Lower frequency of TLR9 variant associated with protection from breast cancer among African Americans

<div><p>Introduction</p><p>Toll-like receptor 9 (TLR9) is an innate immune system DNA-receptor that regulates tumor invasion and immunity <i>in vitro</i>. Low tumor TLR9 expression has been associated with poor survival in Caucasian patients with triple negative breast cancer (TNBC). African American (AA) patients with TNBC have worse prognosis than Caucasians but whether this is due to differences in tumor biology remains controversial. We studied the prognostic significance of tumor Toll like receptor-9 (TLR9) protein expression among African American (AA) triple negative breast cancer (TNBC) patients. Germline <i>TLR9</i> variants in European Americans (EAs) and AAs were investigated, to determine their contribution to AA breast cancer risk.</p><p>Methods</p><p>TLR9 expression was studied with immunohistochemistry in archival tumors. Exome Variant Server and The Cancer Genome Atlas were used to determine the genetic variation in the general EA and AA populations, and AA breast cancer cases. Minor allele frequencies (MAFs) were compared between EAs (n = 4300), AAs (n = 2203), and/or AA breast cancer cases (n = 131).</p><p>Results</p><p>Thirty-two <i>TLR9</i> variants had a statistically significant MAF difference between general EAs and AAs. Twenty-one of them affect a CpG site. Rs352140, a variant previously associated with protection from breast cancer, is more common in EAs than AAs (p = 2.20E-16). EAs had more synonymous alleles, while AAs had more rare coding alleles. Similar analyses comparing AA breast cancer cases with AA controls did not reveal any variant class differences; however, three previously unreported <i>TLR9</i> variants were associated with late onset breast cancer. Although not statistically significant, rs352140 was observed less frequently in AA cases compared to controls. Tumor TLR9 protein expression was not associated with prognosis.</p><p>Conclusions</p><p>Tumor TLR9 expression is not associated with prognosis in AA TNBC. Significant differences were detected in <i>TLR9</i> variant MAFs between EAs and AAs. They may affect TLR9 expression and function. Rs352140, which may protect from breast cancer, is 1.6 X more common among EAs. These findings call for a detailed analysis of the contribution of TLR9 to breast cancer pathophysiology and health disparities.</p></div

Comparison of allele counts between AA cases and controls for different variant classes.

<p>Comparison of allele counts between AA cases and controls for different variant classes.</p

Comparison of allele counts between AA cases and controls for different variant classes.

<p>Comparison of allele counts between AA cases and controls for different variant classes.</p

Comparison of allele counts between ethnicities for different variant classes.

<p>Comparison of allele counts between ethnicities for different variant classes.</p

Variants with a statistically significant MAF in EAs.

<p>Variants with a statistically significant MAF in EAs.</p

Patient outcomes stratified by tumor TLR9 expression status.

<p>a) Breast cancer recurrence probability stratified by median tumor TLR9 expression status, b) survival probability stratified by ipsilateral breast cancer (IBTR) and tumor TLR9 expression status and c) breast cancer specific-survival probability stratified by tumor TLR9 expression status (n = 43).</p

Examples of immunohistochemical TLR9 stainings in tissue sections of paraffin-embedded TNBC blocks from African American women.

<p>The staining examples represent a) negative, b) low, c) intermediate and d) high TLR9 expressing TNBC tumors. Staining results in a & b represent the low TLR9-group (TLR9 score ≤ 8), whereas c & d represent the high tumor TLR9-group (TLR9 score > 8).</p

Variants with a statistically significant MAF in AAs.

<p>Variants with a statistically significant MAF in AAs.</p

TLR9 germline coding variants detected in 131 AA BC-affected individuals in the TCGA.

<p>TLR9 germline coding variants detected in 131 AA BC-affected individuals in the TCGA.</p