Prognostic molecular markers of childhood leukemia

During the last decades there has been a dramatic increase in survival rates for childhood leukemia, resulting today in an overall survival close to 90 % for children with acute lymphoblastic leukemia (ALL). This accomplishment is largely due to treatment protocols based on careful risk group assessment guiding the type and intensity of the anti-leukemic treatment. Relapse remains still the most important cause of treatment failure and the outcome is then much more dismal. The aim of this thesis was to increase the understanding how genetic aberrations and alterations in cell cycle regulating proteins in the leukemic cells, contribute to pathogenesis and prognosis of childhood ALL.

On behalf of the Nordic Society of Pediatric Hematology and Oncology (NOPHO), we investigated the frequency and clinical implications of the genetic aberration dic(9;20)(p13.2;q11.2), which easily escapes detection by chromosome banding analysis alone. We obtained material and performed interphase FISH on more than half (53%) of all Nordic pediatric patients diagnosed with B-cell precursor (BCP) ALL during 2001-2006. 4.6% (25/542) had dic(9;20), making it the third most common genetic subgroup of BCP ALL after high hyperdiploidy and t(12;21)(p13;q22). 5-year event-free survival and overall survival were 0.68 and 0.83, respectively, which were significantly worse than for t(12;21)- positive cases. We conclude that that dic(9;20) is twice as common as previously surmised and that it is a non-standard risk abnormality, which should not receive standard risk therapy.

Neonatal dried blood spots (DBS), or Guthrie cards, have been used to demonstrate the prenatal origin of clonal leukemia-specific aberrations in several genetic subgroups of childhood ALL. In addition, the ETV6/RUNX1 fusion transcript has been detected in 1 % of normal cord blood samples. We report that transcripts encoding beta-actin are readily detectable in RNA isolated from neonatal DBS stored for up to 20 years and that no significant loss of transcripts appeared to occur over time. The lack of significant decay of RNA in DBS stored for up to 20 years, suggests that such filters are useful for studies of RNA determinants of diseases with an onset, not only in childhood, but also in adult life. We also investigated DBS from 14 children, who later were diagnosed with leukemia (8 with t(12;21) ALL and six with t(9;22) ALL or chronic myeloid leukemia) and 14 age-matched controls, for the presence of leukemia-specific fusion transcripts. Transcripts for ETV6/RUNX1 and BCR/ABL could not be detected by RT-PCR, using clone specific primers, in any of the samples.

PTEN and SHP1 are tumor suppressor genes involved in the regulation of cell cycle control and apoptosis. Disturbance in the expression and signaling pathways of VEGF has been linked to pathogenesis of hematological malignancies. We have investigated the expression of PTEN, SHP 1, VEGF and two of its receptors, VEGFR-1 and VEGFR-2, in childhood BCP ALL and non malignant controls by immunohistochemistry. PTEN was over expressed while SHP1 showed a low expression in diagnostic ALL samples, compared to non-malignant controls. ALL samples had significantly increased expression of VEGFR-1 compared to no expression in the non-malignant group, indicating a link between VEGFR-1 protein expression and pre-B ALL. Our sample size was small and the potential role of these proteins in diagnosis and prognosis of childhood ALL needs to be further investigated.

List of scientific papers

I. Forestier E, Gauffin F, Andersen MK, Autio K, Borgström G, Golovleva I, Gustafsson B, Heim S, Heinonen K, Heyman M, Hovland R, Johannsson JH, Kerndrup G, Rosenquist R, Schoumans J, Swolin B, Johansson B, Nordgren A; on behalf of NOPHO, SCLSG and NLCSG (2008). "Clinical and cytogenetic features of pediatric dic(9;20)(p13.2;q11.2)-positive B-cell precursor acute lymphoblastic leukemias: a Nordic series of 24 cases and review of the literature." Genes Chromosomes Cancer 47(2): 149-58
https://pubmed.ncbi.nlm.nih.gov/17990329

II. Zacharidis V, Gauffin F, Kuchinskaya E, Heyman M, Schoumans J, Blennow E, Gustafsson B, Barbany G, Golovleva I, Ehrencrona H, Cavalier Franco L, Palmqvist L, Lönnerholm G, Nordenskjöld M, Johansson B, Forestier E, Nordgren A, for NOPHO and SCLSG. (2010). "The dic(9;20)(p13.2;q11.2) is an emerging non-standard risk aberration in childhood B-cell precursor acute lymphoblastic leukemia: results from the NOPHO ALL-2000 trial." (Manuscript)

III. Gauffin F, Nordgren A, Barbany G, Gustafsson B, Karlsson H (2009). "Quantitation of RNA decay in dried blood spots during 20 years of storage." Clin Chem Lab Med 47(12): 1467-9
https://pubmed.ncbi.nlm.nih.gov/19863301

IV. Gauffin F, Barbany G, Karlsson H, Heyman M, Thörn I, Nordenskjöld M, Gustafss B, Nordgren A (2010). "ETV6/RUNX1 and BCR/ABL are not found on RNA transcript level in neonatal dried blood spots from children with leukemia." (Manuscript)

V. Gauffin F, Diffner E, Gustafsson B, Nordgren A, Wingren AG, Sander B, Persson JL, Gustafsson B (2009). "Expression of PTEN and SHP1, investigated from tissue microarrays in pediatric acute lymphoblastic, leukemia." Pediatr Hematol Oncol 26(1): 48-56
https://pubmed.ncbi.nlm.nih.gov/19206008

VI. Diffner E, Gauffin F, Anagnostaki L, Nordgren A, Gustafsson B, Sander B, Gustafsson B, Persson JL (2009). "Expression of VEGF and VEGF receptors in childhood precursor B-cell acute lymphoblastic leukemia evaluated by immunohistochemistry." J Pediatr Hematol Oncol 31(9): 696-701
https://pubmed.ncbi.nlm.nih.gov/19707156