Jan-Lukas Robertus

miRNA profiling of B-cell subsets: specific miRNA profile for germinal center B cells with variation between centroblasts and centrocytes

MicroRNAs (miRNAs) are an important class of small RNAs that regulate gene expression at the post-transcriptional level. It has become evident that miRNAs are involved in hematopoiesis, and that deregulation of miRNAs may give rise to hematopoietic malignancies. The aim of our study was to establish miRNA profiles of naïve, germinal center (GC) and memory B cells, and validate their expression patterns in normal lymphoid tissues. Quantitative (q) RT-PCR profiling revealed that several miRNAs were elevated in GC B cells, including miR-17-5p, miR-106a and miR-181b. One of the most abundant miRNAs in all three B-cell subsets analyzed was miR-150, with a more than 10-fold lower level in GC B cell as compared with the other two subsets. miRNA in situ hybridization (ISH) in tonsil tissue sections confirmed the findings from the profiling work. Interestingly, gradual decrease of miR-17-5p, miR-106a and miR-181b staining intensity from the dark to the light zone was observed in GC. A strong cytoplasmic staining of miR-150 was observed in a minority of the centroblasts in the dark zone of the GC. Inverse staining pattern of miR-150 against c-Myb and Survivin was observed in tonsil tissue sections, suggesting possible targeting of these genes by miR-150. In line with this, the experimental induction of miR-150 lead to reduced c-Myb, Survivin and Foxp1 expression levels in the Burkitts lymphoma cell line, DG75. In conclusion, miRNA profiles of naïve, GC and memory B cells were established and validated by miRNA ISH. Within the GC cells, a marked difference was observed between the light and the dark zone.

Specific expression of miR-17-5p and miR-127 in testicular and central nervous system diffuse large B-cell lymphoma.

Recent studies have shown that certain non-coding short RNAs, called miRNAs, play an important role in diffuse large B-cell lymphomas. Patients with diffuse large B-cell lymphoma have great diversity in both clinical characteristics, site of presentation and outcome. The aim of our study is to validate the differential expression in germinal center and non-germinal center diffuse large B-cell lymphoma,s and to study to the extent to which the primary site of differentiation is associated with the miRNA expression profile. We studied 50 cases of de novo diffuse large B-cell lymphoma for the expression of 15 miRNAs (miR-15a, miR-15b, miR-16, miR-17-3p, miR-17-5p, miR-18a, miR-19a, miR-19b, miR-20a, miR-21, miR-92, miR-127, miR-155, miR-181a and miR-221). Apart from 19 nodal cases without extranodal dissemination (stages I and II), we selected two groups with unambiguous stages I and II extranodal presentation; 9 cases of primary central nervous system, 11 cases of primary testicular and 11 cases of other primary extranodal diffuse large B-cell lymphomas. All cases were analyzed with qRT-PCR. In situ hybridization for the most differentially expressed miRNAs was performed to show miRNA expression in tumor cells, but not in background cells. MiR-21 and miR-19b showed the highest expression levels. No significant differences were seen between germinal center and non-germinal center diffuse large B-cell lymphomas in either the total or the nodal group for any of the 15 miRNAs. Two miRNAs showed significant differences in expression levels for diffuse large B-cell lymphoma subgroups according to the site of presentation. MiR-17-5p showed a significant higher expression level in the central nervous system compared with testicular and nodal diffuse large B-cell lymphomas (P<0.05). MiR-127 levels were significantly higher in testicular than in central nervous system and in nodal diffuse large B-cell lymphomas (P<0.05). We conclude that the location of diffuse large B-cell lymphoma is an important factor in determining the differential expression of miRNAs.

Cutaneous leiomyosarcoma arising in a smallpox scar

BackgroundCutaneous leiomyosarcoma (CLM) is a very rare smooth muscle tumour that accounts for about 2–3% of all superficial soft tissue sarcomas. Although the development of various malignancies in scar tissue is well known, we report the first case of a CLM developing in a small pox scar.Case presentationA 66-year-old man presented with a painless, slow-growing lump in a small pox scar on his left shoulder. Histological biopsies showed the lesion to be a primary, well-differentiated cutaneous leiomyosarcoma. A CT scan of the thorax was conducted, which showed no signs of metastases. The complete lesion was then surgically excised, and histopathological examination revealed a radically excised cutaneous type leiomyosarcoma After 13 months’ review the patient was doing well with no evidence of tumour recurrence.ConclusionsThis is the first report of a CLM arising in a small pox scar. Although the extended time interval between scarring and malignant changes makes it difficult to advise strict follow-up for patients with small pox scars, one should be aware that atypical changes and/or symptoms occurring in a small pox scar could potentially mean malignant transformation.

MiRNA profiling in B non-Hodgkin lymphoma: a MYC-related miRNA profile characterizes Burkitt lymphoma: Correspondence

Several studies have indicated the importance of miRNAs in B cell maturation and in the development of B cell lymphomas. The oncogene MYC plays an important role in B cell lymphomagenesis, particularly in Burkitt lymphoma (BL). Several recent publications have shown that MYC regulates expression of up to 60 miRNAs (Tables I and SI). The impact of the translocation and overexpression of MYC on the miRNA profile in BL has not yet been explored. We determined the miRNA expression profile of paediatric t(8;14) positive and high MYC expressing BL in comparison to MYC translocation negative mantle cell lymphoma (MCL), follicular lymphoma (FL) and chronic lymphocytic leukaemia (CLL). As a control we included normal B cell subsets obtained from hyperplastic tonsils. Hierarchical clustering showed that the B cell subsets and the non-Hodgkin lymphomas (NHLs) formed two distinct sub-clusters (Fig 1). Unsupervised clustering of the 23 miRNAs significantly differentially expressed between the four B cell subsets (>4-fold) revealed one cluster for the naı̈ve and memory B cells and two additional clusters for the germinal centre (GC) B cells and plasma cells. These results were consistent with previously published data (Appendix S1). 76 miRNAs were differentially expressed (>4-fold) between the four NHL subtypes. Most differences were observed between BL and the other NHLs (CLL n = 58, FL n = 32 and MCL n = 36 miRNAs). Unsupervised hierarchical clustering analysis revealed a unique miRNA profile in BL (Appendix S2). In contrast, a maximum of eight miRNAs were differently expressed between MCL, FL and CLL. A list of validated target genes of the differentially expressed miRNAs is presented in Table SII. Comparing each malignancy to its normal counterpart (MCL with naı̈ve, FL and BL with GC B cells and CLL with memory cells), 54–77 miRNAs were differentially expressed (Figure S1). MYC expression has been shown to be a dominant factor in the regulation of many miRNAs. In the present series of lymphomas, quantitative reverse transcription polymerase chain reaction (qRT-PCR) showed a much higher expression in BL than all other NHL (Fig 1C). Comparison of the MYChigh BL to all other MYC-low lymphomas revealed 122 differentially expressed miRNAs (Table SIII), including 39 of the 50 evaluable (78%) known targets of MYC (10 miRNAs were not expressed in our cases) (Fig 1D and Table I). The expression level of these 39 miRNAs was always consistent with the expected upor downregulation, with most miRNAs being downregulated. This indicates a dominant MYC-induced miRNA profile in primary BL. This signature included 39 of the known MYC-regulated miRNAs. The 83 other miRNAs were also differentially expressed between MYC-high BL and MYC-low NHL samples. At present, there are no data supporting a direct MYC-dependent regulation for those 83 miRNAs. A MYC-dependent miRNA signature has also been suggested for diffuse large B cell lymphoma based on a differential expression of MYC targets (Li et al, 2009). Four BL cases (B4, B5, B6 and B7) and two MCL cases (M2 and M3) harboured a 13q31.3 amplification (determined by fluorescence in situ hybridization, results not shown). These six cases showed high expression of the miR-17-92 cluster (Fig 1D). Both in BL and MCL, this association was independent of the MYC expression level (Fig 1C). In line with the findings of Tagawa et al (2007) this indicates that genomic amplification and not MYC overexpression is instrumental in the expression of the miR-17-92 cluster. The miR-17-92 cluster is positively regulated by MYC and acts with MYC to accelerate tumour development (He et al, 2005). MiR-17 and miR-20, two members of this cluster, promote cell cycle progression via E2F1 (O’Donnell et al, 2005). One interesting MYC-repressed gene is MIRLET7A. As MYC is a direct target of MIRLET7A this suggests a positive stimulatory loop for MYC (Sampson et al, 2007). Induction of MIRLET7A in the Namalwa BL cell line resulted in reduced MYC expression and reduced proliferation, whereas downregulation of MYC resulted in increased expression of MIRLET7A. In addition to a significant downregulation of MIRLET7A, we also observed a significant downregulation for MIRLET7E in BL compared to the three other NHL subtypes. MiR-150 was also significantly downregulated in BL and targets MYB, which has an essential role in haematopoietic and lymphoid development and apoptosis (Xiao et al, 2007). Overexpression of miR-150 in BL cells resulted in reduced MYB levels and increased apoptosis (Tan et al, 2009). Interestingly miR-15a has recently also been shown to repress the MYB oncogene in leukemic cell lines (Zhao et al, 2009). Low expression of miR-150 in BL compared to the other three NHL subtypes and of miR-15a in comparison to CLL might thus result in enhanced MYB levels in BL. Ectopic expression of miR-26a in BL cell lines impaired cell cycle progression via its target EZH2, a member of the polycomb-group of genes (Sander et al, 2008). The low expression of miR-26a in BL is thus consistent with the previously observed high expression of correspondence

MiRNA profiling in B non-Hodgkin lymphoma

Several studies have indicated the importance of miRNAs in B cell maturation and in the development of B cell lymphomas. The oncogene MYC plays an important role in B cell lymphomagenesis, particularly in Burkitt lymphoma (BL). Several recent publications have shown that MYC regulates expression of up to 60 miRNAs (Tables I and SI). The impact of the translocation and overexpression of MYC on the miRNA profile in BL has not yet been explored. We determined the miRNA expression profile of paediatric t(8;14) positive and high MYC expressing BL in comparison to MYC translocation negative mantle cell lymphoma (MCL), follicular lymphoma (FL) and chronic lymphocytic leukaemia (CLL). As a control we included normal B cell subsets obtained from hyperplastic tonsils. Hierarchical clustering showed that the B cell subsets and the non-Hodgkin lymphomas (NHLs) formed two distinct sub-clusters (Fig 1). Unsupervised clustering of the 23 miRNAs significantly differentially expressed between the four B cell subsets (>4-fold) revealed one cluster for the naı̈ve and memory B cells and two additional clusters for the germinal centre (GC) B cells and plasma cells. These results were consistent with previously published data (Appendix S1). 76 miRNAs were differentially expressed (>4-fold) between the four NHL subtypes. Most differences were observed between BL and the other NHLs (CLL n = 58, FL n = 32 and MCL n = 36 miRNAs). Unsupervised hierarchical clustering analysis revealed a unique miRNA profile in BL (Appendix S2). In contrast, a maximum of eight miRNAs were differently expressed between MCL, FL and CLL. A list of validated target genes of the differentially expressed miRNAs is presented in Table SII. Comparing each malignancy to its normal counterpart (MCL with naı̈ve, FL and BL with GC B cells and CLL with memory cells), 54–77 miRNAs were differentially expressed (Figure S1). MYC expression has been shown to be a dominant factor in the regulation of many miRNAs. In the present series of lymphomas, quantitative reverse transcription polymerase chain reaction (qRT-PCR) showed a much higher expression in BL than all other NHL (Fig 1C). Comparison of the MYChigh BL to all other MYC-low lymphomas revealed 122 differentially expressed miRNAs (Table SIII), including 39 of the 50 evaluable (78%) known targets of MYC (10 miRNAs were not expressed in our cases) (Fig 1D and Table I). The expression level of these 39 miRNAs was always consistent with the expected upor downregulation, with most miRNAs being downregulated. This indicates a dominant MYC-induced miRNA profile in primary BL. This signature included 39 of the known MYC-regulated miRNAs. The 83 other miRNAs were also differentially expressed between MYC-high BL and MYC-low NHL samples. At present, there are no data supporting a direct MYC-dependent regulation for those 83 miRNAs. A MYC-dependent miRNA signature has also been suggested for diffuse large B cell lymphoma based on a differential expression of MYC targets (Li et al, 2009). Four BL cases (B4, B5, B6 and B7) and two MCL cases (M2 and M3) harboured a 13q31.3 amplification (determined by fluorescence in situ hybridization, results not shown). These six cases showed high expression of the miR-17-92 cluster (Fig 1D). Both in BL and MCL, this association was independent of the MYC expression level (Fig 1C). In line with the findings of Tagawa et al (2007) this indicates that genomic amplification and not MYC overexpression is instrumental in the expression of the miR-17-92 cluster. The miR-17-92 cluster is positively regulated by MYC and acts with MYC to accelerate tumour development (He et al, 2005). MiR-17 and miR-20, two members of this cluster, promote cell cycle progression via E2F1 (O’Donnell et al, 2005). One interesting MYC-repressed gene is MIRLET7A. As MYC is a direct target of MIRLET7A this suggests a positive stimulatory loop for MYC (Sampson et al, 2007). Induction of MIRLET7A in the Namalwa BL cell line resulted in reduced MYC expression and reduced proliferation, whereas downregulation of MYC resulted in increased expression of MIRLET7A. In addition to a significant downregulation of MIRLET7A, we also observed a significant downregulation for MIRLET7E in BL compared to the three other NHL subtypes. MiR-150 was also significantly downregulated in BL and targets MYB, which has an essential role in haematopoietic and lymphoid development and apoptosis (Xiao et al, 2007). Overexpression of miR-150 in BL cells resulted in reduced MYB levels and increased apoptosis (Tan et al, 2009). Interestingly miR-15a has recently also been shown to repress the MYB oncogene in leukemic cell lines (Zhao et al, 2009). Low expression of miR-150 in BL compared to the other three NHL subtypes and of miR-15a in comparison to CLL might thus result in enhanced MYB levels in BL. Ectopic expression of miR-26a in BL cell lines impaired cell cycle progression via its target EZH2, a member of the polycomb-group of genes (Sander et al, 2008). The low expression of miR-26a in BL is thus consistent with the previously observed high expression of correspondence