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Is human cytomegalovirus associated with breast cancer progression?
© Utrera-Barillas et al.; licensee BioMed Central Ltd. 2013
Received: 13 December 2012
Accepted: 22 March 2013
Published: 4 April 2013
It has been hypothesized that human cytomegalovirus (HCMV) may be associated with breast cancer progression. However, the role of HCMV infection in breast cancer remains controversial. We aimed to assess whether HCMV genes (UL122 and UL83) could be detected in breast carcinomas and reinvestigated their possible association with breast cancer progression. DNA from paraffin-embedded tissues was analyzed by real-time PCR. We investigated 20 fibroadenomas and 27 primary breast carcinomas (stages II, III, and IV).
Two carcinomas were positive for HCMV, one was positive for two TaqMan viral detection probes, and one was positive for a sole TaqMan viral detection probe (UL83), whereas the remainder of the samples was negative.
Samples studied showed no association between HCMV infection and breast cancer progression.
The number of new cancer cases and deaths is expected to increase worldwide. In Mexico, breast cancer has an incidence of 21.2%, mortality of 13.2%, and 5-year prevalence of 30.8% . Cases of breast cancer were recognized from the second decade of life and there was a peak incidence between 40 and 54 years of age. The majority of cases were at advanced stages (II, III, and IV). Metastasis is frequently a final and fatal step in the progression of solid malignancies. The molecular requirements for some of these steps may be specific tissues. Viruses have been central to modern cancer research and provide profound insight into both infectious and non-infectious cancer causes and players in disease progression. This diverse group of viruses reveals unexpected connections among innate immunity, immune sensors, and tumor suppressor signaling that control both viral infection and cancer. Infectious agents can be promoters of neoplastic transformation. Viruses associated with cancer (EBV, KSHV, HPV, HBV, HCV, HTLV-1, SV40, JCV, BKV, and MCV, HIV indirect carcinogen, HCMV oncomodulator, and controversial HMTV/MMTV) have been found to cause 16.1% of human cancers worldwide . Human cytomegalovirus (HCMV) is a widespread opportunistic herpesvirus that causes severe and fatal diseases in immunocompromised individuals including those who are organ transplant recipients, HIV-infected and patients with cancer. In vitro, HCMV can transform cells and deregulate other pathways relevant to adenocarcinoma pathogenesis, especially those affecting the cell cycle, mutagenesis, apoptosis, and angiogenesis when it has an oncomodulator role . The purpose of this study was to determine and investigate a possible association between progression of breast cancer and HCMV infection in our tumor collective of advanced breast carcinomas consisting of 27 primary breast carcinomas and 20 fibroadenomas by real-time PCR in order to compare malignant vs. non-malignant tumors.
Materials and methods
Samples of archived paraffin-embedded breast tissue (years: 2001−2008) were obtained from the Oncology Hospital of the XXI Century National Medical Center, Mexican Social Security Institute. The local ethics and scientific research committees approved the protocol.
The study group was comprised of 27 patients with breast carcinomas (stages II, III, and IV) who had recurrent disease and progression to anthracyclines and taxanes. All samples had a minimum of 70% cancer cells. The control group was comprised of 20 samples of fibroadenomas.
Genomic DNA extraction
To purify DNA from paraffin-embedded tissue, 10-μm-thick slices of carcinomas and fibroadenomas were subsequently treated with 900 μL of xylene to remove residual xylene. The tissue was washed twice with 1000 μL of 100% ethanol and was finally resuspended in 540 μL of RLT buffer (Qiagen). Dewaxed tissues were incubated three times with 60 μL of proteinase K at 56°C/24 h. DNA was extracted with AllPrep DNA/RNA/Protein Mini Kit (Qiagen) according to the manufacturer’s instructions. Briefly, lysated tissues were placed in a column and spun for 1 min/10000 rpm; later, the column was washed twice with AW1 and AW2 buffers. Finally, the DNA was eluted with 25 μL of EB buffer preheated to 70°C. DNA quantification was performed at 260 nm in Nanodrop (Thermo Scientific).
Multiplex PCR for glyceraldehyde-3-phosphate-dehydrogenase
Sequence primers (multiplex PCR for glyceraldehyde-3-phosphate-dehydrogenase amplification)
GTT CCA ATA TGA TTC CAC CC
CTC CTG GAA GAT GGT GAT GG
AGG TGG AGC GAG GCT AGC
TTT TGC GGT GGA AAT GTC CT
AGG TGA GAC ATT CTT GCT GG
TCC ACT AAC CAG TCA GCG TC
ACA GTC CAT GCC ATC ACT GC
GCT TGA CAA AGT GGT CGT TG
AGC CCC TAA GGT CTT CAA GC
CAT GCC TGT AGC TGG GAC TA
GGC TCC CTT GGG TAT ATG GT
GGA GCC AGT CTT GGA TGA
CCC CAC ACA CAT GCA CTT AC
AAT GAA GGG GTC ATT GAT GG
Detection of HCMV by real-time PCR
HCMV detection was performed by real-time PCR (qPCR), amplifying a 96-bp fragment, immediate early gene expression, Ie2 (UL122, forward primer: GGCTCACCTCGTCAATCTTGA; reverse primer: AGAAGGTGCGCAATATCATGAAAGA; TaqMan probe: FAM-CCCCCTTCTGCACACCC) and 84 bp, late gene expression, pp65 (UL83, forward primer: GGGACACAACACCGTAAAGC; reverse primer: GTGGAAGAGGACCTAACGATGAC; TaqMan probe: FAM-CCGCAACCCTTCATGC). qPCR was performed with 100 ng of genomic DNA at a final volume of 8 μL: 1X TaqMan Universal PCR Master Mix (Applied Biosystems), 0.54 μL UL122- or 0.54 μL UL83- TaqMan probe. Amplification was performed in an ABI 7500 thermal cycler as follows: 60°C/2 min and 95°C/10 min, and 40 cycles: 95°C/15 sec and 60°C/1 min, and 60°C/30 sec. Positive control was done with HCMV strain AD-169 (ATCC VR-583). Negative controls were DNA white blood cells from healthy individuals (blood donors) and reaction mix without DNA. All qPCR reactions were carried out in duplicate.
We used Fisher’s exact test to compare the number cytomegalovirus-positive samples between fibroadenomas and breast carcinomas.
The study group included subjects with a mean age of 53.26 ± 11.99 years (range: 27−76 years). Mean overall survival was 44 months (range: 12−84 months). All had progression with active tumor sites affecting bone, soft tissue, liver, lung, and central nervous system. The control group had a mean age of 45 ± 9.27 years (range: 27−67 years). Females with fibroadenoma were younger than females with carcinoma.
Sample 19 was HCMV-positive with both TaqMan probes: one detected an immediate early gene (synonym of viral replication) and the other detected a late gene (presence of virus). The sample ´eight´ only tested positive for a late gene. The remaining samples were HCMV-negative.
Clinicopathological features of breast cancer patients included in the study
n = 27
Infiltrating ductal carcinoma
In the samples analyzed, there was no statistically significant association between malignant vs. non-malignant tumors (p = 0.50) and breast cancer progression vs. HCMV infection (p = 0.50), but we detected HCMV in two malignant tumors samples: one of which was positive for two detection probes, whereas the other sample was positive for one.
The authors acknowledge the assistance of Sharon Morey, Executive Editor, Scientific Communications for English editing. This work was partially supported by grant FIS/IMSS/PROT/G10/820 and FIS/IMSS/PROT/G09/761 from the Mexican Social Security Institute.
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