Capa I

In addition to the recognised clinical and biological factors associated with prognosis, there are several additional features that define individual patient’s outcome. The pharmacogenomic and pharmacokinetics factors, the treatment intensity, scheduling and compliance and other yet unrecognised factors may play a complex interactive role in individual patients – thus limiting the sensitivity of predicting the outcome of the individual patient. As a result, response to leukaemic therapy has emerged as an independent prognostic factor which includes all of the above characteristics.

Modern treatment achieves clinical and morphological remission in most patients. However, with complete clinical remission (CCR) (<5% blasts in BM), the disease burden may not be completely eradicated and as many as 1010 cells may theoretically persist in the marrow (Pui and Campana, 2000). The morphological examination of the bone marrow is not sensitive for monitoring residual disease, as only 1-5 blasts/100 cells can be identified. Furthermore, morphological examination of the bone marrow is difficult to interpret and presence of increased numbers of blasts may merely represent the regenerating bone marrow – therefore, this method is not specific. In addition, the morphologic examination is subjective to the observer’s interpretation; therefore standardization within a laboratory and between laboratories is difficult. Historically, all patients in CCR received the same consolidation treatment irrespective of the extent of submicroscopic disease burden. Some patients in CCR may not have required any treatment at all while others should have received enhanced treatment because of the burden of submicroscopic disease (Figure 1-4). The international Berlin-Frankfurt-Munster (BFM) group has long used 1-week single agent steroid response as a measure for monitoring treatment response (Schrappe et al., 2000). However, more recently, focus has shifted to using more sensitive, specific and reproducible methods for the detection of minimal residual disease (MRD).

Figure 1-4 Principle of MRD monitoring

Prognosis of individual leukaemia is affected by the host characteristics (e.g. age, pharmacogenomics), leukaemia characteristics (e.g. WCC, cytogenetics) and treatment factors (dose intensity, duration). Submicroscopic residual leukaemic burden can exist wtih morphological remission which is responsible for treatment failure or relapse. (Adapted from Campana (2003)).

1.3.5.1 MRD determination by flow cytometry

Lymphocyte development begins in the bone marrow with pluripotent haematopoietic stem cells (HSC). Upon appropriate stimuli, the HSCs differentiate into lymphoid and subsequently B- and T- lymphoid lineage. A number of surface and intracellular proteins (e.g. cluster of differentiation or CD markers) are expressed during specific stages of development. As discussed in section 1.3.2, leukaemic cells tend to mimic the normal stages of B- and T cell differentiation (Figure 1-5). In addition, leukaemic cells express proteins not seen in any of the normal lymphocytes in the same stage of development and may lack some antigens expressed by their normal counterparts. As a result, individual patient’s leukaemic cells can be discriminated from normal developing lymphocytes based on aberrant expression of such markers. This immunophenotypic signature, called Leukaemia associated immunophenotype (LAIP) can be used to monitor residual leukaemic cells over time.

Flow cytometry based MRD detection (flow-MRD) has the ability to detect 1 leukemic cell in a background of 10,000 normal haematopoietic cells (0.01%) (Coustan-Smith et al., 1998, Weir et al., 1999) and can be applied in more than 95% cases of childhood ALL (Weir et al., 1999, Dworzak et al., 2002). However, flow cytometry requires fresh material for analysis and expertise in interpretation of results. In addition, leukaemic relapse may be

Patient factors Disease factors Host factors

Response to therapy Morphology MRD Symptoms Clinical remission Treatment Cure D is ea se b u rd en 100% 5% 1% 0.1% 0.01% 0.001% 0.00%

accompanied by emergence of a different LAIP thus making accurate monitoring difficult. The Children’s oncology group (COG) routinely uses flow cytometry for MRD monitoring (Chen et al., 2012). In one study on 2143 of BCP-ALL patients, MRD at day 29 (end of induction) was able to effectively predict outcome. Importantly, in multivariate anaylsis, MRD at day 29 was the strongest risk factor for event free survival (EFS) while a conventional risk factor ETV6:RUNX1 translocation was not associated with a significant difference in outcome (Borowitz et al., 2008). Efforts at standardization of flow-MRD in the UK in multicentre setting have shown promising results (Irving et al., 2009).

Figure 1-5 Simplified model of (a) B- cell development stages and corresponding BCP-ALL types and (b) V(D)J and VJ rearrangement

Schematic representation of (a) B- cell differentiation stages and expression of relevant CD markers with leukaemic counterparts above. (b)(i) V(D)J rearrangement process with D-J rearrangement followed by a V- DJ rearrangement I (Ig heavy chain (IGH), TCR beta (TCRB), & TCR delta (TCRD) genes (ii) direct V-J rearrangements in case of Ig kappa (IGK), Ig lambda (IGL), TCR alpha (TCRA), and TCR gamma (TCRG) genes. Rearrangement is initiated following expression of recombination activation gene (RAG1 and 2). HSC (Haematopoietic stem cell), LSC (Lymphoid stem cell, CD (Cluster of differentiation) Adapted from (Cobaleda and Sanchez-Garcia, 2009, Bene and Kaeda, 2009).

1.3.5.2 MRD determination by polymerase chain reaction

The main function of normal B- and T cells is to maintain the immune defences by identifying and neutralizing foreign antigens. The ability of lymphocytes to identify potentially unlimited number of antigens is based on the repertoire of antigen receptor molecules expressed by the lymphocytes. This diversity to identify millions of antigens is created by Ig and T-cell receptor (TCR) gene rearrangements. This involves random recombination of variable (V), diversity (D) and junctional (J) region genes (Tonegawa,

1983). In addition, insertion of non-templated (N-) nucleotides provides additional diversity to the Ig and TCR molecules. Consequently, each lymphoid precursor and its progeny differ from the others. This unique identity – a DNA finger-print – forms the basis for monitoring leukaemic cells in the background on normal regenerating bone marrow. The principle underlying real-time quantitative PCR (qPCR) monitoring of MRD is the specific amplification of leukaemic DNA sequences in the background of predominantly normal DNA from the bone marrow. Using DNA from diagnostic bone marrow specimen as a control to generate standard curve, accurate quantification of minimal levels of residual leukaemia is possible. Since the leukaemic cell population carry identical Ig/TCR gene rearrangement, diagnostic bone marrow DNA is used to identify and sequence the junctional regions of the rearranged genes. These sequences are used to design allele specific oligonucleotide (ASO) primers. A laborious standardization phase involves testing for sensitivity. Comprehensive guidelines for optimum DNA extraction, testing for sensitivity and specificity, and data interpretation have been published (van der Velden et al., 2007, Flohr et al., 2008, van der Velden et al., 2003) . In principal, qPCR is capable of detecting a single copy of leukemic DNA. However, practically, a sensitivity of 1 x 10 -5 (1 leukaemic cell/ 100,000 cells) can be achieved in nearly 95% patients with this method. Currently, this method is widely used in many paediatric ALL protocols (van der Velden et al., 2003, Vora et al., 2013, Zhou et al., 2007).

Levels of qPCR MRD after induction therapy are associated with relapse and poor prognosis. In an earlier study, patients with 10-3 residual leukaemic cells at any time point (after induction, consolidation or delayed intensification) were at a very high risk of relapse. MRD levels were the most important prognostic factors followed by immunophenotype and WCC (Cave et al., 1998). Since then, molecular monitoring of MRD is applied in several major treatment protocols with strong association of MRD positivity and relapse, treatment failure and other poor outcome variables (Zhou et al., 2007, van Dongen et al., 1998, Flohr et al., 2008, van der Velden et al., 2003, Vora et al., 2013). Based on MRD results, treatment reduction for some low risk ALL types has shown promise. Treatment reduction of MRD negative low risk children and young adults in the MRC UKALL 2003 trial showed that there was no significant difference in EFS in any of the randomized groups (Vora et al., 2013).

In addition to MRD monitoring for rearranged Ig/TCR genes, qPCR can also be used to identify the fusion genes transcript levels in ALL with recurrent cytogenetic abnormalities (e.g. BCR-ABL1). This method carries the advantage of detection of the genetic mutation

associated with the leukaemia and the potential detection of pre-leukemic clone, irrespective of the clonal evolution and clonal heterogeneity. In addition, this method carries the advantage of a fixed set of standardized primers for the fusion gene transcripts (Gabert et al., 2003). Global use of this technique has been limited due to: a small proportion of suitable patients with fusion genes (40%), issues with RNA stability, the levels of fusion gene transcripts produced /cell may vary even for the same fusion gene type and finally, due to the potential variation in the levels of the house-keeping gene to control for sample quantities (van Dongen et al., 1999). Therefore, currently, this method is in use only for BCR-ABL1 ALL (Jeha et al., 2014).

Despite being a strong indicator of treatment response and prognosis, a negative MRD does not always predict outcome and approximately 5-12% patients with a negative MRD at the end of induction still relapse (van Dongen et al., 1998, Cave et al., 1998, Zhou et al., 2007, Flohr et al., 2008). In addition, MRD monitoring only reliably demonstrates the levels of leukaemia in the bone marrow. Theoretically, leukaemic cells in the so-called sanctuary sites (such as the testes and the CNS) can be the cause of relapses in patients who clear bone marrow MRD. In addition, there is not enough evidence to show whether MRD levels can be used as a surrogate marker while testing new therapies. For instance, in the recent UK ALLR3 trial, significantly improved EFS was observed in children treated with Mitoxantrone compared to Idarubicin despite similar levels of post-induction MRD in the two groups (Parker et al., 2010).