This study is the first to evaluate phenotypic and genotypic predictors of LE in a large cohort of women who had LE diagnosed using BIS rather than self-report. Based on an extensive evaluation of demographic, disease, and treatment characteristics, the factors associated with an increased risk of LE in the bivariate analyses were higher BMI, poorer functional status, having lung disease, a higher stage of disease, increased number of lymph nodes removed, increased number of positive lymph nodes, not having a SLND, having had an ALND, and receipt of CTX or RT. All of these risk factors are consistent with previous reports.
In the initial multivariate analyses, poorer functional status, having lung disease, number of positive nodes, and having had an ALND were not retained in the final model (). However, when genomic estimates of and self-reported race/ethnicity were added to the multivariate logistic regression analysis, receipt of CTX and RT were no longer significant predictors of LE group membership. This final phenotypic model explained only 17.4% of the variance in LE group membership. These findings suggest that complex interactions may exist between phenotypic characteristics and the development of LE. Future studies of LE risk need to evaluate a comprehensive list of phenotypic characteristics as well as interactions among these characteristics.
In the past few years, the complex array of molecular events that regulate the development and maintenance of lymphatic system, as well as contribute to its malfunction have begun to be elucidated in animals and humans 
. In this study, candidate genes were selected that were identified in previous animal and human studies to play a role in lymphatic morphogenesis (). While not all of the candidate genes were associated with the development of LE, the significant associations that were identified provide new information on genomic risk factors and potential therapeutic targets. Of note, each of the SNPs explained between 1.0% (VCAM1 rs3176861) to 3.1% (SYK rs158689) of the variance in the development of LE.
FOXC2 is a transcription factor that appears to be important for the normal development and maintenance of both venous and lymphatic vessels 
. In adults, FOXC2 is highly expressed in developing lymphatic vessels as well as in lymphatic valves 
. While early lymphatic development will proceed normally in the absence of FOXC2, the collecting lymphatic vessels that are formed lack valves and the lymphatic capillaries acquire an ectopic coverage of basement membrane components and smooth muscle cells 
. FOXC2 is the transcription factor associated with lymphedema-distichiasis (LD, OMIM #153400) a monogenic disorder that is characterized by late onset LE, a double row of eyelashes, and varicose veins 
. Therefore, common functional polymorphisms that result in modest alterations in function or expression of the FOXC2 transcription factor may be associated with the development of secondary LE following breast cancer treatment. The FOXC2 A03 haplotype consists of one rare and one common allele in two SNPs located in the immediate early promoter (rs34221221; rare “C” allele) and immediately downstream from the FOXC2 coding region (rs1035550, common “C” allele). SNP rs34221221 occurs in the immediate early promoter of the FOXC2 gene (−514) which is a highly conserved region of the gene. While functional studies need to be done, it is reasonable to suggest that this polymorphism alters transcription factor binding and subsequent gene expression. SNP rs103550 lies downstream from the FOXC2 coding region of the gene and is not likely to have a functional role. Functional studies of this two-SNP haplotype are warranted if the association with LE is replicated in an independent sample.
LCP2 functions in lymphatic vessel development by modulation of the hematopoetic signaling pathway that mediates the separation of the two major vascular networks (i.e., blood, lymphatic) 
. SYK acts on LCP2 as part of a central signaling pathway that regulates separation of these two vascular networks. Variation in either locus could result in their altered interaction with upstream (i.e., SYK) or downstream (i.e., LCP2) members of the signaling cascade. Mice with mutations in SYK develop arterio-venous shunts and abnormal lymphatic-venous connections 
. In addition, genetic ablation of SYK causes the accumulation of leukocytes that is associated with lymphatic proliferation and lymphatic vessel dilation which results in the formation of shunts between the blood and lymphatic vessels (F. Kiefer, personal communication cited in Tammela and Alitalo 
). While LPC2 rs35721 is located in the intronic region of the gene and has no known function, it could be in LD with a functional variation. Although SYK rs158689 is located in an intron, it is predicted to disrupt a putative CCCTC-binding factor (CTCF) termed insulator elements. These insulator elements play a vital role in the regulation of gene expression by limiting the boundary of heterochromatin and by restricting transcriptional factor access 
. Functional studies are needed to determine if the rare “T” allele of rs158689 alters the function of the putative insulator element and SYK gene expression.
Lymphatic vessels participate in inflammatory responses by promoting lymphocyte transport to draining lymph nodes 
. VCAM1 plays a role in the migration of lymphocytes into lymphoid organs. Compared to healthy controls, cytokines levels are increased in lymphatic fluid from patients with obstructive LE. These elevated levels of cytokines may be due to their local production by infiltrating immune cells 
. The elevated levels of these cytokines would be expected to contribute to the chronic inflammation that is typically observed in tissues with poor lymphatic drainage. Allelic variations in VCAM1 could influence the rate of lymphocyte homing to lymph nodes. Like other SNPs in this study, rs3176861, located in the intron of the VCAM gene, has no known function. However, it could be in LD with a functional variation.
NRP1 and NRP2 are transmembrane proteins involved in a number of physiologic processes 
. NRP2 can bind to members of the VEGF family of growth factors and influence lymphagiogenesis. NRP2 is expressed in a subset of lymphatic vessels and acts as a co-receptor for VEGFC 
. VEGFC binds to and activates VEGFR3 and VEGFR2 receptors on the lymphatic epithelium 
. In one study 
knockout mice did not exhibit defects in blood vessels. However, lymphatic development was abnormal, including abnormal patterning and marked reduction in small lymphatic vessels and capillaries. These findings suggest that NRP2 has a role in VEGFC mediated VEGFR3 signaling and lymphangiogenesis. The NRP2 haplotype consists of one rare and two common alleles in three SNPs (rs849530 “G” rare allele, rs950219 “G” common allele, rs3771052 “G” common allele) located in intron 1 of the gene. These SNPs lie in a conserved region of the NRP2 gene. However, this haplotype has no known function which suggest that this haplotype is a surrogate for an unmeasured SNP(s) in LD with the haplotype. The VEGFC haplotype consists of one rare and one common allele in two SNPs (rs3775202 “G” rare allele, rs3775195 “C” common allele) located in intron 4 of the gene. These SNPs lie in a conserved region of the VEGFC gene. However, this haplotype has no known or predicted function which suggests that it is a surrogate for an unmeasured SNP(s) in LD with this haplotype.
It is interesting to note that no associations were found in SNPs for HGF, LYVE1, MET, PROX1, RORC, VEGFB, VEGFC, and VEGFR3. One possible reason for this lack of association is that these genes are involved primarily during embryogenesis in the development of the lymphatic system and are not involved in the mechanisms that underlie the development of secondary LE 
. However, recent evidence suggests that the VEGF pathway is involved in lymphangiogenesis associated with inflammation 
. An alternative explanation for the lack of association is that the occurrence of the minor allele was too infrequent in this sample to identify significant differences between patients with and without LE. Evidence to support this hypothesis is found in , where p-values for some of these genes approached statistical significance.
Several study limitations need to be acknowledged. While the sample size was relatively large, larger samples may identify additional candidate gene associations. In terms of the genetic analyses, additional studies are needed to confirm the associations found in this study. Future studies can evaluate additional candidate genes or perform genome wide association studies to uncover novel molecular pathways. Once candidate genes are confirmed, then DNA sequencing may need to be performed to find the causal variant(s). Despite these limitations, findings from this study suggest a role for a number of lymphatic and angiogenic candidate genes in the development of secondary LE following breast cancer treatment.