Multiple Ways to Detect IDH2 Mutations in Angioimmunoblastic T-Cell Lymphoma from Immunohistochemistry to Next-Generation Sequencing.

Angioimmunoblastic T-cell lymphoma (AITL) is a peripheral T-cell lymphoma associated with chemoresistance and a poor prognosis. Various nonsynonymous mutations in the R172 residue of IDH2 are present in 20% to 30% of AITL patients. In addition to their diagnostic value, these mutations are potentially targetable, especially by isocitrate dehydrogenase (IDH) 2 inhibitor, and therefore their identification in a routine setting is clinically relevant. However, in AITL, the neoplastic cells may be scarce, making the identification of molecular anomalies difficult. We evaluated the diagnostic value of different methods to detect IDH2 mutations in formalin-fixed, paraffin-embedded tumor samples. Immunohistochemistry with an anti-IDH2 R172K antibody, Sanger sequencing, high-resolution melting PCR, allele-specific real-time quantitative PCR, and next-generation sequencing (NGS) were applied to biopsy specimens from 42 AITL patients. We demonstrate that the IDH2 R172K antibody is specific to this amino acid substitution and highly sensitive for the detection of the IDH2R172K variant, the most frequent substitution in this disease. In our study, NGS and allele-specific real-time quantitative PCR displayed a good sensitivity, detecting 96% and 92% of IDH2 mutations, respectively, in contrast to Sanger sequencing and high-resolution melting PCR, which showed a significantly lower detection rate (58% and 42%, respectively). These results suggest that a combination of immunohistochemistry and AS-PCR or NGS should be considered for the identification of IDH2 mutations in AITL in a routine setting.

Diagnosis of different liver fibrosis characteristics by blood tests in non-alcoholic fatty liver disease.

AIMS: Our aim was to develop an accurate, non-invasive, blood-test-based method for identifying the main characteristics of liver fibrosis in non-alcoholic fatty liver disease (NAFLD). METHODS: Fibrosis was staged according to NASH-CRN and Metavir systems in 226 patients with NAFLD. A fully automated algorithm measured the fractal dimension (FD) and the area of fibrosis (AOF). Independent predictors of diagnostic targets were determined using bootstrap methods. RESULTS: (i) Development. Significant fibrosis defined by NASH-CRN F ≥2 was diagnosed by weight, glycaemia, aspartate aminotransferase (AST), alanine aminotransferase (ALT) and prothrombin index [area under the receiver operating characteristic (AUROC)=0.867]; significant fibrosis defined by Metavir F ≥2 was diagnosed by weight, age, glycaemia, AST, ALT, ferritin and platelets (FibroMeter AUROC=0.941, P<0.005). AOF was estimated by the combination of hyaluronic acid, glycaemia, AST, ALT, platelets and prothrombin index ((a) R(2) =0.530), while FD was estimated by hyaluronic acid, glycaemia, AST/ALT, weight and platelets ((a) R(2) =0.529). (ii) Evaluation. Although NASH-CRN was a better system for fibrosis staging, Metavir staging was a better reference for blood test. Thus, the patient rate with predictive values ≥90% by tests was 97.3% with Metavir reference vs. 66.5% with NASH-CRN reference (P<10(-3)). FibroMeter showed a significantly higher AUROC than the NAFLD fibrosis score for significant fibrosis, but not for severe fibrosis or cirrhosis, with both staging systems. Relationships between fibrosis lesions were well reflected by blood tests, e.g., the correlation between histological area and FD of fibrosis (r(s) =0.971, P<10(-3)) was well reflected by the relationship between respective blood tests (r(s) =0.852, P<10(-3)). CONCLUSIONS: Different characteristics of fibrosis in NAFLD can be diagnosed and quantified by blood tests with excellent accuracy.

A non-randomised dose-escalating phase II study of thalidomide for the treatment of patients with low-risk myelodysplastic syndromes: the Thal-SMD-2000 trial of the Groupe Français des Myélodysplasies.

Patients (n=47) with low-risk myelodysplastic syndrome were treated with thalidomide [200 mg/d, increased by 200 mg/d/4 weeks up to week 16]. Responses were evaluated according to the International Working Group criteria at week 16 for 39 patients who received at least 8 weeks of treatment. Twenty-three (59%) patients showed haematological improvement (HI): four major erythroid response (HI-EM), 15 minor erythroid response, six major neutrophil response, two major platelet response. Side effects caused 22/39 to stop thalidomide before week 16. Nine of 23 responders continued thalidomide after week 16 [19% of trial patients] with sustained response in eight of nine. Six reached week 56, including the four HI-EM patients [13% of trial patients]. Nineteen of 36 red blood cell transfusion-dependent patients (53%) showed erythroid response, but only four became transfusion-independent. Among the 23 responders, the median duration of response was 260 d (range 30-650). Responses were sustained in all patients except one, and were observed between week 4 and week 8 in 85% of patients, at doses ranging from 200 to 400 mg. Only two patients responded at 600 mg/d and none at 800 mg/d. No clinical characteristics of responding versus non-responding patients were identified. The erythroid response rate was identical in all cytogenetic subgroups, including 5q31.1 deletions. Pretreatment vascular endothelial growth factor levels were lower in responders compared with non-responders (P=0.004). Microvessel density (MVD) increased and apoptosis decreased in four of six and in all six responders studied respectively whereas MVD and apoptosis were unchanged in three non-responders.

New Doppler ultrasound signs improve the non-invasive diagnosis of cirrhosis or severe liver fibrosis.

OBJECTIVES: To determine whether ultrasound and, particularly, new Doppler signs increased the diagnostic accuracy of the most accurate, currently available markers for the diagnosis of cirrhosis or severe fibrosis. METHODS: Thirty-two clinical (n = 4), biochemical (n = 11) and Doppler ultrasound (n = 17) variables were recorded in 106 patients with compensated chronic liver disease. Diagnostic accuracy was evaluated by discriminant analysis; first, globally, using all variables then by stepwise analysis. RESULTS: (A) Diagnosis of cirrhosis. Using Doppler ultrasound, diagnostic accuracy was 92% (95% confidence interval 81-98) globally, and 89% (76-95) with three variables (spleen length, hepatic vein spectrum and maximum portal vein velocity). Based upon clinical signs, diagnostic accuracy was 86% (77-92) globally, and 85% (76-91) with one variable (firm liver). Based upon biochemical parameters, diagnostic accuracy was 80% (70-88) globally, and 81% (72-88) with two variables (hyaluronate and platelet count). Based upon all parameters, diagnostic accuracy was 91% (79-96.5) globally, and 91% (79-96.5) with four variables (firm liver, hyaluronate, platelet and hepatic vein spectrum). On an intention to diagnose basis, Doppler ultrasound provided a lower independent contribution due to missing data. (B) In the diagnosis of severe fibrosis, diagnostic accuracy was 83% (69-92) globally, and 77% (62-87) with one variable. CONCLUSIONS: Cirrhosis can be correctly diagnosed in approximately 90% of patients with compensated chronic liver disease using a few Doppler ultrasound signs including a new sign, the hepatic vein spectrum. Doppler ultrasound could be used for the first line diagnosis and biochemical markers, such as hyaluronate, in patients with missing Doppler ultrasound data.