Tesi di: Giorgia AimoniMatricola: 707369

Characterization of the genomic breakpoint

for molecular monitoring of residual disease in

Chronic Myeloid LeukemiaRelatore: Prof. Francesco PasqualiCorrelatore: Prof. Giovanni Porta

Anno Accademico 2010/2011

Università degli Studi dell’InsubriaFacoltà di Scienze MM.FF.NN. – VareseCorso di Laurea in Biotecnologie

Dipartimento di Scienze Biomediche Sperimentali e Cliniche

CHRONIC MYELOID LEUKEMIA

CML is a myeloproliferative disease due to an acquired abnormality that involves the clonal expansion of a hematopoietic stem cell.

1-2 cases per 100,000 person/years, 15% of adult leukemias

enlarged spleen, joint pain, anemia, thrombocytopenia, low-grade fever

chronic phase blast crisis

Philadelphia chromosomet(9;22)(q34;q11.2)

BCR and ABL genes

Monitoring of residual disease

Cytogenetic analysis

RNA qRT-PCR

DNA Q-PCR High sensitivity and direct detection

One cell can over-expresses BCR/ABL

Quiescent cells can not be detected

AIM OF THE STUDY

1. Characterization of the genomic breakpoint by long-range PCR and sequencing

2. Molecular monitoring of residual disease by DNA Q-PCR

PHASE 1 long-range PCR

breakpoint previously characterized

DNA extraction from PB DNA quantification (agarose gel and nanodrop) Primers design (DM Ross et al, 2010)

Rw 3b

DNA PCR purification and quantification

Fw 13-Rw 3a4800 bp

Long-range PCR

BCR ABL

Sequencing and bioinformatics

BLAST and Clustal W in silico analysis

Chr. 22 BCR 21.962.205Chr. 9 ABL 132.580.490

The finding was confirmed with the genomic junction previously characterized in order to test this new methodology that involves less time and labor.

monitoring of follow-up (Mattarucchi et al, 2009)

DNA extraction from PB or BM

Sample Sample type Months8E BM 08B PB 68I BM 422 PB 483 PB 554 PB 585 PB 646 PB 70

PHASE 2DNA Q-PCR

LC = 100 x [2 / (2ΔCt + 1)]

DNA quantification (agarose gel and fluorometer) DNA Q-PCR

non-translocated cells leukemic cells

0,0316

0,0010 UND 0,0007 0,0012 0,0002 UND0

0,0050,01

0,0150,02

0,0250,03

0,035

6 42 48 55 58 64 70

Months after Imatinib therapy

% BCR/ABL cells (DNA)

1,82

0 00,234

0 0 00

0,5

1

1,5

2

6 42 48 55 58 64 70Months after Imatinib therapy

Copy number of Bcr/Abl (mRNA)

The discrepancies between the real-time analysis DNA and RNA indicated Ph+ quiescent cells, including leukemic stem cells.

CONCLUSION

Although DNA-based approach requires an initial characterization of the genomic breakpoint, it provides a patient-specific assay that can directly detect the leukemic cells percentage, independent of their state of BCR/ABL expression.

This could help the research and improve the practice of conventional molecular diagnostics.

WORK IN PROGRESS…

DNA Q-PCR analysis on the other patients characterized

Use of KCl22 cell line to validate the method

Ackowledgements

DSBSC - Insubria

Professor Giovanni Porta

Doctor Ilaria Pagani

Professor Francesco Pasquali

Professor Francesco Lo Curto

“Ospedale di Circolo” of Varese and “Ospedali Riuniti” of Bergamo