Relationship of age of subjects to case/control status and BLV presence^*.

<p>Relationship of age of subjects to case/control status and BLV presence^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0179367#t001fn001" target="_blank">*</a>}.</p

Time interval between paired specimens in women who developed versus did not develop breast cancer (BCa) after the first specimen, which was benign^*.

<p>Time interval between paired specimens in women who developed versus did not develop breast cancer (BCa) after the first specimen, which was benign^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0179367#t003fn001" target="_blank">*</a>}.</p

Association of bovine leukemia virus presence in tissue with diagnosis of breast cancer (unadjusted and age-adjusted odds ratios and confidence intervals)^*.

<p>Association of bovine leukemia virus presence in tissue with diagnosis of breast cancer (unadjusted and age-adjusted odds ratios and confidence intervals)^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0179367#t002fn001" target="_blank">*</a>}.</p

Bovine leukemia virus linked to breast cancer in Australian women and identified before breast cancer development

<div><p>Bovine leukemia virus (BLV), a common virus of cattle globally, was believed for decades not to infect humans. More recent techniques (in situ PCR and DNA sequencing) enabled detection of BLV in human breast tissue, and determination of its significant association with breast cancer in a US population. Using similar techniques to study 96 Australian women, we report here detection of retrotranscribed BLV DNA in breast tissue of 40/50(80%) of women with breast cancer versus 19/46(41%) of women with no history of breast cancer, indicating an age-adjusted odds ratio and confidence interval of 4.72(1.71–13.05). These results corroborate the findings of the previous study of US women with an even higher odds ratio for the Australian population. For 48 of the subjects, paired breast tissue samples, removed 3–10 years apart in two unrelated procedures, were available. For 23/31 (74%) of these, in which the first specimen was diagnosed as nonmalignant (benign or premalignant) and the second as malignant, BLV was already present in benign breast tissue years 3–10 years before the malignancy was diagnosed. This is consistent with the supposition of a causative temporal relationship between BLV infection and subsequent development of cancer.</p></div

BLV-specific DNA sequences of the viral long terminal repeat (LTR) promoter region.

<p>Sequences from breast tissues of 3 subjects (4–09, 100–3, 104–12) are compared to the reference sequence (Ref) (GenBank accession no. EF600696) and the positive control cell line FLK. Base pair position in the reference sequence is indicated by the number following “Ref”. Dots indicate a match with the reference sequence. Variations from the reference sequence are indicated by the uppercase letters for the substituting base. A hypen (-) indicates a deletion at that position. A space indicates the sequence was not obtained beyond that point.</p

Probability of developing breast cancer in women whose breast epithelial cells contained BLV in both the first and second specimen versus women whose breast epithelial cells were BLV+ in only one of the paired specimens^*.

<p>Probability of developing breast cancer in women whose breast epithelial cells contained BLV in both the first and second specimen versus women whose breast epithelial cells were BLV+ in only one of the paired specimens^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0179367#t004fn002" target="_blank">*</a>}.</p

BLV-specific DNA in human breast tissue amplified and detected by PCR-in situ hybridization.

<p>A) Breast tissue specimen diagnosed as invasive pleomorphic lobular carcinoma. Three streaks of BLV-positive (brown staining) mammary epithelial cells are seen invading through connective tissue (fibroblasts and adipocytes) that is largely BLV-negative. Positive reactions in mammary epithelial cells are in cytoplasm (diffuse light brown reaction) of most mammary epithelial cells, and nuclei of some cells (diffuse brown reaction as well as dark dots) and as compared to B) Adjacent tissue section without the BLV-specific ISH probe in the hybridization mix. This serves as a background control against which to compare A, and also a control for reactions due to artifacts inherent in some tissues (excess peroxidase and/or melanin). No brown cells are apparent in B. C) Breast tissue specimen diagnosed as benign ductal hyperplasia (normal). Note oval nest of BLV-positive mammary epithelial cells tightly surrounded by fibroblasts that are BLV-negative. Positive reactions in the mammary epithelial cells are intense in the cytoplasm and some cells have evidence of positive nuclear reactions (dark brown dots). D) Background control for C has no apparent brown cells. Light counterstain for all specimens illustrated is Difquick blue and the magnification is x40.</p

Positive EBNA, CD 15 and LMP1 expression in the same invasive ductal carcinoma breast cancer specimen.

<p><b>Panel A.</b> Positive EBNA1 expression. <b>Panel B.</b> Positive CD15 expression. <b>Panel C.</b> Positive LMP1 expression. It is not possible to determine whether these cells are Reed Sternberg or granuloma cells.</p

Putative Reed Sternberg cells in ductal carcinoma <i>in situ</i> (by immunohistochemistry).

<p><b>Panel A.</b> Positive EBNA 1 expression in a putative Reed Sternberg cells. <b>Panel B.</b> Positive CD 15 expression in putative Reed Sternberg cells. It is not possible to determine whether these cells are Reed Sternberg or granuloma cells.</p

HPV and EBV identified by in situ PCR in the same breast cancer cell nuclei – Ductal carcinoma in situ.

<p>A.HPV (inner nested primers X 200). B. HPV (outer nested primers X 400). C. HPV (inner nested primers X 400). D. EBV (inner nested primers X 200). E. EBV (outer nested primers X 400). F. EBV (inner nested primers X 400). G. MMTV negative (inner nested primers X 200). H. Negative control (no primers X 200).</p