TI - Discussion . AB - To provide a better mechanistic understanding of SCID-X1 patient leukemias , we have analyzed five murine T-ALLs with insertional mutations at Lmo2 . In each case these insertions are clonal . Therefore , they must have occurred early in tumor induction , similar to four of the five cases of gene therapy-induced leukemias . Transcriptional profiles of these T-ALLs showed high expression of the Tal1 and Lyl1 oncogenes as well as other Lmo2 -binding partners such as Gata1 and Gata2 . TAL1 and LYL1 are class II bHLH transcription factors that are frequently overexpressed along with LMO2 in human T-ALLs [27] , [31] . Consistent with this , patient #4 , who developed leukemia in the French trial , and patient #8 , who developed leukemia in the English trial , showed rearrangement of the SIL-TAL1 loci along with insertional activation of LMO2[3] , [5] , [32] . Murine T-ALLs with Lmo2 insertions also showed consistent downregulation of genes activated by E2A and upregulation of genes repressed by E2A , consistent with previous studies suggesting that Lmo2 redirects E2A activity by binding it through its partner proteins , Tal1 or Lyl1 [6] . Genes that are activated by the Lmo2/Tal1/E2A/Gata1-containing complex in normal erythrocyte development were also overexpressed in one murine T-ALL with high Gata1 expression . These findings suggest that murine T-ALLs with Lmo2 insertions resemble human T-ALLs that are initiated by LMO2 deregulation . Our high throughput insertion site analysis identified additional disease-related genes that are likely to cooperate with Lmo2 in leukemia induction . We identified a second tumor , in addition to tumor ( 98-031 ) described previously [9] that has an Il2rg insertion . Remarkably , this tumor has two insertions at Il2rg that occurred independently suggesting strong selection for Il2rg deregulation in Lmo2-initiated T-ALL . In addition , tumor has insertions in Notch1 and Il2rg , suggesting that Il2rg might be able to cooperate with other T-cell oncogenes ( ie Notch1 ) in leukemia induction . The newly identified insertions are also close to or within the Med12 gene . Similar to Il2rg , this gene is not activated or downregulated by the insertions . We favor Il2rg as the retroviral target since the RTCGD is replete with common insertions in cytokine receptors that act in the exact same pathway as Il2rg . More recently , we have found Il2rb is a common insertion site in a retroviral insertional mutagenesis screen in Lmo2 transgenic mice ( UP Dave , unpublished observation ) . Other Il2rg binding partners , Il7ra and Jak1 , were insertionally activated in the same screen which has not reached saturation . While still somewhat controversial , two studies provide additional evidence that Il2rg can function as an oncogene under some circumstances [33] , [34] . A statistically significant enrichment of Il2rg -dependent cytokine pathways in murine T-ALLs containing Lmo2 insertions and human LMO2-overexpressing T-ALLs was also observed by Ingenuity pathway analysis ( see Figure S5 ) . A role for these pathways in T-ALL is further supported by studies showing that cytokines , which depend on Il2rg for signaling can induce T-ALL in transgenic mice , and by the murine T-ALL with two Il2rg and two Irs2 insertions . This murine T-ALL is significant since Irs2 encodes a protein adaptor that is PHOSphorylated by JAK3 tyrosine kinase in response to cytokine ligation by Il2rg and its heterodimeric partners such as Il7ra , Il4ra and Il9ra [18] , [35] . The occurrence of leukemias in the SCID-X1 gene therapy trials also lends compelling support to the notion that IL2RG is oncogenic and a cooperating "hit" with LMO2 . Il2rg expression was not upregulated in any of these three tumors but there is precedent for cancer genes being dysregulated without gross overexpression or at specific developmental stages [36] , [37] , [38] . Similarly , the IL2RG transgene was not overexpressed in patients receiving gene therapy or in those developing T-ALL [5] , [11] . We theorize that a gene is less able to be silenced if there is a nearby retroviral insertion . Enforced expression of Il2rg has been shown to cause T-cell leukemias without gross overexpression [33] , [39] . In the study by Woods et al , they specifically remark that transgene levels in thymic lymphomas were comparable to Il2rg expression levels seen in developing thymi [34] . Another compelling possibility is that Il2rg is misexpressed in a target cell population where Il2rg is not normally found such as a cell more primitive than a thymocyte precursor . The study by Shou et al presents an alternate hypothesis that the SCID-X1 background is required for leukemia development perhaps due to altered numbers of hematopoietic precursors or stem cells [33] . It may be that the lack of Il2rg creates a differentiation block that expands a cell type that is susceptible to transformation . Recurrent insertions in genes that are also CIS in the RTCGD were identified by Shou et al but Lmo2 was not one of them . The tumors were not analyzed for Lmo2 overexpression or for Notch1 activating mutations . Our data suggests that leukemia requires many "hits" and in their model somatic mutation in cooperating oncogenes or tumor suppressor genes may be more likely than insertional mutation since their vector was replication-defective . Retroviral insertion site profiles have recently been published for the French and English cohorts of SCID-X gene therapy patients ( n = 14 ) [4] , [5] , [11] , [40] . Presently , there is no statistical difference between the incidence of leukemia in the French ( 4 of 10 ) and English patients ( 1 of 10 ) and with long term follow-up , even the insertion site profiles may be quite similar in the two studies . Comparison of the insertion sites identified in the French patients with those in RTCGD showed there was a statistically significant number of insertions in these patients , which are located at CISs in murine hematopoietic tumors ( 63/554 , Fisher's exact test , p = 45x10-22 ) . Remarkably , patient #10 of the French trial showed clonal insertions in LMO2 and BMI1 ; these two insertions were observed in tumor 98-031 , underscoring the predictive power of our mouse models [11] . In designing safer vectors for transduction , it will be important to consider self-inactivating LTRs and perhaps lentiviral backbones , which have different insertion site preferences than gamma-retroviruses , which may carry a lower risk of insertional mutation [41] . With the high transduction efficiencies achieved in these gene therapy trials , one could also see clonal expansion in cases where the transduced gene is not oncogenic . This , in fact , happened recently in two chronic granulomatous disease patients treated by retroviral gene therapy [42] . In both patients , a clonal expansion of the myeloid compartment was observed that began 3 to 5 months post-transplant .