Insertional mutagenesis combined with acquired somatic mutations causes leukemogenesis following gene therapy of SCID-X1 patients

Abstract

X-linked SCID (SCID-X1) is amenable to correction by gene therapy using conventional gammaretroviral vectors. Here, we describe the occurrence of clonal T cell acute lymphoblastic leukemia (T-ALL) promoted by insertional mutagenesis in a completed gene therapy trial of 10 SCID-X1 patients. Integration of the vector in an antisense orientation 35 kb upstream of the protooncogene LIM domain only 2 (LMO2) caused overexpression of LMO2 in the leukemic clone. However, leukemogenesis was likely precipitated by the acquisition of other genetic abnormalities unrelated to vector insertion, including a gain-of-function mutation in NOTCH1, deletion of the tumor suppressor gene locus cyclin-dependent kinase 2A (CDKN2A), and translocation of the TCR-beta region to the STIL-TAL1 locus. These findings highlight a general toxicity of endogenous gammaretroviral enhancer elements and also identify a combinatorial process during leukemic evolution that will be important for risk stratification and for future protocol design.

Figures

Figure 1. Normal γc surface expression and a lack of constitutive signaling through the IL receptor complex.

(A) Representation of T cell recovery following gene therapy in 10 patients with SCID-X1. The immunological reconstitution of patient 8 (P8) to normal levels is indicated in red up to 3 weeks prior to diagnosis of leukemia. (B) Expression of γc on the surface of leukemic blasts measured by flow cytometry was within the normal range of control peripheral blood T cells. (C) Phosphorylation of the tyrosine residue of STAT5 is indicative of signaling through the JAK-STAT pathway when relevant ILs bind to cell-surface receptor complexes containing γc. Insert shows the presence of phosphorylated STAT5 by flow cytometry in response to IL-7 and IL-15 from the patient and PBMCs from a control. The response to different cytokines and different concentrations of IL-7 is shown in the main panel. Constitutive phosphorylation of STAT5 is not detectable and is also not induced by IL-2 or IL-15. Unstim, unstimulated. (D) Spectratype analysis revealed a dominant TCR Vβ6b clone in both CD4+ and CD8+ cells (d717, at time of leukemia diagnosis), which disappeared following chemotherapy (d735), allowing the emergence of a normal distribution of clones (see also Supplemental Figure 1).

Figure 3. SNP array analysis shows LOH.

A genome-wide SNP array was used to examine gross deletions or rearrangements within the patient’s chromosomes. Signal from the array showing homozygosity (AA/BB) and heterozygosity (AB) of alleles on each chromosome are shown by blue or red dots, respectively. Strikingly, analysis of chromosome 9 revealed a LOH of a large region of the short arm in leukemia cells (top panel) when compared with DNA from pre–gene therapy cells (middle panel). LOH as identified by a hidden Markov model (HMM) is represented graphically in purple to show the area altered between constitutive and leukemic DNA. The tumor suppressor gene CDKN2A, which encodes p14(ARF1) and p16(INK4a), is located at 9p21, shown in the lower panel by an arrow. Alterations within this genomic locus are probably the cause for downregulation of CDKN2A expression (as described in Figure 2A).

Figure 4. FISH analysis reveals a chromosomal rearrangement.

Metaphase cells showing a rearrangement of the TCR-β locus by FISH. (A) Normal copy of chromosome 7 with intact TCR-β (colocalization of a red-green probe) (s). Rearrangement partner chromosome 1 (blue) showing the translocated portion of the probe containing the remainder of the 732-kb region upstream of the TCR-β breakpoint cluster (t). Derivative chromosome 7 with intact portion of the probe (red signal) covering 320 kb downstream of the TCR-β breakpoint cluster region with a residual signal from the probe (green signal) covering the proximal region 732 kb upstream from the TCR-β breakpoint cluster region (u). The insertion was located in the STIL-TAL1 region (1p32–1p36). Additional probes show (B) the red/green signal of the normal TCR-β region probe with the inserted fragment (v, green only), (C) the position of chromosome 1 using the 1p36 (false-colored orange) and 1q25 probe sets (false-colored blue), and (D) an overlay of the 2 previous pictures, locating the insertion proximal to 1p36. (E) Red/green signals of the normal (w) and abnormal (x) TCR-β region probes and normal-sized STIL-TAL1 (y) and large STIL-TAL probe signal apparently encompassing the inserted green TCR-β region probe signal (z). Original magnification, ×100.

Figure 2. Gene expression levels and retroviral insertion site.

(A) Microarray analysis was used to compare levels of gene expression in the patient’s cells to those found in thymocytes or a panel of leukemias. DP1 and DP2 represent arrays performed on 2 different developmental stages of normal human CD4+CD8+ thymocytes. The y axis shows the fold change in expression, with values above 1 representing an increase and those below 1 a decrease. When compared with thymocytes and other leukemias, LMO2, NOTCH1, HES1, STIL, TAL1, and CMPK gene expression is upregulated. In contrast, expression levels of tumor suppressor genes p14(ARF1) and p16(INK4a) were reduced. γc mRNA was not overexpressed relative to thymocytes (consistent with surface expression data), although it was in comparison with the heterogeneous group of leukemias. (B) The vector insertion site is 35087 bp upstream of the LMO2 transcription start site in the opposite orientation (red X). LAM-PCR analysis (inset) of 10 ng and 1 ng of d717 PBMC DNA identified 1 dominant clone. M, 100-bp ladder; –C, 100 ng nontransduced DNA. When using limiting amounts of DNA, the internal control is out-competed by the LMO2 amplicon. The genomic locus and expression levels of genes surrounding LMO2 are shown in comparison with a dataset of arrays performed on other childhood leukemias or DP1 and DP2 human thymocytes. (C) Sequencing of denaturing HPLC analysis revealed a R1599P substitution in the HD-N domain of NOTCH1 in the leukemic cells (d717) but not before. No other NOTCH1 mutations were found.