Using a population-based cohort of 162 DLBCL patients diagnosed from 1998–2000 (pre-rituximab era) and followed through 2008, we show for the first time that germline genetic variation in four LMO2 SNPs (rs1885524, rs10836127, rs750781, rs941940) was associated with overall survival after accounting for clinical factors. The correlation of LMO2 SNP genotype and LMO2 IHC status was more variable, although the SNP rs941940 was significantly correlated with both expression and survival, and several other SNPs trended towards a correlation with both expression and survival. Individually, the SNPs were weak predictors of outcome (c-statistics 0.525–0.579), similar to that of LMO2 IHC (c-statistic 0.573), and weaker than the clinical risk score (c-statistic=0.676). However, when the individual SNPs were combined with the clinical risk score, the c-statistics improved. Further addition of LMO2 IHC to models with SNPs and the clinical risk score only trivially improved the c-statistics. In total, these results suggest that germline genetic variation in LM02 provides as good or slightly better prognostic information than tumor LMO2 IHC status.
Strengths of this study include the population-based ascertainment of newly diagnosed cases, which enhances generalizability. Our genotyping included extensive quality controls. The LMO2 IHC uses a relatively robust antibody, and scoring was highly reliable as assessed by two independent pathologists. Further, the prevalence of LMO2 expression and its association with survival was in the range of previous studies using this antibody [10
There are also several important limitations. We used a retrospective study design and patients were not uniformly treated as in a clinical trial. While this is an important limitation, it needs to be balanced against the limitations of a clinical trial, which are generally based upon a very select subset of generally otherwise healthy patients. We do note that the SNP associations were strongest for patients receiving chemotherapy, which in this setting would be presumably for curative intent. While we did not have data to calculate the IPI, we were able to adjust for clinical and demographic variables, and our clinical risk score predicted with the same robustness as the IPI in other datasets [13
]. We also only had IHC and genotyping data on 38% of the original cohort of patients, although we did not see systematic differences based on inclusion in this analysis. We did not include an analysis of germinal center B-cell like (GCB) phenotype in this report. However, in a prior report, we found that 61% of GCB DLBCLs (as assessed by IHC) were LMO2 IHC positive but that 26% of non-GCB cases were also LMO2 positive. Furthermore, there was no significant association of GCB-phenotype with overall survival [12
], supporting a role for LMO2 independent of GCB phenotype that has been previously reported [10
Our study was unable to enroll patients with rapidly fatal disease, consistent with enrollment of only living cases into the parent case-control study. The impact of this bias is that our observed survival is consistent with SEER estimates conditioned on surviving 12 months after diagnosis [13
]. Thus, the inferences from this study would not apply to early deaths. Nevertheless, LMO2 IHC positivity and the association with overall survival was similar to other published data [10
], suggesting that the association of LMO2 IHC expression as a prognostic marker for overall survival is not particularly impacted by this bias. The number of events was modest (N=52 deaths), and so the models need to be considered in this context, and in the context that we did not have an independent validation sample. Finally, this study was based on patients initially treated in the pre-rituximab era, and thus our findings will need to be evaluated in rituximab era patients.
Our findings that LMO2 expression as measured by IHC in DLBCL is a strong prognostic factor after considering clinical factors replicates in a population-based setting the results of Natkunam and colleagues [10
]. They found that LMO2 IHC was positive in 53% of 263 patients treated with anthracycline-based chemotherapy (pre-rituximab) and that LMO2 positive patients had significantly better progression-free (median 12 versus 49 months, p=0.010) and overall (median 21 versus 80 months, p=0.018) survival. Our results are also consistent with studies that have found higher LMO2
expression as measured by cDNA microarray [7
], and RT-PCR [8
], but not qNPA [26
], is associated with better survival in the pre-rituximab setting. While we did not have any data on R-CHOP treated patients, LMO2 IHC expression [10
] and mRNA expression as assessed by RT-PCR [27
] or qNPA [26
] have all been found to be associated with better survival in R-CHOP treated patients, although LMO2 IHC expression was not statistically significantly associated with survival (63% 5-year survival for LMO2+ and 61% 5-year survival for LMO2− patients, p=0.78) in a study of DLBCL patients age 60 and older [25
]. However, the proportion of LMO2 positive cases in this study was somewhat lower than in previous studies and only 82 cases were analyzed for LMO2 expression.
Our data also strongly suggest that common germline genetic variation in the LMO2
gene is associated with DLBCL prognosis. There were four SNPs in LMO2
that were associated with overall survival in a multivariate model, two were in the promoter (rs750781 and rs941940), one was intronic (rs10836127) but in weak LD with the promoter SNPs, and the fourth (rs1885524) was intronic and not in LD with the promoter SNPs. The latter intronic SNP was in high LD with rs1885523, which is located in a polymerase II binding site. Of these same four SNPs, only one SNP (rs941940) was significantly associated with LMO2 IHC expression, and the allele associated with expression was also associated with better survival. The only other SNP associated with LMO2 IHC expression, rs7941248, was in high LD with multiple conserved transcription factor binding sites, including GATA1, which has been shown to regulate LMO2 expression [24
]. However, rs7941248 was not significantly associated with survival (p-trend=0.33), although patients carrying one or more variant alleles (which was associated with LMO2 IHC expression) did have better survival. The lack of a strong correlation between SNPs predicting survival and SNPs predicting LMO2 IHC expression may be due to variability in LMO2 protein expression, technical issues in LMO2 IHC staining and scoring, or other mechanisms that regulate LMO2 expression (e.g., epigenetics, miRNAs). Nevertheless, taken together, our data support the hypothesis that genetic variation in LMO2
, particularly in the promoter, may play a role in both LMO2 expression and survival in DLBCL patients.
The biologic relevance of LMO2 in DLBCL is not known, but it appears to be involved in several important physiologic and pathologic processes relevant to lymphomagenesis. LMO2 is an important transcription factor and its expression is required for hematopoiesis, [1
] angiogenesis early in development [2
], and vascular endothelial remodeling in adults [2
]. Nuclear LMO2 is also widely expressed in the vasculature of native tissues, including lymphatic vasculature, and is detected in the secretory but not proliferative phase of the endometrial gland, suggesting tissue-specific regulation [29
]. From a pathologic perspective, chromosomal translocations with the T-cell receptor locus or insertional mutations have been associated with T-cell leukemias [5
], although microarray analysis has found ectopic LMO2 expression in many cases without chromosomal changes [31
]. In a transgenic mouse model of LMO2, the mice developed T-cell lymphoblastic lymphomas and associated leukemias [32
]. LMO2 is also uniformly expressed in benign vascular and lymphatic neoplasms and in most malignant vascular neoplasms [29
]. Of note, to date LMO2 expression in DLBCL has not been associated with any somatic genetic alterations [9
]. Physiologic (and aberrant) control of LMO2 expression is only now being unraveled, and early reports support a role for tissue-specific regulatory elements [34
] as well as microRNAs (specifically miR-223) during erythroid differentiation [35
Irrespective of LMO2 biologic functions, our study shows that genetic variation in LMO2 in combination with clinical factors is a robust prognostic factor for DLBCL, and that LMO2 IHC did not add additional predictive ability once these factors were considered. If replicated, future prognostic indices should consider germline genetic risk markers in LMO2 and perhaps other genes known to be prognostic in DLBCL.