Markers for Minimal Residual Disease (MRD) in B-Lineage Acute Lymphoblastic Leukemia (B-ALL): Analysis of IgH Gene Rearrangement Repertoire (AIRR) vs Conventional Strategy.
Authors: Morley Alexander , Brisco Michael , Latham Sue , Wilczek Vicki , Hughes Elisabeth , Budgen Brad , Sutton Rosemary , Bahar Anita , Malek Maria , Marshall Glenn 
Published: October 2019

Abstract
Background: Detection of molecular markers for monitoring MRD in B-ALL conventionally involves searching for a variety of immunoglobulin and T-cell receptor rearrangements. However preliminary studies have suggested that AIRR may have advantages. Aim :To directly compare AIRR to the conventional approach for detection of molecular markers.
Materials and Methods Marrow samples from a consecutive series of 50 children with B-linage ALL were studied by the conventional approach in Sydney and by AIRR in Adelaide. AIRR involves use of primers specific for individual V and J segments of the IgH gene followed by sequencing of amplified products and calculation of the relative abundance of rearrangements from the quantitative PCR data. IgH rearrangements are regarded as suitable for detecting the major leukemic clone if they comprise > 10% of the rearrangements present. Incomplete D-J rearrangements are detected using primers directed towards genomic regions upstream of the D segments.
Results Table 1 shows the number of patients with either 0, 1 or 2 rearrangements suitable for monitoring MRD of the major leukemic clone.
The 2 approaches appeared to be equivalent in terms of the number of markers for the major clone, but, of the rearrangements detected conventionally, 49% were IgH rearrangements and 51% involved IgK or various T-cell receptor genes. All of the IgH rearrangements detected in the 50 patients, grouped by abundance, are shown in Table 2.
Table 1 number of patients no marker 1 marker 2 markers conventional approach 2 9 39 AIRR 2 12 36 Table 2 relative abundance of rearrangement 100-10% 10-1% <1% detected by both analyses 65 6 6 conventional only 4 0 0 AIRR only 28 12 31
High and low abundance rearrangements mark major and minor clones repectively. AIRR detected significantly more rearrangements of both types. Clone sizes and lineage relationships could be inferred from rearrangement abundance and sequence relationships. 68 minor clones were detected at diagnosis in 31 patients. 26 of the 68 showed no sequence relationship to the major clone.
Discussion Use of IgH gene rearrangements as markers enables MRD in B-ALL to be sensitively quantified, down to 10−6. The superior ability of AIRR to identify marker IgH rearrangements is therefore likely to enable more sensitive monitoring of the major leukemic clone. Relapse in B-ALL is sometimes due a minor chemoresistant leukemic clone which is already present at diagnosis but is only identified retrospectively. The ability of AIRR to identify IgH markers for minor clones at diagnosis, together with the ability to sensitively monitor clones marked by IgH rearrangements, may enable prospective identification of these minor clones.
Conclusions
AIRR and the conventional strategy are similar in terms of the number of patients with ALL in whom 1 or 2 molecular markers for the major leukemic clone can be detected. AIRR detects significantly more IgH markers for the major leukemic clone. This should enable more sensitive quantification of the major clone in many patients. AIRR is much superior at identification of markers for minor clones. This may enable chemoresistant minor clones to be detected prospectively. The clonal hierarchies of minor clones at diagnosis resemble those observed in patients who relapse.