Aims
Bile duct adenomas (BDA) and bile duct hamartomas (BDH) are benign bile duct lesions considered neoplastic or secondary to ductal plate malformation, respectively. We have reported previously a ...high prevalence of BRAF V600E mutations detected by allele‐specific polymerase chain reaction assay in BDA, and suggested that BDA may be precursors to a subset of intrahepatic cholangiocarcinomas harbouring V600E mutations. The aim of the present study was to assess the existence of BRAF V600E mutations, using immunohistochemical methods, in additional BDA as well as in BDH.
Methods and results
Fifteen BDA and 35 BDH were retrieved from the archives of the pathology departments of two French university hospitals. All cases were reviewed by two pathologists specialized in liver diseases. BRAF V600E mutational status was investigated by immunohistochemistry. Mutated BRAF mutant protein was detected in 53% of the BDA and in none of the cases of BDH.
Conclusion
Our findings suggest that BDA and BDH are different processes, and that BDA represent true benign neoplasms. They also support the hypothesis that mutated BDA might precede the development of the subset of intrahepatic cholangiocarcinomas harbouring BRAF V600E mutations.
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.
Introduction
Various Peripheral T-cell lymphoma (PTCL) entities are recognized in the World Health Organization (WHO) classification based on clinical, histopathological, phenotypic and molecular ...criteria. Their diagnosis is however often challenging for pathologists, and up to 30% of cases, currently not classifiable, are recognized as not otherwise specified (NOS). Recent gene expression profiling (GEP) studies have significantly improved the molecular and ontogenic characterization of these tumors, but such high-throughput technologies are hardly feasible in the routine clinical setting. There is therefore an important need for alternative diagnostic strategies to allow for the development of specific therapies. Here, we sought to create a parsimonious and robust GEP assay to differentiate the different PTCL entities and to better characterize the heterogeneity of PTCL-NOS.
Methods
A Reverse Transcriptase-Multiplex Ligation dependant Probe Amplification (RT-MLPA) assay addressing 20 markers was applied to a cohort of 227 PTCLs biopsies enriched in PTCL-NOS (n=126). This assay determines the expression of seventeen genes routinely tested as immunohistochemical (IHC) markers or selected from high throughput GEP studies, together with the EBV infection status (EBER1 expression) and the presence of RHOAG17V and IDH2R172K/T mutations.
Results
Unsupervised hierarchical clustering analysis of 101 control cases representing the main PTCL entities other than PTCL-NOS by RT-MLPA accurately classified 28/29 Angioimmunoblastic T-cell lymphomas (AITL), 21/21 Anaplastic large T-cell lymphomas (ALCL) ALK+, 16/16 NK/T-cell lymphomas (NKTCL), 6/6 Hepatosplenic T-cell lymphomas (HSTL) and 12/12 Adult T-cell Leukemia/Lymphomas (ATLL)(Figure). AITL were classified according to the expression of Tfh markers (CXCL13, CXCR5, ICOS, BCL6) and RHOA mutations (n=18); NKTCLs according to EBER1, GZMB and Th1 markers (TBX21, IFNγ); HSTLs to CD56, GATA3, TBX21 and BCL6; ALCL ALK+ according to CD30, ALK and cytotoxic markers (PRF, GZMB); ATLLs to ICOS and Th2 markers (GATA3, CCR4). Interestingly, ALCL ALK- cases (n=17) were divided into 2 subgroups: one, associated with high expression of CD30 and cytotoxic markers (PRF, GZMB), clustered with ALCL ALK+ cases (n=11), the other showed absence of PRF and GZMB, but expression of CD30 and Th2 markers (n=6).
Applied to 126 nodal PTCL-NOS, the RT-MLPA classifier identified 33 AITL-Like cases expressing Tfh markers and often presenting with RHOA mutations (15 cases). It also identified 5 NKTCL-like cases (EBV-cytoxic) and 1 ALCL-like case (cytotoxic-CD30).The CD30-Th2 signature was found in 15 cases, reinforcing the hypothesis that it may delineate a novel PTCL entity, at the frontier between ALCL ALK- and other PTCLs. In agreement with previous GEP studies, 23 cases expressed Th2 markers but no CD30 (often in association with a significant Tfh signature, probably reflecting a contribution from the microenvironment). Twenty-five other cases showed a hybrid cytotoxic-Th1 signature. The remaining 14 cases did not reveal any recognizable gene expression profile.
Finally, we observed a strong correlation between RT-MLPA and IHC for most markers evaluated by both methods (p<10-3 for CD8, CD30, GZMB, PRF, ALK, CD56, CXCL13, ICOS and GATA3), indicating that our assay is reliable and may constitute a valuable alternative to IHC.
Conclusion
This study demonstrates the applicability of a parsimonious RT-MLPA classifier for the classification of PTCLs. Its simplicity of use and its applicability on FFPE samples makes it an attractive alternative to high throughput GEP approaches and IHC. Its implementation in clinical trials, in combination with conventional pathological evaluation, may thus participate to improve the classification of PTCLs and therefore the management of PTCL patients.
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No relevant conflicts of interest to declare.
The biological evaluation of a natural sesquiterpene dimer meiogynin A 1, is described as well as that of five non-natural analogues. Although active on a micromolar range on the inhibition of ...Bcl-xL/Bak and Mcl-1/Bid interaction, meiogynin A 1 is not cytotoxic on three cell lines that overexpress Bcl-xL and Mcl-1. Contrarily, one of its analogues 6 with an inverted configuration on the side chain and an aromatic moiety replacing the cyclohexane ring was active on both target proteins, cytotoxic on a micromolar range and was found to induce apoptosis through a classical pathway.
Background and aim of the study
Primary mediastinal B-cell lymphoma (PMBL) is an entity of aggressive B-cell lymphoma that is clinically and biologically distinct from the other molecular subtypes of ...diffuse large B-cell lymphoma (DLBCL). We recently detected by Whole exome sequencing a recurrent point mutation in the XPO1 (exportin 1) gene (also referred to as chromosome region maintenance 1; CRM1), which resulted in the Glu571Lys (p.E571K) missense substitution in 2 refractory/relapsed PMBL (Dubois et al., ICML 2015; Mareschal et al. AACR 2015). XPO1 is a member of the Karyopherin-b superfamily of nuclear transport proteins. XPO1 mediates the nuclear export of numerous RNAs and cellular regulatory proteins, including tumor suppressor proteins. This mutation is in the hydrophobic groove of XPO1 that binds to the leucine-rich nuclear export signal (NES) of cargo proteins. In this study, we investigated the prevalence, specificity, and biological / clinical relevance of XPO1 mutations in PMBL.
Patients and methods
High-throughput targeted or Sanger sequencing of 117 PMBL patients and 3 PMBL cell lines were performed. PMBL cases were defined either molecularly by gene expression profile (mPMBL cohort) or by standard histological method (hPMBL cohort) and enrolled in various LYSA (LYmphoma Study Association) clinical trials. To assess the frequency and specificity of XPO1 mutations, cases of classical Hodgkin lymphoma (cHL) and primary mediastinal grey zone lymphoma (MGZL) were analysed. Cell experiments were performed to assess the impact of the E571 mutation on the activity of selective inhibitor of nuclear export (SINE) molecules.
Results
XPO1 mutations were present in 28/117 (24%) PMBL cases but were rare in cHL cases (1/19, 5%) and absent from MGZL cases (0/20). A higher prevalence (50%) of the recurrent codon 571 variant (p.E571K) was observed in PMBL cases defined by gene expression profiling (n = 32), as compared to hPMBL cases (n = 85, 13%). No difference in age, International Prognostic Index (IPI) or bulky mass was observed between the PMBL patients harboring mutant and wild-type XPO1 in the overall cohort whereas a female predominance was noticed in the mPMBL cohort. Based on a median follow-up duration of 42 months, XPO1 mutant patients exhibited significantly decreased PFS (3y PFS = 74% CI95% 55-100) compared to wild-type patients (3y PFS = 94% CI95% 83-100, p=0.049) in the mPMBL cohort. In 4/4 tested cases, the E571K variant was also detected in cell-free circulating plasmatic DNA, suggesting that the mutation can be used as a biomarker at the time of diagnosis and during follow-up. Importantly, the E571K variant was detected as a heterozygous mutation in MedB-1, a PMBL-derived cell line, whereas the two other PMBL cell lines tested, Karpas1106 and U-2940, did not display any variants in XPO1 exon 15. KPT-185, the SINE compound that blocks XPO1-dependent nuclear export, induced a dose-dependent decrease in cell proliferation and increased cell death in the PMBL cell lines harbouring wild type or mutated alleles. To test directly if XPO1 mutation from E571 to E571K alters XPO1 inhibition by SINE compounds, the mutated protein was tested in vitro. The E571XPO1 mutated allele was transiently transfected into osteosarcoma U2OS cells which stably express the fluorescently labelled XPO1 cargo REV. Cells were treated with the clinical SINE compound selinexor, which is currently in phase I/II clinical trials and nuclear localization of REV-GFP was analysed in red transfected cells. The results showed that the nuclear export of the mutated XPO1 protein was inhibited by selinexor similarly to the wild-type XPO1 protein (Figure 1).
Conclusion
Although the oncogenic properties of XPO1 mutations remain to be determined, their recurrent selection in PMBL strongly supports their involvement in the pathogenesis of this curable aggressive B-cell lymphoma. XPO1 mutations were primarily observed in young female patients who displayed a typical PMBL molecular signature. The E571K XPO1 mutation represents a novel hallmark of PMBL but does not seem to interfere with SINE activity.
Rev-GFP (green fluorescent) expressing U2OS cells were transfected with wild type XPO1-RFP (red fluorescent protein), XPO1-C528S-RFP, XPO1-E571K-mCherry, and XPO1-E571G-mCherry. The cells were then treated with 1µM KPT-330 for 8 hours.
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Landesman:Karyopharm Therapeutics: Employment. Senapedis:Karyopharm Therapeutics, Inc.: Employment, Patents & Royalties. Argueta:Karyopharm Therapeutics: Employment. Milpied:Celgene: Honoraria, Research Funding.
Combined hepatocellular-cholangiocarcinoma (cHCC-CCA) is a rare primary liver cancer (PLC) associated with a poor prognosis. Given the challenges in its identification and its clinical implications, ...biomarkers are critically needed. We aimed to investigate the diagnostic and prognostic value of the immunohistochemical expression of Nestin, a progenitor cell marker, in a large multicentric series of PLCs.
We collected 461 cHCC-CCA samples from 32 different clinical centers. Control cases included 368 hepatocellular carcinomas (HCCs) and 221 intrahepatic cholangiocarcinomas (iCCAs). Nestin immunohistochemistry was performed on whole tumor sections. Diagnostic and prognostic performances of Nestin expression were determined using receiver-operating characteristic curves and Cox regression modeling.
Nestin was able to distinguish cHCC-CCA from HCC with AUCs of 0.85 and 0.86 on surgical and biopsy samples, respectively. Performance was lower for the distinction of cHCC-CCA from iCCA (AUCs of 0.59 and 0.60). Nestin, however, showed a high prognostic value, allowing identification of the subset of cHCC-CCA (“Nestin High”, >30% neoplastic cells with positive staining) associated with the worst clinical outcome (shorter disease-free and overall survival) after surgical resection and liver transplantation, as well as when assessment was performed on biopsies.
We show in different clinical settings that Nestin has diagnostic value and that it is a useful biomarker to identify the subset of cHCC-CCA associated with the worst clinical outcome. Nestin immunohistochemistry may be used to refine risk stratification and improve treatment allocation for patients with this highly aggressive malignancy.
There are different types of primary liver cancers (i.e. cancers that originate in the liver). Accurately identifying a specific subtype of primary liver cancer (and determining its associated prognosis) is important as it can have a major impact on treatment allocation. Herein, we show that a protein called Nestin could be used to refine risk stratification and improve treatment allocation for patients with combined hepatocellular carcinoma, a rare but highly aggressive subtype of primary liver cancer.
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•Biomarkers for combined hepatocellular-cholangiocarcinoma (cHCC-CCA) are critically needed.•Nestin immunohistochemical expression is able to identify the subset of cHCC-CCA associated with the worst clinical outcome.•cHCC-CCA with >30% of neoplastic cells expressing Nestin are classified “Nestin High”.•Nestin High cHCC-CCA are associated with an adverse outcome after surgical resection and liver transplantation.
The tumor suppressor TP53 is frequently mutated or inactivated in human cancers. Mutations of TP53 are less common in lymphomas than in other tumors, but the protein is often inhibited by the ...overexpression of its main regulator--MDM2. In the past 10 years, major efforts have been made to develop drugs that can reactivate p53 and restore its functions. This review focuses on recent advances in the development of small inhibitors of MDM2, which are potentially relevant for the treatment of B- and T-cell lymphomas. We will describe the current state of development of these drugs and discuss their mechanism of action in these hematological malignancies. Keywords: lymphoma, p53, nutlin, apoptosis, MDM2
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