Julie C. Porcher

Genome-wide analysis reveals recurrent structural abnormalities of TP63 and other p53-related genes in peripheral T-cell lymphomas

Peripheral T-cell lymphomas (PTCLs) are aggressive malignancies of mature T lymphocytes with 5-year overall survival rates of only ∼ 35%. Improvement in outcomes has been stymied by poor understanding of the genetics and molecular pathogenesis of PTCL, with a resulting paucity of molecular targets for therapy. We developed bioinformatic tools to identify chromosomal rearrangements using genome-wide, next-generation sequencing analysis of mate-pair DNA libraries and applied these tools to 16 PTCL patient tissue samples and 6 PTCL cell lines. Thirteen recurrent abnormalities were identified, of which 5 involved p53-related genes (TP53, TP63, CDKN2A, WWOX, and ANKRD11). Among these abnormalities were novel TP63 rearrangements encoding fusion proteins homologous to ΔNp63, a dominant-negative p63 isoform that inhibits the p53 pathway. TP63 rearrangements were seen in 11 (5.8%) of 190 PTCLs and were associated with inferior overall survival; they also were detected in 2 (1.2%) of 164 diffuse large B-cell lymphomas. As TP53 mutations are rare in PTCL compared with other malignancies, our findings suggest that a constellation of alternate genetic abnormalities may contribute to disruption of p53-associated tumor suppressor function in PTCL.

Clonally related follicular lymphomas and langerhans cell neoplasms: Expanding the spectrum of transdifferentiation

The traditional model of hematopoiesis is based on unidirectional maturation of hematopoietic precursors into lineage-committed cells. However, recent studies indicate that mature B lymphocytes may demonstrate significant lineage plasticity. We and others have reported transdifferentiation of follicular lymphomas (FLs) into clonally related histiocytic/dendritic cell neoplasms. Here, we describe 2 patients with FL who developed clonally related Langerhans cell neoplasms. The first was a 52-year-old man diagnosed with FL, grade 1. He received immunochemotherapy and had stable disease for 8 years. He then developed increasing lymphadenopathy, and lymph node biopsy showed Langerhans cell sarcoma with no evidence of FL. The second patient was a 77-year-old woman who presented with lymphadenopathy, an abdominal mass, and pulmonary nodules. Lymph node biopsy showed both Langerhans cell histiocytosis and minimal involvement by FL, grade 1. In each case, a combination of immunoglobulin gene rearrangement and fluorescence in situ hybridization studies provided evidence to support a clonal relationship between the FL and Langerhans cell neoplasm. These cases provide striking examples of neoplastic transdifferentiation and expand the spectrum of lesions clonally identical to otherwise typical FL. Awareness of this phenomenon may aid in diagnosis when histologically dissimilar tumors arise synchronously or metachronously in patients with lymphoma.

Rearrangements and amplification of IER3 (IEX-1) represent a novel and recurrent molecular abnormality in myelodysplastic syndromes

IER3 (formerly IEX-1) encodes a 27-kDa glycoprotein that regulates death receptor-induced apoptosis, interacts with NF-kappaB pathways, and increases expression rapidly in response to cellular stresses such as irradiation. Animal models, gene expression microarray experiments, and functional studies in cell lines have suggested a potential role for IER3 in oncogenesis, but, to date, no abnormalities of IER3 at the DNA level have been reported in patients with neoplasia. Here, we describe breakpoint cloning of a t(6;9)(p21;q34) translocation from a patient with a myelodysplastic syndrome (MDS), facilitated by conversion technology and array-based comparative genomic hybridization, which revealed a rearrangement translocating the IER3 coding region away from critical flanking/regulatory elements and to a transcript-poor chromosomal region, markedly decreasing expression. Using split-signal and locus-specific fluorescence in situ hybridization (FISH) probes, we analyzed 204 patients with diverse hematological malignancies accompanied by clonal chromosome 6p21 abnormalities, and found 8 additional patients with MDS with IER3 rearrangements (translocations or amplification). Although FISH studies on 157 additional samples from patients with MDS and a normal-karyotype were unrevealing, and sequencing the IER3 coding and proximal promoter regions of 74 MDS patients disclosed no point mutations, reverse transcription-PCR results suggested that dysregulated expression of IER3 is common in MDS (61% >4-fold increase or decrease in expression with decreased expression primarily in early MDS and increased expression primarily in later MDS progressing toward leukemia), consistent with findings in previous microarray experiments. These data support involvement of IER3 in the pathobiology of MDS.

Novel TRAF1-ALK fusion identified by deep RNA sequencing of anaplastic large cell lymphoma.

Chromosomal translocations leading to expression of abnormal fusion proteins play a major role in the pathogenesis of various hematologic malignancies. The recent development of high‐throughput, “deep” sequencing has allowed discovery of novel translocations leading to a rapid increase in understanding these diseases. Translocations involving the anaplastic lymphoma kinase (ALK) gene leading to ALK fusion proteins originally were discovered in anaplastic large cell lymphomas (ALCLs). Among ALCLs, NPM1‐ALK fusions are most common and lead to nuclear localization of the fusion protein. Here, we present a 50‐year‐old male with ALCL demonstrating cytoplasmic ALK immunoreactivity only, suggesting the presence of a non‐NPM1 fusion partner. We performed deep RNA sequencing of tumor tissue from this patient and identified a novel transcript fusing Exon 6 of TRAF1 to Exon 20 of ALK. The TRAF1‐ALK fusion transcript was confirmed at the mRNA level by Sanger sequencing and the fusion protein was visualized by Western blot. The discovery of this TRAF1‐ALK fusion expands the diversity of known ALK fusion partners and highlights the power of deep sequencing for fusion transcript discovery.

The oncogenic transcription factor IRF4 is regulated by a novel CD30/NF-κB positive feedback loop in peripheral T-cell lymphoma

Peripheral T-cell lymphomas (PTCLs) are generally aggressive non-Hodgkin lymphomas with poor overall survival rates following standard therapy. One-third of PTCLs express interferon regulatory factor-4 (IRF4), a tightly regulated transcription factor involved in lymphocyte growth and differentiation. IRF4 drives tumor growth in several lymphoid malignancies and has been proposed as a candidate therapeutic target. Because direct IRF4 inhibitors are not clinically available, we sought to characterize the mechanism by which IRF4 expression is regulated in PTCLs. We demonstrated that IRF4 is constitutively expressed in PTCL cells and drives Myc expression and proliferation. Using an inhibitor screen, we identified nuclear factor κB (NF-κB) as a candidate regulator of IRF4 expression and cell proliferation. We then demonstrated that the NF-κB subunits p52 and RelB were transcriptional activators of IRF4. Further analysis showed that activation of CD30 promotes p52 and RelB activity and subsequent IRF4 expression. Finally, we showed that IRF4 transcriptionally regulates CD30 expression. Taken together, these data demonstrate a novel positive feedback loop involving CD30, NF-κB, and IRF4; further evidence for this mechanism was demonstrated in human PTCL tissue samples. Accordingly, NF-κB inhibitors may represent a clinical means to disrupt this feedback loop in IRF4-positive PTCLs.

Aberrant pre-mRNA splicing of a highly conserved cell cycle regulator, CDC25C, in myelodysplastic syndromes

Alternative pre-mRNA splicing alters gene expression and protein function, and aberrant splicing patterns can be associated with neoplasia. The potential role of disordered RNA splicing in myelodysplastic syndrome (MDS) is unexplored. We analysed the splicing repertoire of CDC25C– a gene localised to chromosome 5q31 and encoding a cyclin/cyclin-dependent-kinase regulatory phosphatase critical for cell cycle checkpoint control – in MDS, acute myeloid leukemia, chronic lymphocytic leukemia and healthy tissues. Five novel splicing isoforms were detected, and the splicing patterns were generally distinct in neoplastic samples compared with healthy controls. One of the novel isoforms, which we have termed CDC25C-6, occurred in 58% of the samples in our cohort. The results of this study suggest the possibility of aberrant splicing contributing to the phenotype in MDS and other haematologic malignancies.

Congenital sideroblastic anemia associated with germline polymorphisms reducing expression of FECH

The sideroblastic anemias (SAs) are disorders of ineffective erythropoiesis, collectively characterized by abnormal Prussian blue-positive granules (i.e., iron-stuffed mitochondria) that encircle marrow erythroblast nuclei to form ringed sideroblast cells.[1][1] SAs are usually acquired, but

Prevalence of erythrocyte haemoglobin H inclusions in unselected patients with clonal myeloid disorders

Patients with clonal myeloid disorders, especially myelodysplastic syndromes (MDS), may acquire α‐thalassaemia. To estimate the prevalence of this erythrocyte phenotype, we examined brilliant cresyl blue‐stained blood smears from 201 patients with neoplastic myeloid disorders and 282 controls (195 non‐clonal anaemia, 62 with medical illnesses without anaemia and 25 healthy persons). Haemoglobin H inclusions were detected in 8/100 patients with MDS (8%) and 2/81 (2.5%) patients with myeloproliferative disorders, but in none of the acute leukaemia patients or controls. We conclude that the emergence of thalassaemic clones may be relatively common in the disordered marrow milieu of MDS.

Expression of the chemokine receptor gene, CCR8, is associated With DUSP22 rearrangements in anaplastic large cell lymphoma.

Anaplastic large cell lymphoma (ALCL) is one of the most common T-cell non-Hodgkin lymphomas and has 2 main subtypes: an anaplastic lymphoma kinase (ALK)-positive subtype characterized by ALK gene rearrangements and an ALK-negative subtype that is poorly understood. We recently identified recurrent rearrangements of the DUSP22 locus on 6p25.3 in both primary cutaneous and systemic ALK-negative ALCLs. This study aimed to determine the relationship between these rearrangements and expression of the chemokine receptor gene, CCR8. CCR8 has skin-homing properties and has been suggested to play a role in limiting extracutaneous spread of primary cutaneous ALCLs. However, overexpression of CCR8 has also been reported in systemic ALK-negative ALCLs. As available antibodies for CCR8 have shown lack of specificity, we examined CCR8 expression using quantitative real-time PCR in frozen tissue and RNA in situ hybridization (ISH) in paraffin tissue. Both approaches showed higher CCR8 expression in ALCLs with DUSP22 rearrangements than in nonrearranged cases (PCR: 19.5-fold increase, P=0.01; ISH: 3.3-fold increase, P=0.0008). CCR8 expression was not associated with cutaneous presentation, cutaneous biopsy site, or cutaneous involvement during the disease course. These findings suggest that CCR8 expression in ALCL is more closely related to the presence of DUSP22 rearrangements than to cutaneous involvement and that the function of CCR8 may extend beyond its skin-homing properties in this disease. This study also underscores the utility of RNA-ISH as a paraffin-based method for investigating gene expression when reliable antibodies for immunohistochemical analysis are not available.

Assessment of ATRX expression in patients with myelodysplastic syndromes treated with decitabine

Treatment with one of the DNA methyltransferase inhibitors (DNMTIs, “hypomethylating agents”), azacitidine or decitabine, is now considered standard care for patients with higherrisk myelodysplastic syndromes (MDS) (1) – especially since results were released from the AZA-001 trial (2), which demonstrated a 9-month survival advantage with azacitidine therapy compared to supportive care. However, many patients with MDS do not respond to treatment with DNMTIs as they are currently used, and adverse events are common. It is desirable to be able to predict a priori which patients are most likely to benefit from DNMTI therapy, in order to spare patients unlikely to respond from the risks and cost of treatment, but there are currently no well-established molecular methods for DNMTI response prediction.(3) ATRX is an X-encoded chromatin-associated protein with an evolutionarily conserved Nterminal DNA methyltransferase (DNMT3-like) domain.(4) Germline mutations in ATRX cause a mental retardation-dysmorphology syndrome often associated with mild alpha thalassemia, which gave the gene its name (ATR-X syndrome: alpha thalassemia, retardation, X-linked) (5); in contrast, somatic point mutations in ATRX have been linked to acquired alpha thalassemia arising in the context of MDS, which may be present in as many as 8% of patients. (6-8) Germline mutations in ATRX are associated with widespread and diverse DNA methylation abnormalities across the genome.(9) Recent evidence suggests that the ATRX protein also has an important role in chromosome dynamics during mitosis, as ATRX-depleted cells exhibit defective sister chromatid cohesion and congression at the metaphase plate, as well as abnormal chromosome alignment. (10,11) Circumstantial evidence for a role of ATRX in neoplasia is increasing. In an array-based gene expression profiling study of 42 children with acute myeloid leukemia (AML) associated with somatic FLT3 mutations, the expression ratio of the transcription factor RUNX3 (formerly AML2) to ATRX predicted event-free survival (EFS).(12) In a subsequent study of 132 adults with de novo AML by another investigative group, low ATRX expression correlated with highrisk karyotype and poor clinical outcome.(13) Altered ATRX expression levels have also been reported in gene expression profiling experiments using prostate cancer primary cells (14), esophageal squamous cell carcinoma cell lines (15), chronic lymphocytic leukemia primary cells (Neil Kay, personal communication October 2007), and irradiated breast cancer cell lines (16).