 88%; accuracy increased from 85% to 94%). CONCLUSIONS: EUS-FNA plus KRAS-mutation analysis, using allelic discrimination, is accurate and improves the diagnosis of pancreatic adenocarcinoma when pathology is inconclusive or doubtful.

Successful treatment of fibrosing cholestatic hepatitis with pegylated interferon, ribavirin and sofosbuvir after a combined kidney-liver transplantation.

Fibrosing cholestatic hepatitis (FCH) is a classical but rare and severe form of recurrent hepatitis C virus (HCV) after liver transplantation. Classical anti-HCV therapy, that is pegylated-interferon (peg-interferon) and ribavirin, has been shown to have limited efficacy in treating FCH. Herein, we report on the first case of successful use of peg-interferon, ribavirin, plus sofosbuvir to treat HCV-induced FCH in a combined liver-kidney transplant patient. Antiviral therapy was given for 24 weeks. HCV clearance occurred within 4 weeks after starting therapy and was maintained until 4 weeks after the end of therapy. Antiviral tolerance was good. We conclude that the use of sofosbuvir-based anti-HCV therapy can be successfully used to treat FCH after a liver or combined kidney-liver transplantation.

The pro-inflammatory action of tumour necrosis factor-α in non-alcoholic steatohepatitis is independent of the NSMAF gene product.

BACKGROUND: The role of tumour necrosis factor-α (TNF-α) in the development of non-alcoholic steatohepatitis remains unclear. AIMS: We evaluated the role of TNF-α and NSMAF gene product factor associated with neutral sphingomyelinase activation, a protein adaptor of the TNF-α receptor-1, in a mouse model of non-alcoholic steatohepatitis. METHODS: Mice deficient either for TNF-α or factor associated with neutral sphingomyelinase activation, as well as control animals, were fed a methionine and choline-deficient diet for 5 weeks. Liver histology, serum glucose, triglycerides, cholesterol and alanine aminotransferase levels were compared between groups. RESULTS: Weight loss, decrease of serum triglyceride and glucose levels and increase of alanine aminotransferase levels were attenuated in TNF(-/-) mice. Similarly, we found a significantly lower lobular inflammation in TNF(-/-) mice. Liver expression of transforming growth factor-ß, peroxisome proliferator-activated receptor-γ(1, 2) and monocyte chemoattractant protein-1 was attenuated in TNF(-/-) mice. In addition, the phosphatidylcholine/phosphatidylethanolamine liver ratio decrease was less important in TNF(-/-) mice. The increase in hepatic sphingomyelin and ceramide levels was less pronounced in TNF(-/-) animals. CONCLUSION: Whereas TNF-α modulates the inflammatory process that underlies methionine and choline-deficient diet-induced non-alcoholic steatohepatitis, its effects are not mediated by factor associated with neutral sphingomyelinase activation. Whether changes in liver lipids, like phosphatidylcholine and ceramide, are causally involved in tumour necrosis factor-mediated liver inflammation remains an open issue.

Simplified identification of Lynch syndrome: a prospective, multicenter study.

BACKGROUND: Recommended strategies to screen for Lynch syndrome in colorectal cancer are not applied in daily practice and most of Lynch cases remain undiagnosed. AIMS: We investigated in routine conditions a strategy that uses simplified clinical criteria plus detection of MisMatch Repair deficiency in tumours to identify Lynch carriers. METHODS: Colorectal cancer patients that met at least one of three clinical criteria were included: (1) colorectal cancer before 50 years, (2) personal history of colorectal or endometrial cancer, (3) first-degree relative history of colorectal or endometrial cancer. All tumours underwent an MisMatch Repair test combining microsatellite instability analysis and MisMatch Repair immunohistochemistry. Patients with an MisMatch Repair-deficient tumour were offered germline testing. RESULTS: Of the 307 patients fulfilling the clinical criteria, 46 (15%) had a MisMatch Repair-deficient tumour. Amongst them 27 were identified as Lynch carriers (20 with germline mutation: 12 MLH1, 7 MSH2, 1 MSH6; 7 highly suspected cases despite failure of genetic testing). The simplified clinical criteria selected a population whose MisMatch Repair-deficient status was highly predictive (59%) of Lynch syndrome. CONCLUSION: This bio-clinical strategy based on simplified clinical criteria combined with an MisMatch Repair test efficiently detected LS cases and is easy to use in clinical practice, outside expert centres.

A severe form of abetalipoproteinemia caused by new splicing mutations of microsomal triglyceride transfer protein (MTTP).

Abetalipoproteinemia is a rare autosomal recessive disease characterized by low lipid levels and by the absence of apoB-containing lipoproteins. It is the consequence of microsomal triglyceride transfer protein (MTTP) deficiency. We report two patients with new MTTP mutations. We studied their functional consequences on the triglyceride transfer function using duodenal biopsies. We transfected MTTP mutants in HepG2 and HeLa cells to investigate their association with protein disulfide isomerase (PDI) and their localization at the endoplasmic reticulum. These children have a severe abetalipoproteinemia. Both of them had also a mild hypogammaglobulinemia. They are compound heterozygotes with c.619G>T and c.1237-28A>G mutations within the MTTP gene. mRNA analysis revealed abnormal splicing with deletion of exon 6 and 10, respectively. Deletion of exon 6 (Δ6-MTTP) introduced a frame shift in the reading frame and a premature stop codon at position 234. Despite the fact that Δ6-MTTP and Δ10-MTTP mutants were not capable of binding PDI, both MTTP mutant proteins normally localize at the endoplasmic reticulum. However, these two mutations induce a loss of MTTP triglyceride transfer activity. These two mutations lead to abnormal truncated MTTP proteins, incapable of binding PDI and responsible for the loss of function of MTTP, thereby explaining the severe abetalipoproteinemia phenotype of these children.

IL-6 deficiency attenuates murine diet-induced non-alcoholic steatohepatitis.

BACKGROUND: The role of inflammation in the pathogenesis of non-alcoholic steatohepatitis (NASH), a common cause of liver disease, is still poorly understood. This study aimed at assessing the involvement of a major inflammatory cytokine, IL-6, in NASH. MATERIALS AND METHODS: Steatohepatitis was induced by feeding wild-type or IL-6(-/-) mice for 5 weeks with a methionine and choline-deficient (MCD) diet. RESULTS: Whereas MCD diet-induced weight loss and decreases in serum glucose, cholesterol and triglyceride levels were similar in both genotypes, serum alanine aminotransferase was less elevated in IL-6(-/-) mice than in wild-type animals. Despite having a comparable liver steatosis score, IL-6-deficient mice exhibited less lobular inflammation than their wild-type littermates. Liver gene expression of TGF-beta and MCP-1 was also strongly attenuated in mutant mice; a more modest reduction was observed for PPAR-gamma and F4/80 transcripts as well as proteins. Chromatographic analysis of liver lipids demonstrated that MCD diet induced in normal and mutant mice a similar decrease in the ratio of phosphatidylcholine to phosphatidylethanolamine. However, the diet-induced increase in the levels of sphingomyelin and ceramide was less important in IL-6(-/-) mice. CONCLUSION: Altogether, these results indicate that IL-6 deficiency does not block the development of NASH; yet, IL-6 plays a critical role in the accompanying liver inflammation.

Comparison of liver stiffness, fibrotest and liver biopsy for assessment of liver fibrosis in kidney-transplant patients with chronic viral hepatitis.

To assess the accuracy of the noninvasive tools, fibrotest (FT) and liver stiffness measurement (LSM) for assessing liver fibrosis in kidney-transplant patients with chronic hepatitis virus B (HBV) or C (HCV) infection. Thirty-eight consecutive kidney-transplant patients with HCV (n = 26) or HBV (n = 12) underwent liver biopsies followed by a FT and LSM. Liver biopsies gave the following fibrosis-grade distribution using METAVIR scores: F0/F1, n = 10 (26.9%); F2, n = 14 (36.8%), F3, n = 7 (18.42%); F4, n = 7 (18.4%). The area under the receiver-operating characteristic curve for mild fibrosis stage

Hepatitis E virus and chronic hepatitis in organ-transplant recipients.

Hepatitis E virus (HEV) is considered an agent responsible for acute hepatitis that does not progress to chronic hepatitis. We identified 14 cases of acute HEV infection in three patients receiving liver transplants, nine receiving kidney transplants, and two receiving kidney and pancreas transplants. All patients were positive for serum HEV RNA. Chronic hepatitis developed in eight patients, as confirmed by persistently elevated aminotransferase levels, serum HEV RNA, and histologic features of chronic hepatitis. The time from transplantation to diagnosis was significantly shorter and the total counts of lymphocytes and of CD2, CD3, and CD4 T cells were significantly lower in patients in whom chronic disease developed.