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Biological Risk Prediction in CLL with Emphasis on CLLU1 Expression

Pär Josefsson  


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Accepted by: Faculty of Health Sciences University of Copenhagen
Defended on: June 27, 2008
Official opponents: Lars Bo Nielsen , Ilse Christiansen , Geir Tjønnfjord
Tutors: Anne Mette Buhl , Jesper Jurlander , Niels Borregaard

Published in the PhD Database: July 5, 2008


English abstract
This Ph.D. thesis is based on studies performed at The Leukemia Laboratory, Department of Hematology. Rigshospitalet, University Hospital of Copenhagen, Denmark. It forms part of the investigation of the novel gene CLLU1 identified in The Leukemia Laboratory.
It consists of four studies.

Background: Chronic lymphocytic leukemia (CLL) is the most common adult form of leukemia in the Western World, yet the pathogenesis of the disease remains obscure.
CLL Upregulated gene 1 (CLLU1) is a recently identified gene, which is uniquely overexpressed in CLL and therefore might furnish new information on CLL biology.
CLL is by nature a heterogeneous disease with variable clinical outcome: some patients have rapidly progressive disease with fatal outcome, while others have stable disease for years. Since the finding that absence or presence of mutations in the variable region of immunoglobulin heavy chain (IgVH) genes can predict clinical course and disease outcome in CLL, several other biomarkers have been introduced as prognostic tools in CLL risk prediction aiming at an early recognition of patients likely to need early or aggressive therapeutic intervention. These markers include ZAP-70 expression, CD38 expression and certain chromosomal aberrations.

Aims: The aim of the research project was to investigate the potential clinical usefulness of CLLU1 in CLL risk prediction in context with other known biological prognostic markers.

Results: The first study measured CLLU1 mRNA expression by real-time RT-PCR in a small cohort of untreated CLL patients and demonstrated that CLLU1 expression levels form a continuum and can predict overall survival and time to treatment initiation. The second study validated these findings in a large series of patients, but also found that the prognostic significance seemed restricted to patients with CLL presenting before the age of 70. This study furthermore demonstrated that within this younger age group, CLLU1 expression provides additional prognostic information within all prognostic subgroups defined by IgVH mutational status, ZAP-70 expression, CD38 expression and cytogenetics aberrations, with the sole exception of IgVH unmutated patients. Based on the important finding of this study that the prognostic significance and hence the clinical usefulness of prognostic biomarkers in CLL seems to be age-dependent, a further study of CD38 expression in CLL was performed. This study demonstrated that unlike the other biomarkers the predictive power of CD38 expression seemed restricted to the older patient category. The fourth study concerns investigations of the stability of CLLU1 expression over time and in different hematopoietic tissues and is a contribution to a larger technical study by the CLLU1 research group on the properties of CLLU1.

Discussion: The clinical usefulness of biological markers is founded on the capability of early identification of patients most likely to require early or aggressive treatment. In these studies it was demonstrated that analysis of CLLU1 expression is highly applicable for patients of an age well suited for risk-adapted treatment strategies. It was shown that CLLU1 expression levels, whether high or low at time of diagnosis, are fairly stable over time in untreated CLL patients. The studies confirmed the prognostic significance of other established biomarkers, but also demonstrated a certain age-dependency in their prognostic significance. Some markers like CLLU1, IgVH mutational status and ZAP-70 expression might reflect early events in CLL genesis with a later onset of clinical effects, making them ideal for risk prediction. Conversely, the role of CD38 expression in CLL risk prediction remains unclarified, due to lack of significance in younger CLL patients.



Danish abstract
Denne ph.d. afhandling udgår fra Leukæmilaboratoriet, Hæmatologisk afdeling, Rigshospitalet og udgør en del af udforskningen af det nye gen CLLU1, der er opdaget og klonet i Leukæmilaboratoriet. Det består af fire studier.

Kronisk lymfatisk leukæmi (CLL) er den hyppigste from for leukæmi hos voksne i den vestlige verden. CLL upregulated gene1 (CLLU1) er et nyopdaget gen, der kun er væsentligt udtrykt i CLL. CLL forekommer i to former. Hos nogle patienter er sygdommen stabil og ikke-behandlingskrævende i mange år, mens den hos andre hurtigt får et aggressivt forløb med kort overlevelse. Forekomst eller fravær af mutationer i den variable region af immunglobulinernes tunge kæder (IgVH) kan forudsige sygdommens kliniske forløb allerede på diagnosetidspunktet.

Da mutationsstatus er en dyr og besværlig metode er der fundet andre biologiske markører, der kan forudsige sygdomsforløbet og det kommende behandlingsbehov. Disse markører omfatter ZAP-70 ekspression, CD38 ekspression og kromosomforandringer.

Formålet med forskningsprojektet var at undersøge den prognostiske værdi af CLLU1 og den mulige kliniske anvendelighed af CLLU1 i risiko vurdering af CLL.

Det første studie målte CLLU1 ekspression ved kvantitativ RT-PCR i en test kohorte af ubehandlede CLL patienter sammen med andre etablerede markører, og viste at høj CLLU1 ekspression er forbundet med kort overlevelse og hurtig opstart af behandling hos CLL patienter. Det viste også at CLLU1 er opreguleret i alle prognostiske ugunstige undergrupper. Det andet studie bekræftede dette fund i et stort patientmateriale, men viste også at den prognostiske værdi af CLLU1er knyttet til patienter, der får stillet diagnosen CLL før 70-års alderen. Dette studie viste også at kendskab til CLLU1 ekspression i den yngre aldersgruppe tilføjer yderligere prognostisk information til alle prognostiske undergrupper, med undtagelse af patienter, der har en ugunstig form af CLL med umuteret IgVH. Udfra fundet at den prognostiske værdi og dermed den kliniske anvendelighed af prognostiske markører i CLL synes at være aldersafhængig, blev en særlig undersøgelse af den prognostiske værdi af den allerede etablerede markør CD38 udført. Dette tredje studie viste at den prognostiske værdi CD38, i modsætning til de andre markører, er knyttet til den ældre aldersgruppe. Det fjerde studie bestod i supplerende undersøgelser vedrørende de brugte metoder, samt undersøgelse af om CLLU1 ekspressionen varierer over tid.

Den kliniske anvendelighed af biologiske markører ved risikovurdering bygger på evnen til tidligt at finde de patienter, der kan få et behov af tidlig og aggressiv behandling. Disse studier viste at kendskab til CLLU1 ekspression på diagnosetidspunktet er højst anvendelig for patienter, der aldersmæssigt er mest velegnede til at modtage aggressiv behandling. De viste også at CLLU1 ekspressionen ikke synes at ændre sig væsentligt over tid. Den prognostiske værdi af allerede etablerede biomarkører blev bekræftet, men vi fandt at CD38 bør være forbeholdt risikovurdering hos ældre CLL patienter.