Hematology, R
Contributions
Type: Publication Only
Background
Mantle Cell Lymphoma (MCL) is a B-cell neoplasm that represents 6 % - 9 % of malignant lymphoma in Western Europe. MCL is characterized genetically by the translocation t(11;14) leading to overexpression of the cell cycle protein Cyclin D1 distinguishing MCL from other lymphomas. The outcome is extremely heterogeneous, supporting the need for new molecular markers to clarify the disease cause as well as assisting in diagnosis and prognosis. Chronic lymphocytic leukaemia (CLL) is the most common leukaemia of adults in Western countries. The mutational status of the IgH-V genes envisages here the prognosis along with mutations in a number of genes including p53, notch1, sf3b1, and birc3. Nevertheless, there is still a need for new markers for prediction of survival and treatment strategy in CLL.
Recent next generation sequencing (NGS) studies have pinpointed a number of candidate genes that harbor mutations in clonal B cell neoplasms among others, the ROBO1 gene (1). We likewise identified mutations in the ROBO genes in a pair of monozygotic twins with monoclonal B-cell disorders (2).
The ROBO gene family consists of the genes ROBO1-4 which encode single pass transmembrane proteins involved in neuronal migration across the midline and fetal brain development.
These genes have previously been associated with several malignancies though only a single study, to our knowledge, -directly associated ROBO1 with lymphomas (3).
Aims
The aim of this project is to investigate ROBO1 as a new molecular target in patients suffering from MCL, CLL and acute myeloid leukemia (AML). We explored the mRNA expression patterns of the gene and compared the expression to a cohort of healthy individuals.
Methods
Blood samples from 20 patients with MCL, 20 patients with CLL and 20 patients with AML were collected at diagnosis. In addition blood samples from 20 healthy individuals were employed. The qPCR assay Hs00268049_m1 (Applied Biosystems, Foster City, CA, USA) targeting the specific ROBO1 mRNA was applied in triplicates on a MX3005 (Agilent Technologies, Santa Clare, CA, USA) and the quantitative range of the assays was determined. β-glucoronidase (GUS) and β-2-microglobulin (B2M) were employed as reference genes and the cell line K562 was used as positive control. ΔCq was calculated as Ave(Cq, target gene) – Ave(Cq, reference genes). The non-parametric Mann-Whitney U test was used to evaluate significant difference between the groups (Figure).
Results
We identified a significant difference between the expression of ROBO1 in MCL versus healthy individuals (p = 0.0002), between CLL and healthy individuals (p = < 0.0001) and between AML and healthy individuals (p = < 0.0001). Interestingly, three MCL patients displayed remarkably high expression of ROBO1 compared to the rest of the cohort (Figure).
Summary
Using a qPCR assay, specifically targeting ROBO1 mRNA we found a significantly difference in the expression level in the MCL, CLL and AML cohorts compared to the healthy individuals. There has previously been a report on promoter methylation of ROBO1 in MCL with impact on prognosis (3). Methylation of the promoter CpG islands could explain the significant difference in expression that we observed. However, whether the difference in expression of ROBO1 solely derives from promoter methylation or is due to mutations in the gene needs to be further explored. Perspectives of using the expression profile of genes like ROBO1 as additional tool for prognosis are interesting in these B-cell neoplasms with very heterogeneous outcome.
References:
1. Zhang, J., et al., The genomic landscape of mantle cell lymphoma is related to the epigenetically determined chromatin state of normal B cells. Vol. 123. 2014. 2988-2996.
2. Hansen MC, Nyvold CG, Roug A, Kjeldsen E, Villesen P, Nederby L, Hokland P. Nature and nurture: a case of transcending haematological pre-malignancies in a pair of monozygotic twins adding possible clues on the pathogenesis of B-cell proliferations. 2015. Br J Haem. In press.
3. Enjuanes, A., et al., Identification of Methylated Genes Associated with Aggressive Clinicopathological Features in Mantle Cell Lymphoma. PLoS ONE, 2011. 6(5): p. e19736.
Keyword(s): Chronic lymphocytic leukemia, Expression, Mantle cell lymphoma, Quantitative RT-PCR
Type: Publication Only
Background
Mantle Cell Lymphoma (MCL) is a B-cell neoplasm that represents 6 % - 9 % of malignant lymphoma in Western Europe. MCL is characterized genetically by the translocation t(11;14) leading to overexpression of the cell cycle protein Cyclin D1 distinguishing MCL from other lymphomas. The outcome is extremely heterogeneous, supporting the need for new molecular markers to clarify the disease cause as well as assisting in diagnosis and prognosis. Chronic lymphocytic leukaemia (CLL) is the most common leukaemia of adults in Western countries. The mutational status of the IgH-V genes envisages here the prognosis along with mutations in a number of genes including p53, notch1, sf3b1, and birc3. Nevertheless, there is still a need for new markers for prediction of survival and treatment strategy in CLL.
Recent next generation sequencing (NGS) studies have pinpointed a number of candidate genes that harbor mutations in clonal B cell neoplasms among others, the ROBO1 gene (1). We likewise identified mutations in the ROBO genes in a pair of monozygotic twins with monoclonal B-cell disorders (2).
The ROBO gene family consists of the genes ROBO1-4 which encode single pass transmembrane proteins involved in neuronal migration across the midline and fetal brain development.
These genes have previously been associated with several malignancies though only a single study, to our knowledge, -directly associated ROBO1 with lymphomas (3).
Aims
The aim of this project is to investigate ROBO1 as a new molecular target in patients suffering from MCL, CLL and acute myeloid leukemia (AML). We explored the mRNA expression patterns of the gene and compared the expression to a cohort of healthy individuals.
Methods
Blood samples from 20 patients with MCL, 20 patients with CLL and 20 patients with AML were collected at diagnosis. In addition blood samples from 20 healthy individuals were employed. The qPCR assay Hs00268049_m1 (Applied Biosystems, Foster City, CA, USA) targeting the specific ROBO1 mRNA was applied in triplicates on a MX3005 (Agilent Technologies, Santa Clare, CA, USA) and the quantitative range of the assays was determined. β-glucoronidase (GUS) and β-2-microglobulin (B2M) were employed as reference genes and the cell line K562 was used as positive control. ΔCq was calculated as Ave(Cq, target gene) – Ave(Cq, reference genes). The non-parametric Mann-Whitney U test was used to evaluate significant difference between the groups (Figure).
Results
We identified a significant difference between the expression of ROBO1 in MCL versus healthy individuals (p = 0.0002), between CLL and healthy individuals (p = < 0.0001) and between AML and healthy individuals (p = < 0.0001). Interestingly, three MCL patients displayed remarkably high expression of ROBO1 compared to the rest of the cohort (Figure).
Summary
Using a qPCR assay, specifically targeting ROBO1 mRNA we found a significantly difference in the expression level in the MCL, CLL and AML cohorts compared to the healthy individuals. There has previously been a report on promoter methylation of ROBO1 in MCL with impact on prognosis (3). Methylation of the promoter CpG islands could explain the significant difference in expression that we observed. However, whether the difference in expression of ROBO1 solely derives from promoter methylation or is due to mutations in the gene needs to be further explored. Perspectives of using the expression profile of genes like ROBO1 as additional tool for prognosis are interesting in these B-cell neoplasms with very heterogeneous outcome.
References:
1. Zhang, J., et al., The genomic landscape of mantle cell lymphoma is related to the epigenetically determined chromatin state of normal B cells. Vol. 123. 2014. 2988-2996.
2. Hansen MC, Nyvold CG, Roug A, Kjeldsen E, Villesen P, Nederby L, Hokland P. Nature and nurture: a case of transcending haematological pre-malignancies in a pair of monozygotic twins adding possible clues on the pathogenesis of B-cell proliferations. 2015. Br J Haem. In press.
3. Enjuanes, A., et al., Identification of Methylated Genes Associated with Aggressive Clinicopathological Features in Mantle Cell Lymphoma. PLoS ONE, 2011. 6(5): p. e19736.
Keyword(s): Chronic lymphocytic leukemia, Expression, Mantle cell lymphoma, Quantitative RT-PCR