In hematological myeloid malignancies the accumulation of oncogenic events plays a significant role in disease progression. Therefore, in this work, we studied (i) the mutational landscape in typical chronic myeloid leukemia (CML) patients and (ii) the neoplastic role of Setbp1 mutation in atypical chronic myeloid leukemia (aCML). 1) We evaluated somatic variants in CML patients by Next Generation Sequencing, to study the molecular pathogenesis of cancer. We conducted a mutational analysis on 23 chronic phase BCR-ABL1+ CML patients through exome and RNA sequencing performed on diagnosis samples. A total of 107 non-synonymous variants (range 0-11 per patient) were identified by setting a threshold of mutation frequency >25%, which corresponds to the presence of a heterozygous mutation in >50% of cells, assuming a pure tumoral sample. A positive correlation was observed between number of mutations and patient age, indicating that several events were passenger mutations, being immortalized by the neoplastic transformation. However, when using a newly in-house developed tool (Oncoscore) to weigh the oncogenic potential of each mutation, a significant correlation was observed between the Sokal score and Oncoscore by using linear model statistical analysis. In long term follow-up (>2 years), 21 CML patients achieved complete cytogenetic responses (CCyR) and 2 failed to achieve any cytogenetic response with tyrosine kinase inhibitors. These two patients showed an Oncoscore value of (165.4 ±27.2) which was significantly higher than the one (80.6 ±12.7) in the 21 responding patients. No fusions (other than BCR/ABL1) were identified by RNA Seq, and no chromosomal alterations were observed by using the CEQer software. In conclusion, CML patients at diagnosis carry genetic alterations additional to the BCR/ABL1 fusion, which could be relevant for response to treatment and progression of the disease. 2) We aimed to gain insights into the biological role of Setbp1 mutations found in aCML patients, by invivo genetic manipulation techniques. Recently, by NGS approach, we identified a recurrent SETBP1 missense mutation in aCML patients, associated with poor overall survival. The most frequent SETBP1 mutations identified in various MDS/MPN neoplasms were positioned at D868N, S869G, G870S and I871T. The same mutations identified in myeloid malignancies had previously been observed as de novo recurrent germline mutations responsible for Schinzel-Giedion syndrome. Unfortunately, the biological role of Setbp1 and its activity in leukemic transformation is not exactly known. Therefore, an improved understanding of the molecular mechanism is imperative. So, we applied genetic engineering to construct a conditional knock-in model for dissecting the role of leukemia transforming factors in heterozygous Setbp1G870S mice. For construction, 3 genomic fragments of Setbp1 intron 3 and exons 4 through 6 were subcloned into the conditional replacement vector pDELBOY-3X. The linearised vector was then transfected into murine ES cells. We are currently screening ES cells to identify a correctly targeted clone for blastocyst injection and transplantion into pseudo-pregnant mice. The 1st generation Setbp1WT/floxed mice will express wild type Setbp1 under the control of its endogenous promoter. Thereafter, the expression of Setbp1G870S would be induced in a conditional manner with Cre-mediated recombination. Depending on the type of promoter driving Cre recombinase expression, the mutant allele will be expressed either constitutively (germline) or somatically, and it will be possible to study the oncogenic effects of Setbp1G870S in specific tissues, or in all tissue/cells. Additionally, the molecular interactions and physiological pathways accountable for tumorigenesis and the clonal evolution pattern will be examined by implementing the molecular and functional genomic techniques, which help in better understanding of developing targeted therapies.

(2016). Elucidating the oncogenic role of genetic events in BCR-ABL1 positive chronic myeloid leukemia. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2016).

Elucidating the oncogenic role of genetic events in BCR-ABL1 positive chronic myeloid leukemia

KHANDELWAL, PRAVEEN
2016-02-04

Abstract

In hematological myeloid malignancies the accumulation of oncogenic events plays a significant role in disease progression. Therefore, in this work, we studied (i) the mutational landscape in typical chronic myeloid leukemia (CML) patients and (ii) the neoplastic role of Setbp1 mutation in atypical chronic myeloid leukemia (aCML). 1) We evaluated somatic variants in CML patients by Next Generation Sequencing, to study the molecular pathogenesis of cancer. We conducted a mutational analysis on 23 chronic phase BCR-ABL1+ CML patients through exome and RNA sequencing performed on diagnosis samples. A total of 107 non-synonymous variants (range 0-11 per patient) were identified by setting a threshold of mutation frequency >25%, which corresponds to the presence of a heterozygous mutation in >50% of cells, assuming a pure tumoral sample. A positive correlation was observed between number of mutations and patient age, indicating that several events were passenger mutations, being immortalized by the neoplastic transformation. However, when using a newly in-house developed tool (Oncoscore) to weigh the oncogenic potential of each mutation, a significant correlation was observed between the Sokal score and Oncoscore by using linear model statistical analysis. In long term follow-up (>2 years), 21 CML patients achieved complete cytogenetic responses (CCyR) and 2 failed to achieve any cytogenetic response with tyrosine kinase inhibitors. These two patients showed an Oncoscore value of (165.4 ±27.2) which was significantly higher than the one (80.6 ±12.7) in the 21 responding patients. No fusions (other than BCR/ABL1) were identified by RNA Seq, and no chromosomal alterations were observed by using the CEQer software. In conclusion, CML patients at diagnosis carry genetic alterations additional to the BCR/ABL1 fusion, which could be relevant for response to treatment and progression of the disease. 2) We aimed to gain insights into the biological role of Setbp1 mutations found in aCML patients, by invivo genetic manipulation techniques. Recently, by NGS approach, we identified a recurrent SETBP1 missense mutation in aCML patients, associated with poor overall survival. The most frequent SETBP1 mutations identified in various MDS/MPN neoplasms were positioned at D868N, S869G, G870S and I871T. The same mutations identified in myeloid malignancies had previously been observed as de novo recurrent germline mutations responsible for Schinzel-Giedion syndrome. Unfortunately, the biological role of Setbp1 and its activity in leukemic transformation is not exactly known. Therefore, an improved understanding of the molecular mechanism is imperative. So, we applied genetic engineering to construct a conditional knock-in model for dissecting the role of leukemia transforming factors in heterozygous Setbp1G870S mice. For construction, 3 genomic fragments of Setbp1 intron 3 and exons 4 through 6 were subcloned into the conditional replacement vector pDELBOY-3X. The linearised vector was then transfected into murine ES cells. We are currently screening ES cells to identify a correctly targeted clone for blastocyst injection and transplantion into pseudo-pregnant mice. The 1st generation Setbp1WT/floxed mice will express wild type Setbp1 under the control of its endogenous promoter. Thereafter, the expression of Setbp1G870S would be induced in a conditional manner with Cre-mediated recombination. Depending on the type of promoter driving Cre recombinase expression, the mutant allele will be expressed either constitutively (germline) or somatically, and it will be possible to study the oncogenic effects of Setbp1G870S in specific tissues, or in all tissue/cells. Additionally, the molecular interactions and physiological pathways accountable for tumorigenesis and the clonal evolution pattern will be examined by implementing the molecular and functional genomic techniques, which help in better understanding of developing targeted therapies.
MOLOGNI, LUCA
CML, NGS, aCML, SETBP1
MED/15 - MALATTIE DEL SANGUE
English
Scuola di Dottorato in Scienze Mediche Sperimentali e Cliniche
EMATOLOGIA SPERIMENTALE - 05R
27
2014/2015
(2016). Elucidating the oncogenic role of genetic events in BCR-ABL1 positive chronic myeloid leukemia. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2016).
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/10281/99449
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