Petra Korać

Repurposing the CRISPR-Cas9 system for targeted DNA methylation

Epigenetic studies relied so far on correlations between epigenetic marks and gene expression pattern. Technologies developed for epigenome editing now enable direct study of functional relevance of precise epigenetic modifications and gene regulation. The reversible nature of epigenetic modifications, including DNA methylation, has been already exploited in cancer therapy for remodeling the aberrant epigenetic landscape. However, this was achieved non-selectively using epigenetic inhibitors. Epigenetic editing at specific loci represents a novel approach that might selectively and heritably alter gene expression. Here, we developed a CRISPR-Cas9-based tool for specific DNA methylation consisting of deactivated Cas9 (dCas9) nuclease and catalytic domain of the DNA methyltransferase DNMT3A targeted by co–expression of a guide RNA to any 20 bp DNA sequence followed by the NGG trinucleotide. We demonstrated targeted CpG methylation in a ∼35 bp wide region by the fusion protein. We also showed that multiple guide RNAs could target the dCas9-DNMT3A construct to multiple adjacent sites, which enabled methylation of a larger part of the promoter. DNA methylation activity was specific for the targeted region and heritable across mitotic divisions. Finally, we demonstrated that directed DNA methylation of a wider promoter region of the target loci IL6ST and BACH2 decreased their expression.

Application of the FICTION technique for the simultaneous detection of immunophenotype and chromosomal abnormalities in routinely fixed, paraffin wax embedded bone marrow trephines

The use of interphase fluorescence in situ hybridisation (FISH) to study cytogenetic abnormalities in routinely fixed paraffin wax embedded tissue has become commonplace over the past decade. However, very few studies have applied FISH to routinely fixed bone marrow trephines (BMTs). This may be because of the acid based decalcification methods that are commonly used during the processing of BMTs, which may adversely affect the suitability of the sample for FISH analysis. For the first time, this report describes the simultaneous application of FISH and immunofluorescent staining (the FICTION technique) to formalin fixed, EDTA decalcified and paraffin wax embedded BMTs. This technique allows the direct correlation of genetic abnormalities to immunophenotype, and therefore will be particularly useful for the identification of genetic abnormalities in specific tumour cells present in BMTs. The application of this to routine clinical practice will assist diagnosis and the detection of minimal residual disease.

Immunohistochemical analysis of NOTCH1 and JAGGED1 expression in multiple myeloma and monoclonal gammopathy of undetermined significance.

Notch signaling is implicated in the pathogenesis of multiple myeloma expressing high level of active Notch proteins NOTCH1 and JAGGED1 in tumor plasma cells. We investigated expression of NOTCH1 and JAGGED1 in bone marrow trephine biopsies of 80 newly diagnosed multiple myeloma and 20 monoclonal gammopathy of undetermined significance patients using immunohistochemical methods. The number of positive tumor cells was counted per 1000 tumor cells and the intensity of staining was assessed semi quantitatively. Multiple myelomas expressed NOTCH1 in 92.31% (72/78) and JAGGED1 in 92.21% (71/77) cases. NOTCH1 staining was strong in the majority of cases (59.7%), whereas JAGGED1 was predominately weak (67.6% of cases). In contrast, both markers were negative in all monoclonal gammopathy of undetermined significance cases. However, upon progression of disease from monoclonal gammopathy of undetermined significance to multiple myeloma (seen in 4 patients), analysis of the subsequent bone marrow biopsy showed weak expression of both markers in tumorous plasma cells. Immunohistochemistry results were compared with the pattern of bone marrow infiltration, plasma cell differentiation, and the presence of t(11;14)(q13,q32), t(14;16)(q32;q23),and t(4;14)(p16.3;q23) and overall survival in multiple myeloma patients. A significant correlation was found between strong NOTCH1 staining in multiple myeloma plasma cells and the diffuse type of bone marrow infiltration (P = .002) and an immature morphologic type of plasma cells (P = .043). After a median follow-up of 20.3 months, in multiple myeloma patients no difference in overall survival between NOTCH1 (P = .484) and JAGGED1 (P = .822) positive and negative cases were found. In conclusion, our results indicate importance of NOTCH1 and JAGGED1 expression in plasma cell neoplasia and a possible diagnostic value of their immunohistochemical evaluation of bone marrow infiltrates for multiple myeloma.

Bcl-2 and MALT1 genes are not involved in the oncogenesis of uterine tumors resembling ovarian sex cord tumors

Uterine tumors resembling ovarian sex cord tumors (UTROSCT) are rare entities. They were described by Clement and Scully in 1976 who classified them into groups I and II. Group I comprises typical endometrial stromal neoplasms with focal areas resembling ovarian sex cord elements and group II are predominantly or completely composed of ovarian sex cord-like elements. We report a case of UTROSCT type II with cytogenetic analysis. The tumor occurred in a 76-year-old woman who presented with vaginal bleeding. The tumor was lobulated, firm, yellow and histologically composed of sex cord-like elements. Tumor cells expressed vimentin, CD10, CD99 and alpha-actin. Cytogenetic analysis in a previously reported case detected translocation t(4;18)(q21.1;q21.3) in the majority of cells. Bcl-2 and MALT1 genes are located at or near the translocation breakpoints, and the aim of this study was to determine whether these genes were involved in chromosomal translocation or tumorigenesis. We did not find IgH translocation or the most common MALT translocations. Bcl-2 was also not involved in this oncogenesis.

Unlocking cancer glycomes from histopathological formalin-fixed and paraffin-embedded (FFPE) tissue microdissections

N- and O-glycans are attractive clinical biomarkers as glycosylation changes in response to diseases. The limited availability of defined clinical specimens impedes glyco-biomarker identification and validation in large patient cohorts. Formalin-fixed paraffin-embedded (FFPE) clinical specimens are the common form of sample preservation in clinical pathology, but qualitative and quantitative N- and O-glycomics of such samples has not been feasible to date. Here, we report a highly sensitive and glycan isomer selective method for simultaneous N- and O-glycomics from histopathological slides. As few as 2000 cells isolated from FFPE tissue sections by laser capture microdissection were sufficient for in-depth histopathology-glycomics using porous graphitized carbon nanoLC ESI-MS/MS. N- and O-glycan profiles were similar between unstained and hematoxylin and eosin stained FFPE samples but differed slightly compared with fresh tissue. This method provides the key to unlock glyco-biomarker information from FFPE histopathological tissues archived in pathology laboratories worldwide.

DNA hypomethylation upregulates expression of the MGAT3 gene in HepG2 cells and leads to changes in N-glycosylation of secreted glycoproteins

Changes in N-glycosylation of plasma proteins are observed in many types of cancer, nevertheless, few studies suggest the exact mechanism involved in aberrant protein glycosylation. Here we studied the impact of DNA methylation on the N-glycome in the secretome of the HepG2 cell line derived from hepatocellular carcinoma (HCC). Since the majority of plasma glycoproteins originate from the liver, the HepG2 cells represent a good model for glycosylation changes in HCC that are detectable in blood, which is an easily accessible analytic material in a clinical setting. Two different concentrations of 5-aza-2′-deoxycytidine (5-aza-2dC) differentially affected global genome methylation and induced different glycan changes. Around twenty percent of 84 glyco-genes analysed changed expression level after the 5-aza-2dC treatment as a result of global genome hypomethylation. A correlation study between the changes in glyco-gene expression and the HepG2 glycosylation profile suggests that the MGAT3 gene might be responsible for the glycan changes consistently induced by both doses of 5-aza-2dC. Core-fucosylated tetra-antennary structures were decreased in quantity likely as a result of hypomethylated MGAT3 gene promoter followed by increased expression of this gene.

MALT1, BCL10 and FOXP1 in salivary gland mucosa-associated lymphoid tissue lymphomas.

In view of the certain anatomic site‐dependent frequency of chromosomal translocations involved in extranodal marginal zone B cell lymphoma of mucosa‐associated lymphoid tissue (MALT lymphoma) pathogenesis, 17 salivary gland MALT lymphoma cases were analyzed for MALT1 and FOXP1 translocations. B cell CLL/lymphoma 10 (BCL10) and forkhead box PA (FOXP1) protein expression were studied by immunohistochemistry and translocations identified using fluorescence in situ hybridization (FISH)‐specific probes FOXP1, t(11;18)(q21;q21)/API2‐MALT1 and t(14;18)(q32;q21)/IgH‐MALT1. None of the 11 analyzed cases showed FOXP1 rearrangement or amplification. The t(11;18) was present in five of 13 cases and the t(14;18) in three of 13 cases. MALT1 translocations were mostly mutually exclusive except in a single case. FOXP1 protein expression showed differences in the proportion of tumor cells with nuclear expression but not in their intensity, with the exception of one case where very intense nuclear staining was noted. BCL10 nuclear expression was present in four of 17 cases, two of which lacked t(11;18). Our results suggest that MALT1‐specific translocations and FOXP1 rearrangements are not commonly involved in pathogenesis. A case with strong FOXP1 protein expression indicates the possibility that the upregulation of FOXP1 expression is significant in a small subset of salivary gland MALT lymphomas. Also a single case in which both MALT1 translocations were present indicates that these are not always mutually exclusive.

N- and O-glycomics from minor amounts of formalin-fixed, paraffin-embedded tissue samples

The availability of well-defined samples in sufficient numbers represents a major bottleneck for any biomarker related research. The utilization of preserved, archived and clinically well-described samples therefore holds a great potential to bridge this gap. This chapter describes a universal workflow for the comprehensive characterization of N- and O-glycans released from whole formalin-fixed, paraffin-embedded tissue sections, including an option for further partitioning using laser microdissection of specific tissue areas/cell populations. Glycoproteins are extracted and subsequently immobilized onto a PVDF membrane prior enzymatic release of N-glycans. Following N-glycan retrieval O-glycans are released using reductive β-elimination from the same sample spot, significantly reducing the required amount of starting material. Released and reduced glycan structures are characterized using porous graphitized carbon liquid chromatography online coupled to an electrospray ionization-ion trap mass spectrometer. This technique provides information on the relative abundances of individual glycans along with detailed structural information, including isomer differentiation and functional epitope characterization of N- and O-glycans obtained from minimal amounts of tissue down to a few thousand cells.

Role of MYC in B Cell Lymphomagenesis

B cell lymphomas mainly arise from different developmental stages of B cells in germinal centers of secondary lymphoid tissue. There are a number of signaling pathways that affect the initiation and development of B cell lymphomagenesis. The functions of several key proteins that represent branching points of signaling networks are changed because of their aberrant expression, degradation, and/or accumulation, and those events determine the fate of the affected B cells. One of the most influential transcription factors, commonly associated with unfavorable prognosis for patients with B cell lymphoma, is nuclear phosphoprotein MYC. During B cell lymphomagenesis, oncogenic MYC variant is deregulated through various mechanisms, such as gene translocation, gene amplification, and epigenetic deregulation of its expression. Owing to alterations of downstream signaling cascades, MYC-overexpressing neoplastic B cells proliferate rapidly, avoid apoptosis, and become unresponsive to most conventional treatments. This review will summarize the roles of MYC in B cell development and oncogenesis, as well as its significance for current B cell lymphoma classification. We compared communication networks within transformed B cells in different lymphomas affected by overexpressed MYC and conducted a meta-analysis concerning the association of MYC with tumor prognosis in different patient populations.

Follicular and mantle cell lymphoma characteristics present simultaneously in the same lymph node.

Follicular lymphoma is composed of clonal germinal center B cells. It shows a follicular pattern lacking mantle zones, with a network of interfollicular dendritic cells. Transformation to more aggressive lymphomas is documented, but the only connections to mantle cell lymphoma are described cases of composite lymphoma consisting of these 2 entities. We discuss here a case of a lymph node harboring CD20+, CD10+, BCL2+, BCL6+/−, cyclin D1−, CD5+, Ki67+, and SOX11+ with CD21, showing an almost intact network of dendritic cells in one part of a lymph node, and CD20+, CD5+, SOX11+, BCL6−, cyclin D1−, CD10−, Ki67+, and CD21+ cells restricted to the mantle area in another part of the same lymph node. Both parts of the lymph node had BCL2 rearrangement, a lack of t(11:14)(q13;q32), the presence of SOX11 expression, and the same clonal band. The described case suggests heterogenous development of small cell lymphomas and indicates the possibility of differentiation regression.