Inflammatory breast cancer: high incidence of detection of mixed human cytomegalovirus genotypes associated with disease pathogenesis

Abstract

Inflammatory breast cancer (IBC) is a highly metastatic, aggressive, and fatal form of breast cancer. Patients presenting with IBC are characterized by a high number of axillary lymph node metastases. Recently, we found that IBC carcinoma tissues contain significantly higher levels of human cytomegalovirus (HCMV) DNA compared to other breast cancer tissues that may regulate cell signaling pathways. In fact, HCMV pathogenesis and clinical outcome can be statistically associated with multiple HCMV genotypes within IBC. Thus, in the present study, we established the incidence and types of HCMV genotypes present in carcinoma tissues of infected non-IBC versus IBC patients. We also assessed the correlation between detection of mixed genotypes of HCMV and disease progression. Genotyping of HCMV in carcinoma tissues revealed that glycoprotein B (gB)-1 and glycoprotein N (gN)-1 were the most prevalent HCMV genotypes in both non-IBC and IBC patients with no significant difference between patients groups. IBC carcinoma tissues, however, showed statistically significant higher incidence of detection of the gN-3b genotype compared to non-IBC patients. The incidence of detection of mixed genotypes of gB showed that gB-1 + gB-3 was statistically significantly higher in IBC than non-IBC patients. Similarly, the incidence of detection of mixed genotypes of gN showed that gN-1 + gN-3b and gN-3 + gN-4b/c were statistically significant higher in the carcinoma tissues of IBC than non-IBC. Mixed presence of different HCMV genotypes was found to be significantly correlated with the number of metastatic lymph nodes in non-IBC but not in IBC patients. In IBC, detection of mixed HCMV different genotypes significantly correlates with lymphovascular invasion and formation of dermal lymphatic emboli, which was not found in non-IBC patients.

Keywords: UL55; UL73; glycoproteins; human cytomegalovirus; inflammatory breast neoplasms; lymphovascular invasion; metastasis.

Figure 1

Distribution frequencies of HCMV genotypes in non-IBC and IBC carcinoma tissues are shown. (A) Agarose gel electrophoresis for nested multiplex PCR showed single detection of different HCMV gB genotypes. Lane C− represents negative control; lanes 1 and 2 represent gB-1 (420 bp) in non-IBC and IBC, respectively. Lanes 3 and 4 represent gB-2 (613 bp) in non-IBC and IBC, respectively. Lanes 5 and 6 represent gB-3 (190 bp) in non-IBC and IBC, respectively. Lanes 7 and 8 represent gB-4 (465 bp) in non-IBC and IBC, respectively, and lanes 9 and 10 represent gB-5 (139 bp) in non-IBC and IBC, respectively. (B) Agarose gel electrophoresis for multiplex PCR showed single detection of different HCMV gN genotypes. Lane C− represents negative control; lanes 1 and 2 represent gN-1 (283 bp) in non-IBC and IBC, respectively. Lanes 3 and 4 represent gN-2 (380 bp) in non-IBC and IBC, respectively. Lanes 5 and 6 represent gN-3b (214 bp), in non-IBC and IBC, respectively, and lanes 7 and 8 represent gN-4b/c (244 bp) in non-IBC and IBC, respectively. (C) Bars represent distribution frequencies of single infection with different HCMV gB genotypes in non-IBC versus IBC carcinoma tissues. (D) Bars represent distribution frequencies of single infection with different HCMV gN genotypes non-IBC versus IBC carcinoma tissues. *Indicates a significant p value as determined by Fisher’s exact test.

Figure 2

Incidence of detection of mixed HCMV genotypes in non-IBC and IBC carcinoma tissues is shown. Bars represent the incidence of mixed presence of different HCMV gB and gN genotypes in non-IBC and IBC carcinoma tissues as detected by multiplex PCR IBC carcinoma tissues characterized by high incidence of mixed HCMV genotypes compared to non-IBC carcinoma tissues.*Indicates a significant p value as determined by Fisher’s exact test.

Figure 3

Incidence of detection of mixed HCMV different gB and gN genotypes in non-IBC and IBC carcinoma tissues is shown. (A) Agarose gel electrophoresis for nested multiplex PCR showed mixed presence of different HCMV gB genotypes. Lane C− represents negative control; lanes 1 and 2 represent gB-1 + gB-2 (420 bp + 613 bp) in non-IBC and IBC, respectively. Lanes 3 and 4 represent gB-1 + gB-3 (420 bp + 190 bp) non-IBC and IBC, respectively. Lanes 5 and 6 represent gB-1 + gB-4 (420 bp + 465 bp) in non-IBC and IBC, respectively. Lanes 7 and 8 represent gB-2 + gB-3 (613 bp + 190 bp) in non-IBC and IBC, respectively. Lanes 9 and 10 represent gB-2 + gB-4 (613 bp + 465 bp) in non-IBC and IBC, respectively. (B) Agarose gel electrophoresis for multiplex PCR showed mixed infection with different HCMV gN genotypes. Lane C− represents negative control, lanes 1 and 2 represent gN-1 + gN-2 (283 bp + 380 bp) in non-IBC and IBC, respectively, lanes 3 and 4 represent gN-1 + gN-3b (283 bp + 214 bp) in non-IBC and IBC, respectively, lanes 5 and 6 represent gN-1 + gN-4b/c (283 bp + 244 bp) in non-IBC and IBC, respectively, lanes 7 and 8 represent gN-2 + gN-4b/c (380 bp + 244 bp) in non-IBC and IBC, respectively, and lanes 9 and 10 represent gN-3b + gN-4b/c (214 bp + 244 bp) in non-IBC and IBC, respectively. (C) Bars represent the incidence of mixed infection with different HCMV gB genotypes in non-IBC and IBC carcinoma tissues as detected by multiplex PCR. (D) Bars represent the incidence of mixed infection with different HCMV gN genotypes. Non-IBC bar represents the percentage of mixed gN genotypes infections in non-IBC and IBC carcinoma tissues as detected by multiplex PCR. *Indicates a significant p value as determined by Fisher’s exact test.

Figure 4

Number of positive metastatic lymph nodes among HCMV positive non-IBC and IBC patients by HCMV gB or gN genotypes is shown. (A) Dots represent the numbers of metastatic lymph nodes in non-IBC and IBC patients containing different HCMV gB genotypes. (B) Dots represent the numbers of metastatic lymph nodes in non-IBC and IBC patients containing different HCMV gN genotypes. *Indicates a significant p value as determined by Student’s T-test.

Figure 5

Association between lymph node metastasis and mixed presence of HCMV genotypes in IBC and non-IBC patients is shown. (A) Dots represent the numbers of metastatic lymph nodes in non-IBC and IBC patients containing mixed HCMV gB genotypes. (B) Dots represent the numbers of metastatic lymph nodes in non-IBC and IBC patients with mixed HCMV gN genotypes. Results showed that detection of mixed HCMV different gB or gN genotypes statistically correlates with lymph node metastasis in non-IBC. *Indicates a significant p value as determined by Pearson’s correlation coefficient.

Figure 6

Microscopic images of H and E stained paraffin embedded tissue sections in breast carcinoma tissues are shown. (A) Representative of paraffin embedded tissue section of non-IBC carcinoma tissues showing invasion of carcinoma cells to lymphatic vessels. (B) Representative of paraffin-embedded tissue section of IBC carcinoma tissues showing tumor emboli formation due to carcinoma cells invasion into lymphatic vessels (magnification: left panel, 10× and right panel, 40×).