Minimal residual disease in acute lymphoblastic leukemia

Hélène Cavé
Laboratoire de Biochimie Génétique,
Hôpital Robert Debré, 48 Boulevard Sérurier, 75019 Paris, France




Minimal residual disease




When acute lymphoblastic leukemia is diagnosed in a patient, the total number
of leukemia cells is approximated to 1012 to 1013. A majority
of patients reach complete remission (CR) after about 4 weeks of chemotherapy.
Complete remission does not mean that leukemia cells are totally eradicated from
the body but that their level is beyond the sensitivity level of classical cytomorphologic
methods (e.g. 1 to 5%). At this time, up to 1010 malignant cells can
still remain in the patient. They represent the minimal residual disease (MRD).
Techniques which are more sensitive than cytomorphology are now available that
permit to detect these residual blasts (reviewed in (Campana and Pui, 1995; Foroni
et al., 1999)).

Detection of residual cells allows a longer follow-up of the tumor burden
during chemotherapy and thus, permits to better appreciate the sensitivity of
leukemia cells to treatment. It is now established that the level of MRD
represents a powerful prognostic factor. Besides, the detection of an increase
of the MRD level enables to anticipate impending relapse. 


Techniques for the follow-up of the residual disease




Techniques aimed at studying MRD rely on the detection of a leukemia
cell specific marker which enable to distinguish blasts from normal marrow
cells. Such markers have to be detected with high sensitivity, to be present in
all leukemia cells and to be stable during disease evolution. Two kinds of
markers are currently used: genetic markers, which can be detected by PCR, and
immunophenotypic markers, which can be detected by flow cytometry. 

PCR-bases techniques




PCR based strategies can be directed to 2 types of genetic targets: breakpoints
of leukemia-related chromosome aberrations, and antigen-receptor gene
rearrangements. The extreme sensitivity of polymerase chain reaction (PCR)
makes possible the consistent detection of 1 leukemia cell among 105 normal
cells (10-5).

Chromosome aberrations


Recurrent chromosome translocations are found in 30 to 40% of ALL (figure 1) (Pui.
and Evans, 1998). The molecular counterpart of these translocations can be detected
by PCR using primers located on each side of the breakpoint (Pui. and Evans, 1998).
DNA amplification can only be used for chromosome aberrations in which breakpoints
cluster in a small area, such as Tal-1 deletions or Ig-cMyc fusions. In most cases,
breakpoints spread over large intronic regions, but translocations give rise to
fusion transcripts suitable for PCR amplification after a reverse transcription
step (RT-PCR). All fusion transcripts can be used as markers for MRD follow-up.
Their detection in remission samples is achieved, as for diagnosis, by RT-PCR.
The detection sensitivity depends upon both the amount of RNA studied and the
level of expression of the transcript. When 1-2 ug of RNA is studied, fusion transcripts
associated to ALL permit to detect residual blasts with a sensitivity of 10-4
to 10-6. 

The main advantage of such markers is to be directly involved in
leukemogenesis. Accordingly, their presence is constant all over disease
evolution. However, variations in their expression level during the disease and
particularly during chemotherapy cannot be excluded. This makes difficult to
correlate the level of detection of a transcript to the amount of leukemia
cells.

Given the frequency of each translocation, each fusion transcript permits to
study only a subset of patients (figure 1). Another inconvenient is that,
because they are not patient specific, these PCR targets are much more prone to
false positive results due to carry-over than antigen-receptor genes.






Figure 1: Frequencies of the main fusion genes which can be used as PCR
targets for MRD monitoring in ALL



Antigen-receptor gene rearrangements


The use of antigen-receptor gene rearrangements for MRD detection has been
developed in order to overcome the lack of recurrent chromosomal abnormalities
in most of the patients with lymphoid malignancies. In ALL, T-cell receptor
(TcR) and immunoglobulin (Ig) loci undergo somatic rearrangement by V(D)J
recombination without strict lineage specificity. Provided the extreme
diversity created by V(D)J rearrangements, each malignant clone will present a
specific configuration and the sequence of the junctional region(s) (N-region)
is highly clone-specific.  For technical convenience, rearrangements studied
for MRD follow up are those of TcRgamma-delta, Ig heavy chain (IgH) and Igkappa. The frequency
of these rearrangements in ALL is indicated in table 2. A combined study of
these 4 loci permits to identify one or more rearrangement in virtually all
cases of ALL.


Table 1: Frequency of TcR and Ig recombination in childhood ALL (For some
rearrangements, frequencies are slightly different in adult ALL)




-B-lineage ALLT-lineage ALL
IgH >90% 10-15%
Igkappa* 50%0% 
TcRdelta**55%50%
TcRgamma55%90% 
 *Igkappa deletional rearrangements (Kde)

  **The more frequent TcRdelta rearrangements are Vdelta2-Ddelta3 and Ddelta2-Ddelta3
  in B-lineage ALL and Vdelta2-Ddelta3, Vdelta1-J1delta and Ddelta2-Jdelta1 in
  T-ALL.



  All rearrangement-based PCR techniques use the same general strategy (fig.2)
  (Hanssen-Hagge et al., 1989; Macintyre et al., 1990). The presence of rearrangements
  in leukemia blasts is searched in a marrow sample obtained at diagnosis. PCR
  reactions are conducted using different combinations of V and D or J specific
  primers. When a rearrangement is present in leukemia cells, an intense and one-sized
  PCR signal is obtained. PCR products are then sequenced to derive either an
  oligonucleotidic probe or a primer specific for the junctional sequence of each
  specific clone. Test for MRD is conducted by PCR amplification of DNA from remission
  marrow cells, with the use of primer sets corresponding to the clonal rearrangement
  identified at the time of diagnosis. PCR products are then hybridized to the
  radiolabeled clono-specific probe. Alternatively, a primer specific of the junctional
  region can be used for nested PCR. A positive signal corresponds to the presence
  of residual blasts in the remission sample. This strategy permits to reach a
  sensitivity of 10-4 to 10-5 when 1-2 ug of DNA from the
  remission sample is studied. Performing PCR replicates permits to study a higher
  amount of DNA and thus, to reach a higher sensitivity (Roberts et al., 1997).
  


 
 Figure 2: General strategy for MRD assessment using antigen receptor gene
  rearrangements as clonal markers.

  



  The major advantage of antigen-receptor gene rearrangements is that they permit
  to study MRD in almost of cases of ALL. However, the need to sequence one or
  more rearrangement(s) in each patient makes the technique time-consuming and
  costly. Rather than by their sequence, rearrangements can be characterised by
  their size, and detected by resolutive electrophoresis on an automated sequence
  analyser. This simplification makes easier routine detection of MRD but leads
  to an important loss of sensitivity. It is thus mostly suited to the follow-up
  during the early steps of treatment.

  A second advantage is that DNA is less fragile than RNA and makes easier the
  standardisation of the amount of material studied. This is important to determine
  the sensitivity of the detection in case of a negative result.

  However, the use of clonal markers has a main limitation: the stability of the
  markers during the disease course is not absolute and initial markers may be
  absent on blasts responsible for relapse. Marker instability is usually due
  to the fact that the marker identified at diagnosis was only present on a sub-clone
  of blasts, which is not responsible for the relapse. This phenomenon is termed
  " oligoclonality " (Deane and Norton, 1991). Another possibility is
  that the clonal marker is replaced by a secondary rearrangement. This is termed
  " clonal evolution " (Bird et al., 1988). Comparison of clono-specific
  markers present at diagnosis and at relapse reveals changes in rearrangement
  patterns in up to 40% of IgH rearrangements and 7-20% of TcRgamma-delta rearrangements.
  However, at least one allele remains stable in about 90% of cases (Marshall
  et al., 1995). In this respect, the use of several markers is recommended to
  prevent false negative results. Although MRD detection can be hampered by markers
  instability, it is noteworthy that the probability of changes in rearrangement
  pattern increases with time (Marshall et al., 1995). Thus, the risk of a false
  negative result is very low when MRD is studied at early stages of the treatment.

Quantitative aspects


  It is well establish now that quantitative MRD data have a higher predictive
  value than qualitative ones (Brisco et al., 1994; Cave et al., 1998; van Dongen
  et al., 1998). Moreover, sensitivity of the assay, which varies with the marker
  used and the specimen studied, has to be accurately determined in order to be
  able to interpret negative results. Quantitative techniques are thus required.
  Strategies such as limiting dilutions (Roberts et al., 1997; Brisco et al.,
  1994) or competitive PCR (Cave et al., 1994; van Rhee et al., 1995) have been
  used to make PCR-based assays quantitative. However, these techniques have been
  restricted to specialised laboratories. It is now possible to detect PCR product
  formation in " real-time " during amplification reaction and to determine
  PCR kinetics, which is related to the amount of targets in the tube. The recent
  development of semi-automated quantitative assays based on this technology (or
  " real-time PCR ") (Heid et al., 1996) encouraged many laboratories
  to develop quantitative MRD assays using either fusion transcripts, or antigen-receptor
  gene rearrangements.

Immunologicla techniques


Although leukemia cells are believed to be the malignant counterpart of normal
cells at different stages of differentiation, they often display aberrant or unusual
antigen expression. Multiparameter-flow cytometry permits to detect various combinations
of surface membrane, cytoplasmic and nuclear molecules expressed or overexpressed
by leukemia cells but not by normal bone marrow cells. Depending on the strategy
used, 60 to 80% of childhood ALL present a leukemia-specific immunophenotype (Coustan-Smith
et al., 1998; Farahat et al., 1998). Multiparameter-flow cytometry enables to
detect residual blasts with a sensitivity level of 10-4 (Coustan-Smith
et al., 1998; Farahat et al., 1998), which is slightly below the sensitivity of
molecular methods. Unlike molecular methods, flow cytometry discriminates between
viable and dead cells. The main advantages of flow cytometry is that it directly
give quantitative data and is very rapid (results can be obtained in 24h). The
stability of immunological markers over the course of the disease is yet to be
determined.



Clinical significance of minimal residual disease in ALL



Sequential follow-up of MRD in childhood ALL


The clinical significance of MRD assessment has remained controversial for a
long time mostly because the first clinical studies were conducted
retrospectively on small and heterogeneous groups of patients undergoing
different treatments. More recently, large prospective studies of serial bone
marrow specimen unequivocally demonstrated that MRD assessment give clinically
relevant insight into the effectiveness of treatment.

Most studies investigating the value of measuring MRD in bone marrow used antigen-receptor
gene rearrangements as PCR targets. The first large study was retrospective and
included 88 children enrolled in 2 therapeutic trials (Australian and New Zealand
Children's Cancer Study IV and V) (Brisco et al., 1994). The level of residual
blasts was quantified using limiting dilution analysis. More recently, results
of two multicenter European prospective studies have been reported. One studied
MRD on a semi-quantitative basis in 240 children treated according to the national
protocols of the BFM (Berlin-Frankfurt-Munchen) protocol (Cave et al., 1998).
The other studied MRD in 178 children treated according to the EORTC (European
Organisation for research and treatment of Cancer) protocol 58 881 (van Dongen
et al., 1998). In this latter study, residual blasts were quantified using a competitive
PCR assay. The largest study using immunological markers report the follow-up
of 65 patients (Coustan-Smith et al., 1998). 

Although differences were noted according to treatment intensity, conclusions
of these studies were virtually the same: 
  • Residual blasts are still detected after completion of induction therapy in
  approximately 50% of children treated for ALL. This percentage can vary according
  to the intensity of the chemotherapy regimen and MRD results have to be interpreted
  in relationship with the type of treatment (Brisco et al., 1994). 
    • 

  • MRD is closely related to the risk of relapse during all the course of
chemotherapy and can be used as a prognostic factor as soon as induction
therapy is completed. This suggests that the sensitivity to drugs administered
during induction therapy correlates closely with the sensitivity to agents
during later therapy. 
    • 

  • The risk of relapse rises steeply with the amount of residual blasts, and
  the extent of MRD predict the outcome more precisely than simple presence or
  absence. More than 70% of children with MRD levels above 10-2 at
  the end of induction therapy relapsed, as compared to less than 10% of the children
  with MRD levels below 10-4 (Cave et al., 1998; van Dongen et al.,
  1998).
    • 

  • MRD is independent from other prognostic factors such as immunophenotype,
white blood count, age, and corticosteroid response. Thus, MRD provides a
predictive factor in virtually all subgroups of patients with ALL.
    • 

  • The occurrence of individual relapse is not always predicted, especially for
  extra-medullary and off-therapy relapses. This is not due to a lack of sensitivity
  of MRD detection since a large proportion of children who are not going to relapse
  have weakly positive signals. Other factors must be involved.
  
    • 

Sequential follow-up of MRD in adult ALL


    MRD has been less extensively studied in adult than in childhood ALL and studies
    have only been conducted on few patients. As for children, the level of MRD
    at the end of induction therapy predicts clinical outcome. Comparison of MRD
    results found in children who received similar induction therapy indicates
    that adult ALL are more resistant to treatment even in the absence of a t(9 ;22)
    (Brisco et al., 1996; Foroni et al., 1997). This is consistent with the fact
    that adult patients have a worse overall outcome than children do. It is not
    clear whether the increased resistance to chemotherapy observed in adults
    is age- or leukemia related.

MRD measurement in blood samples


    The level of residual blasts in peripheral blood is proportional to that in
    bone marrow but is about ten times lower (Marshall et al., 1995; van Rhee
    et al., 1995; Brisco et al., 1997). For this reason, bone marrow is considered
    as the best tissue source to study MRD.

MRD evaluation in bone marrow transplantation


    Very few data concerns the prognostic information provided by MRD assessment
    for patients who underwent bone marrow transplantation (BMT). Recently, one
    group retrospectively studied MRD status before (Knechtli et al., 1998) and
    after (Knechtli et al., Br J Haematol, 1998) allo-BMT in a series of 64 patients.
    Their result suggests that pre-and post-transplant MRD levels correlate significantly
    with the risk of medullar relapse. Unfortunately, data were presented separately,
    which makes impossible to evaluate the individual relationship between pre-
    and post-BMT status of the patients. Moreover, transplantation with T-depleted
    unrelated donors were largely over-represented and results might not have
    a general significance. However, MRD assessment should be useful to monitor
    of emerging therapies such as donor lymphocyte infusion or immunotherapy.
    As in the case of first-line therapy, quantitative data might help to predict
    with more accuracy patient outcome after allo-BMT (Knechtli et al., Br J Haematol,
    1998).

 Conclusion

  

  Although certain presenting features are statistically correlated with the prognosis
  of ALL, the outcome remains largely unpredictable for most of the patients whatever
  the initial seriousness of the disease. The level of MRD provides a measure
  of kinetics of tumor reduction, giving insights into the in-vivo resistance
  of the overall leukemic population to the chemotherapeutic agents used. In-vivo,
  drug resistance appears partly independent from the prognostic factors at presentation
  an probably involves additional constitutional factors determining drug metabolism
  for instance. 


  Early response predicts long-term outcome. In this respect, MRD assessment complements
  other methods for early evaluation of the response to chemotherapy such as corticoresistance
  or D15 bone marrow aspirate examination. It has been shown that MRD at the end
  of induction therapy provides a prognostic factor independently of corticoresistance.
  Probably because at this time, more drugs have been administered. The benefit
  of MRD assessment over D15 bone marrow aspiration has not been evaluated. D15
  bone marrow aspiration is less costly but difficult to estimate reliably and
  reproducibly.

  New therapeutic trials already integrate MRD results to therapeutic decisions.
  The problem is now to determine what is the best to do for patients who are
  resistant to treatment. 


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Written1999-09Hélène Cavé
de Biochimie Génétique, Hôpital Robert Debré, 48 Boulevard Sérurier, 75019 Paris, France
Citation
This paper should be referenced as such : 
Cav_, H
Minimal residual disease in acute lymphoblastic leukemia
Atlas Genet Cytogenet Oncol Haematol. 1999;3(3):168-172.
Free journal version : [ pdf ]   [ DOI ]
On line version : http://AtlasGeneticsOncology.org/Deep/MinResidDisID20007.htm