Francesco Acquadro

DNA Methylation Profiles and Their Relationship with Cytogenetic Status in Adult Acute Myeloid Leukemia

Background Aberrant promoter DNA methylation has been shown to play a role in acute myeloid leukemia (AML) pathophysiology. However, further studies to discuss the prognostic value and the relationship of the epigenetic signatures with defined genomic rearrangements in acute myeloid leukemia are required. Methodology/Principal Findings We carried out high-throughput methylation profiling on 116 de novo AML cases and we validated the significant biomarkers in an independent cohort of 244 AML cases. Methylation signatures were associated with the presence of a specific cytogenetic status. In normal karyotype cases, aberrant methylation of the promoter of DBC1 was validated as a predictor of the disease-free and overall survival. Furthermore, DBC1 expression was significantly silenced in the aberrantly methylated samples. Patients with chromosome rearrangements showed distinct methylation signatures. To establish the role of fusion proteins in the epigenetic profiles, 20 additional samples of human hematopoietic stem/progenitor cells (HSPC) transduced with common fusion genes were studied and compared with patient samples carrying the same rearrangements. The presence of MLL rearrangements in HSPC induced the methylation profile observed in the MLL-positive primary samples. In contrast, fusion genes such as AML1/ETO or CBFB/MYH11 failed to reproduce the epigenetic signature observed in the patients. Conclusions/Significance Our study provides a comprehensive epigenetic profiling of AML, identifies new clinical markers for cases with a normal karyotype, and reveals relevant biological information related to the role of fusion proteins on the methylation signature.

Keratinocytic epidermal nevi are associated with mosaic RAS mutations

Background Activating RAS mutations in the germline cause rare developmental disorders such as Costello syndrome. Somatic RAS mutations are found in approximately 30% of human cancers. Keratinocytic epidermal nevi (KEN) represent benign congenital skin lesions arranged along Blaschkos lines. A subgroup of KEN is caused by hotspot oncogenic FGFR3 and PIK3CA mutations in mosaicism, but the majority lack these mutations. Methods This study screened 72 KEN for activating mutations in RAS genes and other oncogenes. Results Activating RAS mutations were identified in 28/72 (39%) of KEN. HRAS was the most commonly affected oncogene (86%), with the HRAS p.G13R substitution representing a new hotspot mutation. Conclusion These results indicate that activating RAS somatic mutations leading to mosaicism result in benign KEN of the skin. Given the prevalence of KEN, mosaic HRAS mutations appear to be more common in patients than germline ones. These findings identify KEN as a mosaic RASopathy and lend further support to the notion that genetic mosaicism is an important contributor to disease.

Multiple oncogenic mutations and clonal relationship in spatially distinct benign human epidermal tumors

Malignant tumors result from the accumulation of genetic alterations in oncogenes and tumor suppressor genes. Much less is known about the genetic changes in benign tumors. Seborrheic keratoses (SK) are very frequent benign human epidermal tumors without malignant potential. We performed a comprehensive mutational screen of genes in the FGFR3-RAS-MAPK and phosphoinositide 3-kinase (PI3K)-AKT pathways from 175 SK, including multiple lesions from each patient. SK commonly harbored multiple bona fide oncogenic mutations in FGFR3, PIK3CA, KRAS, HRAS, EGFR, and AKT1 oncogenes but not in tumor suppressor genes TSC1 and PTEN. Despite the occurrence of oncogenic mutations and the evidence for downstream ERK/MAPK and PI3K pathway signaling, we did not find induction of senescence or a DNA damage response. Array comparative genomic hybridization (aCGH) analysis revealed that SK are genetically stable. The pattern of oncogenic mutations and X chromosome inactivation departs significantly from randomness and indicates that spatially independent lesions from a given patient share a clonal relationship. Our findings show that multiple oncogenic mutations in the major signaling pathways involved in cancer are not sufficient to drive malignant tumor progression. Furthermore, our data provide clues on the origin and spread of oncogenic mutations in tissues, suggesting that apparently independent (multicentric) adult benign tumors may have a clonal origin.

ASXL1 , TP53 and IKZF3 mutations are present in the chronic phase and blast crisis of chronic myeloid leukemia

ASXL1 , TP53 and IKZF3 mutations are present in the chronic phase and blast crisis of chronic myeloid leukemia

CSF3R T618I co-occurs with mutations of splicing and epigenetic genes and with a new PIM3 truncated fusion gene in chronic neutrophilic leukemia

Mutations in CSF3R have been recently defined as the common genetic event in patients with myeloid neoplasms, including the rare entity known as chronic neutrophilic leukemia (CNL),1, 2, 3 becoming a potentially useful biomarker for diagnosing and therapy target.4 CSF3R encodes the transmembrane receptor for granulocyte colony-stimulating factor (G-CSF; CSF3), which provides the proliferative and survival signal for granulocytes and also contributes to their differentiation and function.5 Although there are several studies on massive next-generation sequencing of myeloid disorders, not a single comprehensive study has been reported in CNL. Here, we used whole-exome sequencing (WES) and RNA sequencing (RNA-seq) to identify new candidate genes to the disease pathogenesis of an index CNL patient. A 66-year-old man was diagnosed with CNL, according to the 2008 World Health Organization (WHO) classification. At diagnosis, the patient presented peripheral blood leukocytosis (66 × 109/l), segmented neutrophils and band forms were 91.5% of the white blood cells counts (WBCs), immature granulocytes were <10% of WBCs and myeloblasts were <1%. The aspirate showed a hypercellular bone marrow (BM) with neutrophilic granulocytes increased in number and percentage and myeloblasts 0.5% of WBCs. No dysplastic features were observed in the myeloid lineages. His Zubrod Performance Status (ECOG) was 1. A GTG-banding chromosome analysis revealed a normal karyotype (46,XY[20]), and molecular biology studies were negative for BCR-ABL1 transcripts and JAK2 V617F mutation. The patient was treated with hydroxyurea but, unfortunately, died 7 months after the diagnosis due to an intensification of the disease. To improve our understanding of the genes involved in the pathogenesis of CNL, WES was performed on matched tumor and oral mucosa cell (germline) samples from the patient. Candidate somatic mutations were identified using RUbioSeq software.6 The bioinformatics analysis and the filtering steps to identify the coding variants are detailed in the Supplementary Material. In total, we found 1437 candidate variants; among them, 797 were somatic mutations (412 were intronic, intergenic, affecting non-conding-RNA or untranscribed regions and 385 were exonic). From the 385 exonic variants, we selected only those variants within coding regions that, after passing sequencing depth and quality filters, were, frameshift, stop gain/loss and non-synonymous amino acid changes predicted to produce a deleterious effect in the protein structure, resulting in 56 single-nucleototide variants (SNVs) and small insertions/deletions (indels). We selected 24 for further validation by Haloplex/Ion Torrent. In addition to the CSF3R p.Thr618Ile mutation, we validated mutations in U2AF1, TET2, LUC7L2 and ASXL1 (Figure 1a and Table 1). Figure 1 Molecular characterization of CNL. (a) In addition to the CSF3R T618I mutation, WES revealed mutations in U2AF1, TET2, LUC7L2 and ASXL1. LUC7L2 mutation was found in homozygosis; (b) LOH of 53.2 Mb in chromosome 7q including the locus of LUC7L2 ... Table 1 Description of validated mutations in the CNL patient The current study, first, confirms the observations by Maxon et al.1 and Pardanani et al.2 regarding the association between CNL and CSF3R mutations. Second, it presents a complete picture of the mutational profiling of CNL, certainly more complex than expected from these previous reports. In fact, we found and validated mutations affecting both splicing machinery and epigenetic genes. Kosmider et al.,3 very recently, showed that CSF3R somatic mutations can be identified in ∼4% of chronic myelomonocytic leukemias. These mutations, which affect distinct residues in CSF3R as compared with CNL, are frequently associated with mutations in the ASXL1 gene and have a poor prognostic impact on overall and acute myeloid leukemia (AML)-free survival. Together, these data indicate that CNL genome had a combination of few mutations with a pattern of cooperation with a strong biological relationship among genes and categories, similar to AML.7 Along this line of cooperating mutations on epigenetic genes, we also found in our CNL patient mutated copies of ASXL1 and TET2 genes. The variant allelic frequency of LUC7L2 mutation was found to be high (more than 95%). We previously described mutations of this gene in myelodysplastic syndrome (MDS).8 As this gene is located in the 7q region, a frequently deleted chromosomal region in myeloid leukemias,9 we decided to investigate whether a critical deletion or a loss of heterozygosity (LOH) affecting this genomic region was also present in the patient. To study this phenomenon, we interrogate our WES data for the LOH across the whole genome of the sample. Interestingly, we found an LOH of 53.2 Mb in chromosome 7q including the locus of the LUC7L2 gene. Allele frequencies of each SNP along chromosome 7 are shown in Figure 1b and Supplementary Figure 4. As no del(7q) was detected with metaphase cytogenetics, our study demonstrates for the first time mutations in LUC7L2 accompanied by a copy-neutral LOH (uniparental disomy) in 7q in a patient with an aggressive CNL phenotype. To further evaluate the biological consequences of this homozygous mutation, we explored LUC7L2 expression in the BM cells of the patient and in some myeloid leukemia cell lines (Figure 1c). By using real-time PCR, we observed a downregulation in the expression of LUC7L2 in the patient cells compared with normal granulocytes, as well as a general downregulation in myeloid leukemia cell lines. To determine the functional consequences of the mutations in LUC7L2 and U2AF1, genes involved in the splicing machinery, in the proper splicing process, we performed RNA-seq in our index patient as well as in CD34+ cells from a normal control BM. Although no clear genome-wide increase in intron retention was observed in the patient, as previously reported by us10 in some MDS cases, we found an altered pattern of splicing in the mRNA species transcribed from the RUNX1 gene. At the 3′ splice site of RUNX1 intron 5, the un-spliced reads were almost three times more frequent in the mutated patient than in the normal control (Supplementary Figure 1). Because of the large numbers of diverse mutations in the splicing machinery, larger studies will be needed to fully evaluate the impact of these mutations in splicing. In relation with the effects of an aberrant RNA splicing due to the presence of two mutations in the genes responsible for these processes, we used RNA-seq data to investigate the presence of aberrant fusion transcripts in our CNL patient. In fact, we identified a chimeric transcript involving the PIM3 and SCO2 genes (both were located on 22q13.33), which was the result of an intrachromosomal inversion of approximately 0.6 Mb in chromosome 22 (Figure 1d). The PIM3 oncogene belongs to the Ser/Thr protein kinase family and PIM subfamily. This gene is overexpressed in hematological and epithelial tumors and is associated with MYC co-expression. It has a role in the regulation of signal–transduction cascades, contributing to both cell proliferation and survival, and provides a selective advantage in tumorigenesis.11 Interestingly, the inhibition of PIM kinases by pim kinase inhibitors in Myc-induced lymphoma resulted in cell death.12 Functional studies are needed to elucidate the role of this fusion gene in leukemogenesis. Regarding the global expression profile of CNL, we obtained 2022 genes upregulated and 1884 genes downregulated in CNL compared with the control (FDR=0.05). Functional classification of genes differently expressed between CNL cells and CD34+, according to the Ingenuity Systems Analysis (Ingenuity Systems, www.ingenuity.com), revealed an enrichment of categories like cell signaling, cell death and survival, as well as gene expression (B-H P-value <0.05; Supplementary Figure 2). These findings reconfirmed the significant pathophysiology of such several synergetic unique genetic defects in the general CNL cohort, as well as in our index case. The oncogenic CSF3R mutations T618I strongly activate the JAK/STAT pathway and are sensitive to inhibitors of SRC family TNK2 and JAK kinases and may provide a new avenue for therapy.4 In contrast, pim kinase inhibition could be a viable treatment strategy in certain human leukemias that rely on the PIM3 kinase expression.12 In addition, epigenetic modifiers provide new targets for therapeutic intervention, and targeting these enzymatic activities is currently being explored from a therapeutic standpoint in several types of leukemia.13, 14 Although Pardanani et al.,2 considering 35 cases of clinically suspected CNL, did not found alteration in the survival on the basis of the presence or absence of CSF3R mutations, our reported patient had a rapid disease progression and died 7 months after diagnosis, which was probably explained by the profoundly aberrant landscape of gene mutations and rearrangements with functional effects on the biology of the tumor cells. In summary, our study provides, for the first time, a massive molecular and expression data, revealing a large amount of genomic alterations in CNL. In this complex scenario, a combination of new target therapies may be considered as reasonable options for the therapeutic management of this aggressive and rare subtype of leukemia.

A child with mild X-linked intellectual disability and a microduplication at Xp22.12 including RPS6KA3.

Multiplex ligation-dependent probe amplification (MLPA) and array- comparative genomic hybridization analysis have been proven to be useful in the identification of submicroscopic copy-number imbalances in families with nonsyndromic X-linked intellectual disability (NS-XLID). Here we report the first description of a child with mild intellectual disability and a submicroscopic duplication at Xp22.12 identified by MLPA with a P106 MRX kit (MRC-Holland, Amsterdam, Netherlands) and further confirmed and characterized with a custom 244-k oligo-array, fluorescence in situ hybridization, quantitative polymerase chain reaction (qPCR), and immunoblotting. This 1.05-megabase duplication encompasses 7 genes, RPS6KA3 being the only of these genes known to be related to ID. The proband was an 8-year-old boy referred to the genetics unit for psychomotor retardation and learning disabilities. Both maternal brothers also showed learning difficulties and delayed language during childhood in a similar way to the proband. These boys also carried the duplication, as did the healthy mother and grandmother of the proband. The same duplication was also observed in the 5-year-old younger brother who presented with features of developmental delay and learning disabilities during the previous year. Increased RPS6KA3/RSK2 levels were demonstrated in the proband by qPCR and immunoblotting. To our knowledge, this is the first family identified with a submicroscopic duplication including the entire RPS6KA3/RSK2 gene, and our findings suggest that an increased dose of this gene is responsible for a mild form of NS-XLID.

FIP1L1/RARA with breakpoint at FIP1L1 intron 13: a variant translocation in acute promyelocytic leukemia

Acute promyelocytic leukemia (APL) is a distinct subtype of acute myeloid leukemia that is characterized by three distinct features: i) accumulation in the bone marrow (BM) of tumor cells with promyelocytic phenotype; ii) association with specific translocations which involve chromosome 17 at the

DEK oncogene is overexpressed during melanoma progression.

DEK is an oncoprotein involved in a variety of cellular functions, such as DNA repair, replication, and transcriptional control. DEK is preferentially expressed in actively proliferating and malignant cells, including melanoma cell lines in which DEK was previously demonstrated to play a critical role in proliferation and chemoresistance. Still, the impact of this protein in melanoma progression remains unclear. Thus, we performed a comprehensive analysis of DEK expression in different melanocytic tumors. The immunostaining results of 303 tumors demonstrated negligible DEK expression in benign lesions. Conversely, malignant lesions, particularly in metastatic cases, were largely positive for DEK expression, which was partially associated with genomic amplification. Importantly, DEK overexpression was correlated with histological features of aggressiveness in primary tumors and poor prognosis in melanoma patients. In conclusion, our study provides new insight into the involvement of DEK in melanoma progression, as well as proof of concept for its potential application as a marker and therapeutic target of melanoma.

Abrogation of RUNX1 gene expression in de novo myelodysplastic syndrome with t(4;21)(q21;q22)

The disruption of RUNX1 function is one of the main mechanisms of disease observed in hematopoietic malignancies and the description of novel genetic events that lead to a RUNX1 loss of function has been accelerated with the development of genomic technologies. Here we describe the molecular characterization of a new t(4;21)(q21;q22) in a de novo myelodysplastic syndrome that resulted in the deletion of the RUNX1 gene. We demonstrated by quantitative real-time RT-PCR an almost complete depletion of the expression of the RUNX1 gene in our t(4;21) case compared with CD34+ cells that was independent of mutation or DNA methylation. More importantly, we explored and confirmed the possibility that this abrogation also prevented transactivation of RUNX1 target genes, perhaps confirming the genetic origin of the thrombocytopenia and the myelodysplastic features observed in our patient, and certainly mimicking what has been observed in the presence of the RUNX1/ETO fusion protein.

Braided pattern in a dermatofibrosarcoma protuberans: a potential mimicker of neural neoplasms.

Dermatofibrosarcoma protuberans (DFSP) is a dermal and subcutaneous slow-growing tumor of intermediate malignancy. Different histological variants of DFSP have been described, depending on cellular and stromal peculiarities. Here, we report the histological features of a DFSP in which cells were frequently arrayed in cords and fascicles that were interweaved, conforming a peculiar braided pattern. This finding might pose difficulties in the differential diagnosis with neural neoplasms and expands the morphological spectrum of DFSP.