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

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

Variable sequence region of Epstein-Barr viral PCR products compared to the EBV genome (B95-8 strain).

<p>EBV, EBV genome; Raji DNA used as a positive control; A1-4 sequences are based on DNA extracts from archival formalin fixed invasive ductal carcinoma (<i>idc</i>) breast cancer specimens; B1-6 sequences are based on DNA extracts from fresh frozen <i>idc</i> breast cancer specimens. M1-2 sequences are from normal breast epithelial cell (milk) DNA extracts. The alignment of sequences demonstrates the high level of nucleotide homology between the EBV genome, the Raji EBV positive control and EBV identified by standard PCR in fixed, fresh <i>idc</i> breast cancer specimens, and normal breast specimens.</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

EBV, HPV, and MMTV positive and negative according to patient and breast cancer characteristics.

<p>These data are based on DNA extracts from 50 fresh frozen invasive breast tumours. There are small variations in the numbers of specimens analysed for each virus because of inadequate outcomes of some analyses based on standard PCR. The p values based on the Chi – square test are for differences in the presence of viruses between grades of breast cancer. The p values are not included for data based on immunohistochemistry because of low numbers.</p><p>These data indicate: (i) patients with HPV, EBV and MMTV positive breast cancer are significantly younger than patients with viral negative breast cancer, (ii) there is a non-significant increase in grade of EBV and MMTV positive breast cancer, (iii) the expression of apoptopic p53 protein is not inhibited in HPV positive breast cancer as it is in HPV positive cervical cancer.</p

EBV, HPV and MMTV identified in the same ductal carcinoma <i>in situ</i> specimen by <i>in situ</i> PCR.

<p>A.EBV positive, B. HPV positive, C. MMTV positive, D. Negative control – no primer, E. Negative control -no Taq. HPV associated koilocytes are present in B.All photographs were taken with a 20X objective and have been cropped. Variations in colour detection can be seen when the <i>in-situ</i> PCR was done at a different time.</p

Typical nested PCR amplification for EBV, HPV and MMTV.

<p>A. EBV PCR for the second round of amplification of a 209 bp fragment from DNA extracted from 9 breast cancer specimens, B is the no DNA control, Raji DNA as a positive control (+) and M is a size ladder from Puc Hinf1. (12). B. HPV PCR for the second round amplification of a 148 bp fragment from 10 breast DNA extractions. RB is the reagent control,(+ ) is the positive control from Hela DNA containing HPV 18, B1 is a first round no DNA control for the PCR, B2 is a second round no DNA control. C. MMTV PRC for the second round of amplification of a 643 bp fragment from DNA extracted from 8 patient samples. RB is the reagent blank. (+) is the positive control of DNA extracted from mouse tails. B1 is the first round no DNA control, subjected to a second round of PCR. D. Typical <i>b-globin</i> PCR (single amplification) for 6 breast archival specimens showing the integrity of the DNA. + is a positive control from Hela DNA, B is the no DNA control.</p

Identification of EBV, HPV and MMTV in cancer (invasive ductal carcinomas (n = 13) and ductal carcinoma <i>in situ</i> ( n = 14)) and normal specimens by <i>in situ</i> PCR on formalin fixed specimens.

<p>Identification of EBV, HPV and MMTV in cancer (invasive ductal carcinomas (n = 13) and ductal carcinoma <i>in situ</i> ( n = 14)) and normal specimens by <i>in situ</i> PCR on formalin fixed specimens.</p

Table_1_Association of Mouse Mammary Tumor Virus With Human Breast Cancer: Histology, Immunohistochemistry and Polymerase Chain Reaction Analyses.docx

Purpose<p>The purpose of this study is to determine whether mouse mammary tumor virus (MMTV)-associated human breast cancer has the same or similar histology to MMTV-associated mouse mammary tumors. Such associations may indicate a role for MMTV in human breast cancer.</p>Methods<p>Immunohistochemical techniques (using antibodies directed against the signal peptide p14 of the envelope precursor protein of MMTV) and polymerase chain reaction (PCR) analyses were used to identify MMTV proteins and MMTV-like envelope gene sequences in a series of breast cancers from Australian women. The histological characteristics of these human breast cancer specimens were compared with MMTV positive mouse mammary tumors. The same methods were used to study benign breast tissues which 1–11 years later developed into breast cancer.</p>Results<p>MMTV p14 proteins were identified in 27 (54%) of 50 human breast cancers. MMTV env gene sequences were identified by PCR in 12 (27%) of 45 human breast cancers. There was a significant correlation between the presence of MMTV (identified by p14 immunohistochemistry) in human breast cancers and histological characteristics similar to MMTV positive mouse mammary tumors (p = 0.001). There was a non-significant correlation between the presence of MMTV env gene sequences (identified by PCR) in human breast cancers and histological characteristics similar to MMTV positive mouse mammary tumors (p = 0.290). MMTV p14 proteins were identified in 7 (54%) of 13 benign breast specimens that later developed into human breast cancers. MMTV by PCR was identified in two benign specimens one of whom later developed MMTV positive breast cancer.</p>Discussion<p>These observations offer evidence that MMTV may be associated with characteristic human breast cancer histology. p14-based immunohistochemistry appears to be a more reliable technique than PCR for the identification of MMTV in human breast cancer. Identification of MMTV-associated p14 proteins in benign breast tissues confirms prior PCR-based studies that MMTV infection occurs before the development of MMTV positive breast cancer.</p>Conclusion<p>Many MMTV positive human breast cancers have similar histology to MMTV positive mouse mammary tumors. MMTV infection identified in benign breast tissues precedes development of MMTV positive human breast cancer. When considered in the context of prior studies, these observations indicate a likely role for MMTV in human breast cancer.</p