Quality-Assured Analysis of PIK3CA Mutations in Hormone Receptor-Positive/Human Epidermal Growth Factor Receptor 2-Negative Breast Cancer Tissue: A Story About the Need for Proficiency Testing for High-Quality Molecular Biomarker Reporting in Precision Medicine.

In precision oncology, reliable testing of predictive molecular biomarkers is a prerequisite for optimal patient treatment. Interlaboratory comparisons are a crucial tool to verify diagnostic performance and reproducibility of one's approach. Herein is described the design and results of the first recurrent, internationally performed PIK3CA (phosphatidylinositol-4,5-bisphosphate 3 kinase catalytic subunit α) breast cancer tissue external quality assessment (EQA), organized by German Quality in Pathology GmbH and started in 2021. After the internal pretesting phase performed by the (lead) panel institutes, in both 2021 and 2022, each EQA test set comprised n = 10 tissue samples of hormone receptor-positive, human epidermal growth factor receptor 2-negative invasive breast cancer that had to be analyzed and reported by the participants. In 2021, the results were evaluated separately for German-speaking countries (part 1) and international laboratories (part 2). In 2022, the EQA was performed across the European Union. The EQA success rates were 84.6% (n = 11/13), 88.6% (n = 39/44), and 87.9% (n = 29/33) for EQA 2021 part 1, part 2, and EQA 2022, respectively. The most commonly used methods were next-generation sequencing and mutation-/allele-specific qualitative PCR-based assays. In summary, this recurrent PIK3CA EQA proved to be a suitable approach to obtain an international overview of methods used for PIK3CA mutation analysis, to evaluate them qualitatively, and identify the strengths and weaknesses of individual methods.

Inter-identity amnesia in dissociative identity disorder resolved: A behavioural and neurobiological study.

INTRODUCTION: Dissociative identity disorder (DID) is characterised by, among others, subjectively reported inter-identity amnesia, reflecting compromised information transfer between dissociative identity states. Studies have found conflicting results regarding memory transfer between dissociative identity states. Here, we investigated inter-identity amnesia in individuals with DID using self-relevant, subject specific stimuli, and behavioural and neural measures. METHODS: Data of 46 matched participants were included; 14 individuals with DID in a trauma-avoidant state, 16 trauma-avoiding DID simulators, and 16 healthy controls. Reaction times and neural activation patterns related to three types of subject specific words were acquired and statistically analysed, namely non-self-relevant trauma-related words (NSt), self-relevant trauma-related words from a trauma-avoidant identity state (St), and trauma-related words from a trauma-related identity state (XSt). RESULTS: We found no differences in reaction times between XSt and St words and faster reaction times for XSt over NSt. Reaction times of the diagnosed DID group were the longest. Increased brain activation to XSt words was found in the frontal and parietal regions, while decreased brain activity was found in the anterior cingulate cortex in the diagnosed DID group. DISCUSSION: The current study reproduces and amalgamates previous behavioural reports as well as brain activation patterns. Our finding of increased cognitive control over self-relevant trauma-related knowledge processing has important clinical implications and calls for the redefinition of "inter-identity amnesia" to "inter-identity avoidance".

Implementation of circulating tumour DNA multi-target mutation testing in plasma: a perspective from an external quality assessment providers' survey.

Demand for large-scale tumour profiling across cancer types has increased in recent years, driven by the emergence of targeted drug therapies. Analysing alternations in plasma circulating tumour DNA (ctDNA) for cancer detection can improve survival; ctDNA testing is recommended when tumour tissue is unavailable. An online survey of molecular pathology testing was circulated by six external quality assessment members of IQN Path to registered laboratories and all IQN Path collaborative corporate members. Data from 275 laboratories across 45 countries were collected; 245 (89%) perform molecular pathology testing, including 177 (64%) which perform plasma ctDNA diagnostic service testing. The most common tests were next-generation sequencing-based (n = 113). Genes with known stratified treatment options, including KRAS (n = 97), NRAS (n = 84), and EGFR (n = 130), were common targets. The uptake of ctDNA plasma testing and plans to implement further testing demonstrates the importance of support from a well-designed EQA scheme.

Update on quality assurance in neuropathology: Summary of the round robin trials on TERT promoter mutation, H3-3A mutation, 1p/19q codeletion, and KIAA1549::BRAF fusion testing in Germany in 2020 and 2021.

We previously reported on the first neuropathological round robin trials operated together with Quality in Pathology (QuIP) GmbH in 2018 and 2019 in Germany, i.e., the trials on IDH mutational testing and MGMT promoter methylation analysis [1]. For 2020 and 2021, the spectrum of round robin trials has been expanded to cover the most commonly used assays in neuropathological institutions. In addition to IDH mutation and MGMT promoter methylation testing, there is a long tradition for 1p/19q codeletion testing relevant in the context of the diagnosis of oligodendroglioma. With the 5th edition of the World Health Organization (WHO) classification of the central nervous system tumors, additional molecular markers came into focus: TERT promoter mutation is often assessed as a molecular diagnostic criterion for IDH-wildtype glioblastoma. Moreover, several molecular diagnostic markers have been introduced for pediatric brain tumors. Here, trials on KIAA1549::BRAF fusions (common in pilocytic astrocytomas) and H3-3A mutations (in diffuse midline gliomas, H3-K27-altered and diffuse hemispheric gliomas, H3-G34-mutant) were most desired by the neuropathological community. In this update, we report on these novel round robin trials. In summary, success rates in all four trials ranged from 75 to 96%, arguing for an overall high quality level in the field of molecular neuropathological diagnostics.

Proficiency testing of PIK3CA mutations in HR+/HER2-breast cancer on liquid biopsy and tissue.

Precision oncology based on specific molecular alterations requires precise and reliable detection of therapeutic targets in order to initiate the optimal treatment. In many European countries-including Germany-assays employed for this purpose are highly diverse and not prescribed by authorities, making inter-laboratory comparison difficult. To ensure reproducible molecular diagnostic results across many laboratories and different assays, ring trials are essential and a well-established tool. Here, we describe the design and results of the ring trial for the detection of therapeutically relevant PIK3CA hotspot mutations in HR+/HER2-breast cancer tissue and liquid biopsy (LB). For PIK3CA mutation detection in tissue samples, 43 of the 54 participants (80%) provided results compliant with the reference values. Participants using NGS-based assays showed higher success rate (82%) than those employing Sanger sequencing (57%). LB testing was performed with two reference materials differing in the length of the mutated DNA fragments. Most participants used NGS-based or commercial real-time PCR assays (70%). The 167 bp fragments led to a successful PIK3CA mutation detection by only 31% of participants whereas longer fragments of 490 bp were detectable even by non-optimal assays (83%). In conclusion, the first ring trial for PIK3CA mutation detection in Germany showed that PIK3CA mutation analysis is broadly established for tissue samples and that NGS-based tests seem to be more suitable than Sanger sequencing. PIK3CA mutation detection in LB should be carried out with assays specifically designed for this purpose in order to avoid false-negative results.

A neurostructural biomarker of dissociative amnesia: a hippocampal study in dissociative identity disorder.

BACKGROUND: Little is known about the neural correlates of dissociative amnesia, a transdiagnostic symptom mostly present in the dissociative disorders and core characteristic of dissociative identity disorder (DID). Given the vital role of the hippocampus in memory, a prime candidate for investigation is whether total and/or subfield hippocampal volume can serve as biological markers of dissociative amnesia. METHODS: A total of 75 women, 32 with DID and 43 matched healthy controls (HC), underwent structural magnetic resonance imaging (MRI). Using Freesurfer (version 6.0), volumes were extracted for bilateral global hippocampus, cornu ammonis (CA) 1-4, the granule cell molecular layer of the dentate gyrus (GC-ML-DG), fimbria, hippocampal-amygdaloid transition area (HATA), parasubiculum, presubiculum and subiculum. Analyses of covariance showed volumetric differences between DID and HC. Partial correlations exhibited relationships between the three factors of the dissociative experience scale scores (dissociative amnesia, absorption, depersonalisation/derealisation) and traumatisation measures with hippocampal global and subfield volumes. RESULTS: Hippocampal volumes were found to be smaller in DID as compared with HC in bilateral global hippocampus and bilateral CA1, right CA4, right GC-ML-DG, and left presubiculum. Dissociative amnesia was the only dissociative symptom that correlated uniquely and significantly with reduced bilateral hippocampal CA1 subfield volumes. Regarding traumatisation, only emotional neglect correlated negatively with bilateral global hippocampus, bilateral CA1, CA4 and GC-ML-DG, and right CA3. CONCLUSION: We propose decreased CA1 volume as a biomarker for dissociative amnesia. We also propose that traumatisation, specifically emotional neglect, is interlinked with dissociative amnesia in having a detrimental effect on hippocampal volume.

Normal amygdala morphology in dissociative identity disorder.

Studies investigating the structure of the amygdala in relation to dissociation in psychiatric disorders are limited and have reported normal or preserved, increased or decreased global volumes. Thus, a more detailed investigation of the amygdala is warranted. Amygdala global and subregional volumes were compared between individuals with dissociative identity disorder (DID: n = 32) and healthy controls (n = 42). Analyses of covariance did not show volumetric differences between the DID and control groups. Although several unknowns make it challenging to interpret our findings, we propose that the finding of normal amygdala volume is a genuine finding because other studies using this data-set have presented robust morphological aberrations in relation to the diagnosis of DID.

No Self Without Salience: Affective and Self-relevance Ratings of 552 Emotionally Valenced and Neutral Dutch Words.

It is unknown how self-relevance is dependent on emotional salience. Emotional salience encompasses an individual's degree of attraction or aversion to emotionally-valenced information. The current study investigated the interconnection between self and salience through the evaluation of emotional valence and self-relevance. 56 native Dutch participants completed a questionnaire assessing valence, intensity, and self-relevance of 552 Dutch nouns and verbs. One-way repeated-measures ANCOVA investigated the relationship between valence and self, age and gender. Repeated-measures ANCOVA also tested the relationship between valence and self with intensity ratings and effects of gender and age. Results showed a significant main effect of valence for self-relevant words. Intensity analyses showed a main effect of valence but not of self-relevance. There were no significant effects of gender and age. The most important finding presents that self-relevance is dependent on valence. These findings concerning the relationship between self and salience opens avenues to study an individual's self-definition.

Acquired resistance to DZNep-mediated apoptosis is associated with copy number gains of AHCY in a B-cell lymphoma model.

BACKGROUND: Enhancer of zeste homolog 2 (EZH2) is considered an important driver of tumor development and progression by its histone modifying capabilities. Inhibition of EZH2 activity is thought to be a potent treatment option for eligible cancer patients with an aberrant EZH2 expression profile, thus the indirect EZH2 inhibitor 3-Deazaneplanocin A (DZNep) is currently under evaluation for its clinical utility. Although DZNep blocks proliferation and induces apoptosis in different tumor types including lymphomas, acquired resistance to DZNep may limit its clinical application. METHODS: To investigate possible mechanisms of acquired DZNep resistance in B-cell lymphomas, we generated a DZNep-resistant clone from a previously DZNep-sensitive B-cell lymphoma cell line by long-term treatment with increasing concentrations of DZNep (ranging from 200 to 2000 nM) and compared the molecular profiles of resistant and wild-type clones. This comparison was done using molecular techniques such as flow cytometry, copy number variation assay (OncoScan and TaqMan assays), fluorescence in situ hybridization, Western blot, immunohistochemistry and metabolomics analysis. RESULTS: Whole exome sequencing did not indicate the acquisition of biologically meaningful single nucleotide variants. Analysis of copy number alterations, however, demonstrated among other acquired imbalances an amplification (about 30 times) of the S-adenosyl-L-homocysteine hydrolase (AHCY) gene in the resistant clone. AHCY is a direct target of DZNep and is critically involved in the biological methylation process, where it catalyzes the reversible hydrolysis of S-adenosyl-L-homocysteine to L-homocysteine and adenosine. The amplification of the AHCY gene is paralleled by strong overexpression of AHCY at both the transcriptional and protein level, and persists upon culturing the resistant clone in a DZNep-free medium. CONCLUSIONS: This study reveals one possible molecular mechanism how B-cell lymphomas can acquire resistance to DZNep, and proposes AHCY as a potential biomarker for investigation during the administration of EZH2-targeted therapy with DZNep.

Sleep, trauma, fantasy and cognition in dissociative identity disorder, post-traumatic stress disorder and healthy controls: a replication and extension study.

Background: Two aetiology models for dissociative identity disorder (DID) have been proposed, namely a childhood Trauma Model and an iatrogenic or Fantasy model. A recent study indicated that sleep disturbances underlie dissociative symptomatology. Objective: Our current study aims to test whether this finding can be replicated in an independent sample and to investigate if this finding still holds after correcting for childhood and adult traumatization. An experimental working memory task is included to investigate how sleep disturbance, traumatization, dissociation, and fantasy proneness impact cognitive functioning. Methods: Three groups of participants were included - individuals with DID, individuals with post-traumatic stress disorder (PTSD), and matched healthy controls. Sleep disturbances were measured and compared between the groups along with measures of childhood and adult traumatization, psychoform and somatoform [psychological and somatic] dissociative symptoms, and fantasy proneness. Cognitive capacity was assessed using a working memory task. Results: When controlled for traumatic experiences, sleep disturbances did not predict dissociative symptoms. When controlled for sleep disturbance and fantasy proneness, childhood traumatization did predict dissociative symptoms. Psychoform dissociative symptoms correlated with traumatic experiences more than with fantasy proneness. Working memory performance was similar among the participating groups. Propensity to fantasy did not discriminate individuals with DID and PTSD, and was a weak predictor of dissociative symptoms. Conclusion: Whereas DID and PTSD are associated with sleep disturbances, these features do not statistically predict dissociative symptoms in these disorders when traumatic experiences are taken into account. Fantasy proneness is not excessive in DID and PTSD. Hence, we found no evidence that sleep disturbances, propensity to fantasy and abnormal working memory capacity explain dissociative symptoms in DID and PTSD. In sum, the relationship between sleep and dissociative symptoms disappeared when potentially traumatizing events were controlled for.

Antecedentes: Se han propuesto dos modelos etiológicos para el trastorno de identidad disociativo (TID), a sabre, un modelo de trauma infantil y un modelo iatrogénico o de fantasía. Un estudio reciente indicó que las alteraciones del sueño subyacen en la sintomatología disociativaObjetivo: Nuestro actual estudio tiene como objetivo evaluar si este hallazgo se puede replicar en una muestra independiente e investigar si este hallazgo aún se mantiene después de corregir la traumatización en niños y adultos. Se incluye una tarea experimental de memoria de trabajo para investigar cómo la alteración del sueño, la traumatización, la disociación y la propensión a la fantasía afectan el funcionamiento cognitivo.Métodos: Se incluyeron tres grupos de participantes: individuos con TID, individuos con trastorno de estrés postraumático (TEPT) y controles sanos pareados. Las alteraciones del sueño se midieron y compararon entre los grupos junto con medidas de traumatización infantil y adulta, síntomas disociativos psicomorfos y somatomorfos [psicológicos y somáticos] y propensión a la fantasía. La capacidad cognitiva se evaluó mediante una tarea de memoria de trabajoResultados: Cuando se controlaron las experiencias traumáticas, los trastornos del sueño no predijeron síntomas disociativos. Cuando se controlaron las alteraciones del sueño y la propensión a la fantasía, la traumatización infantil predijo los síntomas disociativos. Los síntomas disociativos psicomorfos se correlacionan más con las experiencias traumáticas que con la propensión a la fantasía. El rendimiento de la memoria de trabajo fue similar entre los grupos participantes. La propensión a la fantasía no discriminaba a los individuos con TID y TEPT, y era un predictor débil de síntomas disociativosConclusión: Mientras que el TID y el TEPT están asociados con trastornos del sueño, estas características no predicen estadísticamente los síntomas disociativos en estos trastornos cuando se tienen en cuenta las experiencias traumáticas. La propensión a la fantasía no es excesiva en DID y PTSD. Por lo tanto, no encontramos evidencia de que los trastornos del sueño, la propensión a la fantasía y la capacidad anormal de la memoria de trabajo expliquen los síntomas disociativos en el TID y el TEPT. En resumen, la relación entre el sueño y los síntomas disociativos desapareció cuando se controlaron los eventos potencialmente traumáticos.

DZNep-mediated apoptosis in B-cell lymphoma is independent of the lymphoma type, EZH2 mutation status and MYC, BCL2 or BCL6 translocations.

Enhancer of zeste homolog 2 (EZH2) tri-methylates histone 3 at position lysine 27 (H3K27me3). Overexpression and gain-of-function mutations in EZH2 are regarded as oncogenic drivers in lymphoma and other malignancies due to the silencing of tumor suppressors and differentiation genes. EZH2 inhibition is sought to represent a good strategy for tumor therapy. In this study, we treated Burkitt lymphoma (BL) and diffuse large B-cell lymphoma (DLBCL) cell lines with 3-deazaneplanocin-A (DZNep), an indirect EZH2 inhibitor which possesses anticancer properties both in-vitro and in-vivo. We aimed to address the impact of the lymphoma type, EZH2 mutation status, as well as MYC, BCL2 and BCL6 translocations on the sensitivity of the lymphoma cell lines to DZNep-mediated apoptosis. We show that DZNep inhibits proliferation and induces apoptosis of these cell lines independent of the type of lymphoma, the EZH2 mutation status and the MYC, BCL2 and BCL6 rearrangement status. Furthermore, DZNep induced a much stronger apoptosis in majority of these cell lines at a lower concentration, and within a shorter period when compared with EPZ-6438, a direct EZH2 inhibitor currently in phase II clinical trials. Apoptosis induction by DZNep was both concentration-dependent and time-dependent, and was associated with the inhibition of EZH2 and subsequent downregulation of H3K27me3 in DZNep-sensitive cell lines. Although EZH2, MYC, BCL2 and BCL6 are important prognostic biomarkers for lymphomas, our study shows that they poorly influence the sensitivity of lymphoma cell lines to DZNep-mediated apoptosis.

Identification of ADGRE5 as discriminating MYC target between Burkitt lymphoma and diffuse large B-cell lymphoma.

BACKGROUND: MYC is a heterogeneously expressed transcription factor that plays a multifunctional role in many biological processes such as cell proliferation and differentiation. It is also associated with many types of cancer including the malignant lymphomas. There are two types of aggressive B-cell lymphoma, namely Burkitt lymphoma (BL) and a subgroup of diffuse large cell lymphoma (DLBCL), which both carry MYC translocations and overexpress MYC but both differ significantly in their clinical outcome. In DLBCL, MYC translocations are associated with an aggressive behavior and poor outcome, whereas MYC-positive BL show a superior outcome. METHODS: To shed light on this phenomenon, we investigated the different modes of actions of MYC in aggressive B-cell lymphoma cell lines subdivided into three groups: (i) MYC-positive BL, (ii) DLBCL with MYC translocation (DLBCLpos) and (iii) DLBCL without MYC translocation (DLBCLneg) for control. In order to identify genome-wide MYC-DNA binding sites a chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-Seq) was performed. In addition, ChIP-Seq for H3K4me3 was used for determination of genomic regions accessible for transcriptional activity. These data were supplemented with gene expression data derived from RNA-Seq. RESULTS: Bioinformatics integration of all data sets revealed different MYC-binding patterns and transcriptional profiles in MYC-positive BL and DLBCL cell lines indicating different functional roles of MYC for gene regulation in aggressive B-cell lymphomas. Based on this multi-omics analysis we identified ADGRE5 (alias CD97) - a member of the EGF-TM7 subfamily of adhesion G protein-coupled receptors - as a MYC target gene, which is specifically expressed in BL but not in DLBCL regardless of MYC translocation. CONCLUSION: Our study describes a diverse genome-wide MYC-DNA binding pattern in BL and DLBCL cell lines with and without MYC translocations. Furthermore, we identified ADREG5 as a MYC target gene able to discriminate between BL and DLBCL irrespectively of the presence of MYC breaks in DLBCL. Since ADGRE5 plays an important role in tumor cell formation, metastasis and invasion, it might also be instrumental to better understand the different pathobiology of BL and DLBCL and help to explain discrepant clinical characteristics of BL and DLBCL.

Senescence-associated reprogramming promotes cancer stemness.

Cellular senescence is a stress-responsive cell-cycle arrest program that terminates the further expansion of (pre-)malignant cells. Key signalling components of the senescence machinery, such as p16INK4a, p21CIP1 and p53, as well as trimethylation of lysine 9 at histone H3 (H3K9me3), also operate as critical regulators of stem-cell functions (which are collectively termed 'stemness'). In cancer cells, a gain of stemness may have profound implications for tumour aggressiveness and clinical outcome. Here we investigated whether chemotherapy-induced senescence could change stem-cell-related properties of malignant cells. Gene expression and functional analyses comparing senescent and non-senescent B-cell lymphomas from Eµ-Myc transgenic mice revealed substantial upregulation of an adult tissue stem-cell signature, activated Wnt signalling, and distinct stem-cell markers in senescence. Using genetically switchable models of senescence targeting H3K9me3 or p53 to mimic spontaneous escape from the arrested condition, we found that cells released from senescence re-entered the cell cycle with strongly enhanced and Wnt-dependent clonogenic growth potential compared to virtually identical populations that had been equally exposed to chemotherapy but had never been senescent. In vivo, these previously senescent cells presented with a much higher tumour initiation potential. Notably, the temporary enforcement of senescence in p53-regulatable models of acute lymphoblastic leukaemia and acute myeloid leukaemia was found to reprogram non-stem bulk leukaemia cells into self-renewing, leukaemia-initiating stem cells. Our data, which are further supported by consistent results in human cancer cell lines and primary samples of human haematological malignancies, reveal that senescence-associated stemness is an unexpected, cell-autonomous feature that exerts its detrimental, highly aggressive growth potential upon escape from cell-cycle blockade, and is enriched in relapse tumours. These findings have profound implications for cancer therapy, and provide new mechanistic insights into the plasticity of cancer cells.

Comprehensive Metaboproteomics of Burkitt's and Diffuse Large B-Cell Lymphoma Cell Lines and Primary Tumor Tissues Reveals Distinct Differences in Pyruvate Content and Metabolism.

Burkitt's lymphoma (BL) and diffuse large B-cell lymphoma (DLBCL) are pathologically and clinically distinct subtypes of aggressive non-Hodgkin B-cell lymphoma. To learn more about their biology, we employed metabolomic and proteomic methods to study both established cell lines as well as cryopreserved and formalin-fixed paraffin-embedded (FFPE) tissue sections of BL and DLBCL. Strikingly, NMR analyses revealed DLBCL cell lines to produce and secrete significantly (padj = 1.72 × 10-22) more pyruvic acid than BL cell lines. This finding could be reproduced by targeted GC/MS analyses of cryopreserved tissue sections of BL and DLBCL cases. Enrichment analysis of an overlapping set of N = 2315 proteins, that had been quantified by nanoLC-SWATH-MS in BL and DLBCL cultured cells and cryosections, supported the observed difference in pyruvic acid content, as glycolysis and pyruvate metabolism were downregulated, while one-carbon metabolism was upregulated in BL compared to DLBCL. Furthermore, 92.1% of the overlapping significant proteins showed the same direction of regulation in cryopreserved and FFPE material. Proteome data are available via ProteomeXchange with identifier PXD004936.

Inactivation of RUNX3/p46 Promotes Cutaneous T-Cell Lymphoma.

The key role of RUNX3 in physiological T-cell differentiation has been extensively documented. However, information on its relevance for the development of human T-cell lymphomas or leukemias is scarce. Here, we show that alterations of RUNX3 by either heterozygous deletion or methylation of its distal promoter can be observed in the tumor cells of 15 of 21 (71%) patients suffering from Sézary syndrome, an aggressive variant of cutaneous T-cell lymphoma. As a consequence, mRNA levels of RUNX3/p46, the isoform controlled by the distal promoter, are significantly lower in Sézary syndrome tumor cells. Re-expression of RUNX3/p46 reduces cell viability and promotes apoptosis in a RUNX3/p46low cell line of cutaneous T-cell lymphoma. Based on this, we present evidence that RUNX3 can act as a tumor suppressor in a human T-cell malignancy and suggest that this effect is predominantly mediated through transcripts from its distal promoter, in particular RUNX3/p46.

MINCR is a MYC-induced lncRNA able to modulate MYC's transcriptional network in Burkitt lymphoma cells.

Despite the established role of the transcription factor MYC in cancer, little is known about the impact of a new class of transcriptional regulators, the long noncoding RNAs (lncRNAs), on MYC ability to influence the cellular transcriptome. Here, we have intersected RNA-sequencing data from two MYC-inducible cell lines and a cohort of 91 B-cell lymphomas with or without genetic variants resulting in MYC overexpression. We identified 13 lncRNAs differentially expressed in IG-MYC-positive Burkitt lymphoma and regulated in the same direction by MYC in the model cell lines. Among them, we focused on a lncRNA that we named MYC-induced long noncoding RNA (MINCR), showing a strong correlation with MYC expression in MYC-positive lymphomas. To understand its cellular role, we performed RNAi and found that MINCR knockdown is associated with an impairment in cell cycle progression. Differential gene expression analysis after RNAi showed a significant enrichment of cell cycle genes among the genes down-regulated after MINCR knockdown. Interestingly, these genes are enriched in MYC binding sites in their promoters, suggesting that MINCR acts as a modulator of the MYC transcriptional program. Accordingly, MINCR knockdown was associated with a reduction in MYC binding to the promoters of selected cell cycle genes. Finally, we show that down-regulation of Aurora kinases A and B and chromatin licensing and DNA replication factor 1 may explain the reduction in cellular proliferation observed on MINCR knockdown. We, therefore, suggest that MINCR is a newly identified player in the MYC transcriptional network able to control the expression of cell cycle genes.

Classical Hodgkin's lymphoma shows epigenetic features of abortive plasma cell differentiation.

BACKGROUND: Epigenetic changes are involved in the extinction of the B-cell gene expression program of classical Hodgkin's lymphoma. However, little is known regarding epigenetic similarities between cells of classical Hodgkin's lymphoma and plasma cell myeloma, both of which share extinction of the gene expression program of mature B cells. DESIGN AND METHODS: Global histone H3 acetylation patterns were determined in cell lines derived from classical Hodgkin's lymphoma, plasma cell myeloma and B-cell lymphoma by chromatin immunoprecipitation and subsequent hybridization onto promoter tiling arrays. H3K27 trimethylation was analyzed by chromatin immunoprecipitation and real-time DNA polymerase chain reaction for selected genes. Epigenetic modifications were compared to gene expression data. RESULTS: Characteristic B-cell genes were hypoacetylated in classical Hodgkin's lymphoma and plasma cell myeloma cell lines as demonstrated by comparison of their histone H3 acetylation patterns to those of B-cell lines. However, the number of genes jointly hyperacetylated and expressed in classical Hodgkin' lymphoma and plasma cell myeloma cell lines, such as IRF4/MUM1 and RYBP, is limited. Moreover, H3K27 trimethylation for selected characteristic B-cell genes revealed that this additional epigenetic silencing is much more prevalent in classical Hodgkin's lymphoma than in plasma cell myeloma. CONCLUSIONS: Our epigenetic data support the view that classical Hodgkin's lymphoma is characterized by abortive plasma cell differentiation with a down-regulation of characteristic B-cell genes but without activation of most genes typical of plasma cells.

Potent inhibitory activity of chimeric oligonucleotides targeting two different sites of human telomerase.

Suppression of telomerase activity in tumor cells has been considered as a new anticancer strategy. Here, we present chimeric oligonucleotides (chimeric ODNs) as a new type of telomerase inhibitor that contains differently modified oligomers to address two different sites of telomerase: the RNA template and a suggested protein motif. We have shown previously that phosphorothioate-modified oligonucleotides (PS ODNs) interact in a length-dependent rather than in a sequence-dependent manner, presumably with the protein part of the primer-binding site of telomerase, causing strong inhibition of telomerase. In the present study, we demonstrate that extensions of these PS ODNs at their 3'-ends with an antisense oligomer partial sequence covering 11 bases of the RNA template cause significantly increased inhibitory activity, with IC(50) values between 0.60 and 0.95 nM in a Telomeric Repeat Amplification Protocol (TRAP) assay based on U-87 cell lysates. The enhanced inhibitory activity is observed regardless of whether the antisense part is modified (phosphodiester, PO; 2'-O-methylribosyl, 2'-OMe/PO; phosphoramidate, PAM). However, inside intact U-87 cells, these modifications of the antisense part proved to be essential for efficient telomerase inhibition 20 hours after transfection. In particular, the chimeric ODNs containing PAM or 2'-OMe/PO modifications, when complexed with lipofectin, were most efficient telomerase inhibitors (ID(50) = 0.04 and 0.06 microM, respectively). In conclusion, ODNs of this new type emerged as powerful inhibitors of human telomerase and are, therefore, promising candidates for further investigations of the anticancer strategy of telomerase inhibition.