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.

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.

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.

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

Expression of the transcription factor Klf6 in cirrhosis, macronodules, and hepatocellular carcinoma.

BACKGROUND AND AIMS: Macronodules (MN) occurring in cirrhosis are considered to be precursor lesions for hepatocellular carcinoma (HCC). However, early molecular events in hepatocellular carcinogenesis are poorly understood. The aim of this study was to compare gene expression profiling between cirrhotic tissues, MN, and HCC, to identify genes early involved in liver carcinogenesis. METHODS: Tissues were obtained from explanted livers: nine cirrhosis, 10 MN, and seven HCC. Total RNAs were extracted by RNeasy and reverse transcribed with labelled [(33)P]-alpha ATP. Hybridations were performed on Atlas Human Cancer 1.2 membranes (1176 genes). RESULTS: A two-way hierarchical clustering algorithm successfully isolated specific gene expression profiles when comparing MN, cirrhosis, and HCC. A total of 16 and 14 genes were up- and down-expressed, respectively, in HCC as compared to cirrhotic tissues. The molecular signature of MN was characterized by the down-expression of 23 and 42 genes as compared to cirrhosis and HCC, respectively. Among them, Klf6 was down-expressed in all MN samples whereas it was over-expressed in cirrhosis and HCC. This result was confirmed at RNA level by quantitative real time-polymerase chain reaction and at protein level by Western blotting. However, no mutation in the exon 2 of Klf6 was detected. CONCLUSION: We identified a molecular signature of MN characterized by a down-expression of several genes. One of them, Klf6 was found to be down-expressed in all MN without evidence of somatic mutations in the exon 2. This gene could be involved at an early stage of hepatocarcinogenesis.

Syphilis-related hepatitis in a liver transplant patient.

We herein describe a case of secondary syphilis hepatitis in a liver transplant patient. This homosexual man presented 15 years after an orthotopic liver transplant with nonsquamous papillomacular rash, mild cytolysis, and anicteric cholestasis. Laboratory tests showed syphilis seroconversionwith a VDRL test titer of 1/256, a Treponema pallidum hemagglutination assay of 1/5120, and a positive immunoglobulin M fluorescent Treponemal antibody absorbance. A liver biopsy performed 13 months after the diagnosis showed low-grade hepatitis with a METAVIR score of A1F1; it also showed moderate, nonspecific portal inflammation consisting primarily of neutrophils, with no evidence of cholestasis. The patient was given benzathine-penicillin (2400000 IU) with a transient increase in prednisolone dosages. Cytolysis rapidly, and cholestasis progressively, disappeared. Results of an immunoglobulin M fluorescent Treponemal antibody absorbance test became negative, whereas the VDRL test and the Treponema pallidum hemagglutination assay titers decreased slightly over time.

Contribution of microdissection for the detection of microsatellite instability in colorectal cancer.

The determination ofmicrosatellite instability (MSI) is an important step in the identification of familial colorectal cancer such as hereditary nonpolyposis colon cancer. It could also be of interest in the therapeutic management of sporadic cancer. International criteria for the determination of MSI have been published, recommending the use of microdissection. The aim of this work was to evaluate the impact of contaminant normal DNA in tumor samples for MSI assessment in colorectal cancer using a microdissection technique. We performed a comparative analysis of the microsatellite status between total DNA (DNA extracted from whole tumor samples) and microdissected DNA in 3 different regions from 23 cases of colorectal cancer. Six microsatellites were amplified using fluorescent polymerase chain reaction. We analyzed 9 cases with MSI and 14 cases without instability, with similar results between total DNA and microdissected DNA. Moreover, within a same tumor, the MSI phenotype was observed regardless of the region analyzed. Thus, this work shows the reproducibility of the MSI phenotype throughout a tumor. However, we observed a regional heterogeneity of the MSI profile, consisting of variations in the number and the size of unstable alleles within different regions. This result reflects the genetic heterogeneity of colorectal cancer with MSI. In the 14 cases without instability, we observed an increase of more than 60% in the loss of heterozygosity detection rate after microdissection. Thus, this work confirms the contribution of microdissection for loss of heterozygosity assessment.

[Diagnostic value of anti-terminal deoxynucleotidyl transferase antibody (TdT) in hematologic pathology]. / Intérêt diagnostique de l'anticorps anti-terminal désoxynucleotidyl transférase (TdT) en pathologie hématologique.

AIMS: The diagnosis of primitive hematologic malignancies depends on a panel of monoclonal antibodies which is growing over time. The distinction between immature (lymphoblastic lymphoma/acute lymphoblastic leukaemia) and mature lymphoma is sometimes difficult. In this study, we evaluated anti-TdT antibody in the diagnosis and classification of these proliferations. MATERIALS AND METHODS: 13 lesions were examined by immunohistochemistry: 4 B and T lymphoblastic lymphomas, 2 Burkitt's lymphomas, 5 B and T acute lymphoblastic leukemias and 2 acute monoblastic leukemias. RESULTS: TdT expression is specific of immature lymphoid proliferations (T or B lymphoblasts). TdT is not expressed by mature B or T cell lymphomas such as Burkitt's lymphomas. Significant numbers of cases of acute myeloblastic leukemias are TdT positive but could be easily distinguished from lymphoblastic proliferations. CONCLUSION: Anti-TdT antibody represents a useful marker for differentiating lymphoma/acute lymphoblastic leukemia from other lymphomas. This marker, available in routine diagnosis should be systematically included in the panel of antibodies used for immunophenotyping hematologic malignancies.

[Necrolytic migratory erythema revealing glucagonoma without diabetes]. / Erythème nécrolytique migrateur révélateur d'un glucagonome sans diabète.

We report a case of glucagonoma syndrome, revealed by a necrolytic migratory erythema that had developed for four Years, associated with anorexia, severe weight loss, anemia, hypoprotidemia, and hypoaminoacidemia. The fasting blood glucose level tended paradoxically to be low (0.6 g/l). Elevated plasma glucagon levels confirmed our diagnosis. The absence of diabetes was explained by an independent insulin secretion derived from this composite pancreatic tumor, authenticated by the histological analysis and the proinsulin level. This level was similar to those typically observed in insulinomas. Six Months after a complete surgical exeresis, symptoms disappeared and biological results returned to normal values.