Cancer Letters

Cancer Letters

Volume 408, 1 November 2017, Pages 92-101
Cancer Letters

Original Article
Differential expression of MUC4, GPR110 and IL2RA defines two groups of CRLF2-rearranged acute lymphoblastic leukemia patients with distinct secondary lesions

https://doi.org/10.1016/j.canlet.2017.08.034Get rights and content

Highlights

  • CRLF2-r cases associated with a Ph-like signature are enriched for activating JAK2 and CRLF2 mutations.

  • These CRLF2-r cases can be distinguished by increased expression of MUC4, GPR110 and IL2RA.

  • Non-Ph-like CRLF2-r leukemias may harbor alternate secondary lesions including RAS pathway mutations.

Abstract

CRLF2-rearrangements (CRLF2-r) occur frequently in Ph-like B-ALL, a high-risk ALL sub-type characterized by a signaling profile similar to Ph + ALL, however accumulating evidence indicates genetic heterogeneity within CRLF2-r ALL.

We performed thorough genomic characterization of 35 CRLF2-r cases (P2RY8-CRLF2 n = 18; IGH-CRLF2 n = 17). Activating JAK2 mutations were present in 34% of patients, and a CRLF2-F232C mutation was identified in an additional 17%. IKZF1 deletions were detected in 63% of cases. The majority of patients (26/35) classified as Ph-like, and these were characterized by significantly higher levels of MUC4, GPR110 and IL2RA/CD25. In addition, Ph-like CRLF2-r samples were significantly enriched for IKZF1 deletions, JAK2/CRLF2 mutations and increased expression of JAK/STAT target genes (CISH, SOCS1), suggesting that mutation-driven CRLF2/JAK2 activation is more frequent in this sub-group. Less is known about the genomics of CRLF2-r cases lacking JAK2-pathway mutations, but KRAS/NRAS mutations were identified in 4/9 non-Ph-like samples. This work highlights the heterogeneity of secondary lesions which may arise and influence intracellular-pathway activation in CRLF2-r patients, and importantly presents distinct therapeutic targets within a group of patients harboring identical primary translocations, for whom efficient directed therapies are currently lacking.

Introduction

Acute lymphoblastic leukemia (ALL) is the most common malignancy in children. Due to improved chemotherapeutic regimens over the past five decades, childhood ALL now has long-term survival rates that approach 85% in developed countries; however, disease relapse remains one of the leading causes of cancer-related death in children and young adults [1], [2]. Furthermore, the prognosis is significantly worse in older patients (>39 years) with 5-year survival rates of 40–45%, in part due to increased incidence of high-risk genomic lesions [3], [4].

In recent years, genomic studies and next-generation sequencing have provided insight into the complexity and constellations of structural rearrangements and submicroscopic lesions present in ALL, including alterations resulting in deregulated expression of cytokine receptor-like factor 2 (CRLF2). Increased expression of this receptor is observed in 5–15% of pediatric and adult B-cell precursor (BCP)-ALL, and up to 60% of Down Syndrome associated (DS)-ALL [5], [6], [7], [8]. CRLF2 genomic aberrations commonly involve a focal upstream deletion (pseudo-autosomal region1; PAR1) that juxtaposes CRLF2 to the promoter of the G-protein coupled purinergic receptor P2RY8 gene (P2RY8-CRLF2), or the translocation of CRLF2 to the immunoglobulin heavy chain locus (IGH-CRLF2), bringing CRLF2 under the control of IGH enhancer elements. Both cases result in the overexpression of CRLF2 by linking its full-length coding region to alternate transcriptional regulators.

Normally, CRLF2 functions as a heterodimeric receptor with the interleukin-7 receptor alpha subunit (IL-7Rα) and binding of its ligand, thymic stromal lymphopoietin (TSLP), elicits key signaling responses important in normal lymphopoiesis, allergy and inflammation [9], [10]. CRLF2 overexpression however, requires cooperating lesions for leukemic transformation, and approximately 50% of CRLF2-rearranged (CRLF2-r) cases harbor activating mutations in JAK2 [5], [6], [8], [11]. The JAK2 mutations in BCP-ALL are predominantly missense mutations that cluster in exon 16 within the pseudokinase domain. These lesions are distinct from the JAK2 V617F mutations associated with myeloproliferative neoplasms, and the coexpression of CRLF2 with the JAK2 mutations observed in ALL induces factor-independent transformation of cell lines in vitro [6], [7], [12]. Interestingly, the CRLF2 rearranged cases that lack JAK2 mutations commonly harbor CRLF2 or IL7R mutations that promote constitutive receptor dimerization and downstream JAK/STAT signaling [13], [14].

To date, the prognostic significance of CRLF2 rearrangements has varied between cohorts; while the predictive effect of CRLF2 overexpression on prognosis is only moderate in standard risk (SR) and DS-associated ALL, there is a clear association of CRLF2-r with increased incidence of relapse and poor outcome in adult and high-risk (HR) pediatric cohorts of BCP-ALL [6], [11], [15], [16], [17]. In addition, recent reports demonstrate that CRLF2-r are enriched in Ph-like ALL [13], [18], a high-risk sub-type of BCP-ALL characterized by a gene expression profile analogous to that of Ph + ALL, in the absence of the BCR-ABL1 fusion. Deletions (Δ) and alterations of the lymphoid transcription factor gene IKZF1 (encoding IKAROS) which are hallmarks of both BCR–ABL1 + ALL and Ph-like ALL and associated with poor outcomes, also co-occur with CRLF2-r in HR cohorts.

In this study, we have used a combination of genomic approaches to investigate the heterogeneity present in CRLF2-r ALL. We have developed a rapid system to identify CRLF2-r samples flagged by high expression of CRLF2 via flow cytometry or Taqman Low Density Arrays (TLDA), and a customized 9-gene signature for determination of Ph-like status. We demonstrate that Ph-like CRLF2-r ALL cases compose a distinct sub-group enriched for activating JAK2/CRLF2 mutations, and high expression of IL2Rα/CD25, likely reflecting key underlying differences in disease pathogenesis. Our data suggests that independent dissection of Ph-like versus non-Ph-like CRLF2-r cases may be critical to elucidate the prognostic significance and targetable pathways of CRLF2-r patients that have been conventionally grouped in one class.

Section snippets

Patient samples

Six hundred and thirty BCP-ALL cryopreserved or fresh samples from patients aged between 0.5 and 75 years were screened for CRLF2 rearrangements as described below. Informed consent for tissue banking and research studies was obtained from patients and/or their guardians according to the Declaration of Helsinki.

Cell culture

The human B-precursor ALL cell lines MHH-CALL-4 and MUTZ-5 were purchased from DSMZ. MHH-CALL-4 have an IGH-CRLF2 rearrangement and a JAK2 I682F mutation and MUTZ-5 have an IGH-CRLF2

Identification and characterization of B-ALL patients with CRLF2 rearrangements

We screened for CRLF2 rearrangements in a cohort of 630 BCP-ALL samples, of patients aged between 0.5 and 75 years. Samples with high expression of CRLF2 were initially identified by TLDA and flow cytometry, and analyzed for the presence of P2RY8-CRLF2 and IGH-CRLF2 rearrangements by RT-PCR and FISH respectively. We identified 35 CRLF2-r patients (P2RY8-CRLF2 n = 18 and IGH-CRLF2 n = 17, Table 1 and Supplementary Table 2), including 5 with matched diagnosis and relapse samples. Consistent with

Discussion

Following the initial description of recurrent CRLF2 abnormalities in BCP-ALL in 2009, a range of studies have explored the functional and clinical significance of these rearrangements, and there is now ample evidence to suggest that high CRLF2 expression delineates a group of adult and HR pediatric ALL cases with a high rate of relapse and poor prognosis [11], [16], [17]. In addition, overexpression of CRLF2 is observed in approximately 50% of Ph-like ALL cases, a now well recognized high-risk

Authorship

T.S. and D.L.W. critically analyzed the data and wrote the manuscript. T.S. and S.L.H performed experiments and analyzed patient data. P.D., E.N., B.J.M., K.M.G., W.M., and N.C.V. provided experimental assistance. R.S. provided IKZF1 analysis and interpretation. C.H.K. performed bioinformatics analysis. S.M. performed FISH experiments. M.O., T.R., and A.S.M provided patient material and clinical input. T.P.H. and D.Y. were involved in data interpretation and critical review of the manuscript.

Acknowledgments

The authors acknowledge funding from the National Health and Medical Research Council, Australia (APP1057746, APP1044884); The Cancer Council of South Australia and Leukaemia Foundation, Australia.

References (41)

  • R.C. Harvey et al.

    Development and validation of a highly sensitive and specific gene expression classifier to prospectively screen and identify B-precursor acute lymphoblastic leukemia (ALL) patients with a Philadelphia chromosome-like (“Ph-like” or “BCR-ABL1-Like”) signature for therapeutic targeting and clinical intervention

    Blood

    (2013)
  • T. Akasaka et al.

    Five members of the CEBP transcription factor family are targeted by recurrent IGH translocations in B-cell precursor acute lymphoblastic leukemia (BCP-ALL)

    Blood

    (2007 Apr 15)
  • J. Zhang et al.

    Key pathways are frequently mutated in high-risk childhood acute lymphoblastic leukemia: a report from the Children's Oncology Group

    Blood

    (2011 Sep 15)
  • N. Jain et al.

    Ph-like acute lymphoblastic leukemia: a high-risk subtype in adults

    Blood

    (2017 Feb 02)
  • R.C. Harvey et al.

    Identification of novel cluster groups in pediatric high-risk B-precursor acute lymphoblastic leukemia with gene expression profiling: correlation with genome-wide DNA copy number alterations, clinical characteristics, and outcome

    Blood

    (2010 Dec 2)
  • M.E. Van Meter et al.

    K-RasG12D expression induces hyperproliferation and aberrant signaling in primary hematopoietic stem/progenitor cells

    Blood

    (2007 May 1)
  • C.H. Pui et al.

    Childhood acute lymphoblastic leukemia: progress through collaboration

    J. Clin. Oncol. Off. J. Am. Soc. Clin. Oncol.

    (2015 Sep 20)
  • A. Ronson et al.

    Treatment of relapsed/refractory acute lymphoblastic leukemia in adults

    Curr. Oncol. Rep.

    (2016 Jun)
  • O. Al Ustwani et al.

    Clinical updates in adult acute lymphoblastic leukemia

    Crit. Rev. Oncol. Hematol.

    (2016 Mar)
  • C.G. Mullighan et al.

    Rearrangement of CRLF2 in B-progenitor- and down syndrome-associated acute lymphoblastic leukemia

    Nat. Genet.

    (2009 Nov)
  • Cited by (20)

    • Molecular classification improves risk assessment in adult BCR-ABL1–negative B-ALL

      2021, Blood
      Citation Excerpt :

      We describe, for the first time, CD25+ CD10−/dim pro-B ZNF384-R/-like blasts. Although overexpression of CD25 and its gene, interleukin-2 receptor α, are negative prognostic factors in BCR-ABL1 ALL and BCR-ABL1− ALL,51-53 CD25 did not affect outcome in patients who were ZNF384-R/-like (not shown). This supports the notion that antigen profiles affect outcome only when analyzed in conjunction with their underlying molecular counterparts.

    • The application of RNA sequencing for the diagnosis and genomic classification of pediatric acute lymphoblastic leukemia

      2020, Blood Advances
      Citation Excerpt :

      Larger cohorts and a clearer picture of the relationship between other IKZF1 deletions and the transcripts they generate would allow the detection of the full range of IKZF1 deletions. The B-ALL gene expression classifier, although not dissimilar in principal to other published techniques that detect Ph+ or Ph-like ALL,41 uses RNA-seq to measure gene abundance and can also call the probability of other B-ALL subtypes. With this tool, 2 ERG-deleted/DUX4-rearranged cases were identified that had not been picked up by standard-of-care testing or fusion calling.24,42

    • Mutation accumulation in cancer genes relates to nonoptimal outcome in chronic myeloid leukemia

      2020, Blood Advances
      Citation Excerpt :

      Transcriptional changes taking place at CML were identified by means of differential gene expression analysis, accounting for confounding factors. These analyses revealed deregulation of genes reported to be altered in previous CML investigations, such as RXFP1,43 PIEZO2,55 and CD26,56 and in leukemia studies, such as CD69,57 ST18,58 and MUC4.59 Expression analysis also suggested that DNA damage could be related to the downregulation of DNA-repair machinery genes and that dysregulation events in genes, including DNMT1,60 SEPP1,61 NEIL1,62 and WT1,63,64 may have contributed to the high occurrence of DNA damage-associated variants in our cohort.

    • Precision medicine approaches may be the future for CRLF2 rearranged Down Syndrome Acute Lymphoblastic Leukaemia patients

      2018, Cancer Letters
      Citation Excerpt :

      Usp9x normally functions to stabilise phosphorylated Jak2 to confer active signalling, however, it is possible that activating JAK mutations are compensating for the loss of USP9X in these cases [32]. Non-DS-ALL frequently harbour polysomy 21 indicating that the gene dosage of chromosome 21 may be associated with the development of ALL [15,21,24,33]. A subset of patients with a gene signature similar to that of BCR-ABL1 have been termed Philadelphia like (Ph-like).

    View all citing articles on Scopus
    View full text