A Mouse Mammary Tumor Virus env-Like Exogenous Sequence Is Strictly Related to Progression of Human Sporadic Breast Carcinoma

• DOI: 10.1016/j.ajpath.2011.06.046
• Publication Date: 2011-08-19T17:28:31Z
• Authors: Chiara Maria Mazz...; Mohammad Al Hamad; Giovanni Fanelli; Cristian Scatena; Francesca Zammarchi; Katia Zavaglia; Francesca Lessi; Mauro Pistello; Antonio Giuseppe ...; Generoso Bevilacqua
• Keywords: Mammary carcinoma; Breast carcinoma

A viral etiology of human breast cancer (HBC) has been postulated for decades since the identification mouse mammary tumor virus (MMTV). The detection MMTV env-like exogenous sequences (MMTVels) in 30% to 40% invasive HBCs increased attention this hypothesis. Looking MMTVels during progression may contribute a better understanding their role HBC. Herein, we analyzed HBC preinvasive lesions presence MMTVels. Samples were obtained by laser microdissection FFPE tissues: 20 usual-type ductal hyperplasias, 22 atypical hyperplasias (ADHs), 49 carcinomas situ (DCISs), infiltrating (IDCs), and 26 normal epithelial cells collateral DCIS or an IDC. Controls included reductive mammoplastic tissue, thyroid colon carcinoma, blood samples from healthy donors. detected fluorescence-nested PCR. DNA tissues nine patients real-time quantitative PCR, revealing different load correlated with stage progression. Furthermore, as never previously described, was investigated chromogenic hybridization. found 19% IDC, 27% ADHs, 82% DCISs, 35% IDCs. No control samples. Quantitative PCR hybridization confirmed these results. These data could our See related Commentary on page 1588 Despite fact that carcinoma represents most frequent women it largely studied worldwide many decades, its is unknown. hereditary transmission pathogenetic mutations some predisposing genes, such BRCA1 BRCA2, only factor recognized causative disease.1Petruccelli N. Daly M.B. Feldman G.L. Hereditary ovarian due BRCA2.Genet Med. 2010; 12: 245-259Abstract Full Text PDF PubMed Scopus (228) Google Scholar Although estrogens well demonstrated, sound evidence etiological effect still lacking.2Russo J. Russo I.H. initiation cancer.J Steroid Biochem Mol Biol. 2006; 102: 89-96Crossref (435) minimal percentage cases are caused radiation.3Ronckers C.M. Erdmann C.A. Land C.E. 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Salmons Rouault infects 65: 6651-6659Crossref (89) if really carcinogenesis, act promoting agent etiological/pathogenic cofactor linked progression, infiltration metastasis. determination where appear help unveil This study analyzes [ie, usual hyperplasia (UDH), (ADH), (DCIS), (IDC)]. contemporary epithelium, lesions, cancer, all same patient, investigated. Results demonstrate early appearance Real-time (qPCR) fully confirms data. For first time, knowledge, report (CISH) experiments, signals nuclei IDC All tissues, formalin fixed paraffin embedded, collected archived (from January 1, 2005 December 31, 2009) at Division Surgical, Molecular, Ultrastructural Pathology, University Pisa, Italy. Epithelial (normal, hyperplastic, dysplastic, neoplastic) microdissection. extracted microdissected sequenced.20Zammarchi cases, qPCR performed. adjective used indicate (NECs) histological section, hosting lesion carcinoma. UDH indicates benign proliferative characterized secondary lumens streaming central ADH cytological architectural features low-grade one ducts aggregate exceeding 0.2 cm. can high, intermediate, low grade: i) pleomorphic large abundant mitoses variable architecture, often necrotic debris; ii) uniform small frequently cribriform micropapillary configuration; iii) neoplastic nuclei, showing pleomorphism degree high- DCIS.24Page D.L. Dupont Anatomic markers premalignancy risk cancer.Cancer. 1990; 66: 1326-1335Crossref (194) 25Tavassoli F.A. Norris H.J. comparison long-term follow-up intraductal breast.Cancer. 518-529Crossref (341) 26Pinder S.E. Ellis I.O. diagnosis management pre-invasive disease: (DCIS) (ADH)–current definitions classification.Breast 5: 254-257Crossref (111) specimens NECs IDC; UDHs noncollateral ADHs Of 15 poorly differentiated, 33 moderately differentiated. six papillary carcinomas, adenocarcinomas, infected positive control. Leica ASLMD automatic microdissector (Leica Microsystems, Wetzlar, Germany) select cell population studied. Sections (6-μm thick) cut each case new microtome blade slide, obtaining total 10,000 15,000 Stromal inflammatory carefully excluded. Because experience laboratory method, no difficulty selecting areas interest.20Zammarchi Microdissected kept overnight lysis buffer containing proteinase processed amplification next day. To avoid cross contamination, blank (water) parallel quality amount evaluated agarose gel electrophoresis UV spectrophotometry (NanoDrop 2000, Thermoscientific, Wilmington, DE). checked absence inhibitors amplifying HERV-K10 provirus target templates controls. conditions follows: 94°C 10 minutes; 30 cycles seconds, 50°C 72°C 45 seconds; final extension 7 minutes 30-μL reaction mixture 1× Buffer [500 mmol/L KCl, 150 Tris-HCl (pH 8.0), 1.5 MgCl2, 200 μmol/L 2′-deoxyribonucleoside 5′-triphosphate, 0.5 forward reverse primer, 2.5 AmpliTaq Gold; Applied Biosystems]. free amplicons clearly visible 1.8% gel. Fluorescence-nested detect sequence. Generated sized sequencer. pairs primer basis available GenBank (accession no. AF243039). Slight changes introduced previous procedure20Zammarchi adapt paraffin-embedded tissues. They consisted decreasing length maximum 250 bp. outer yield 248-bp fragment positions 231 480 env, inner 202-bp (nucleotide 431). Sequences forward, 5′-GATGGTATGAAGCAGGATGG-3′; reverse, 5′-CCTCTTTTCTCTATATCTATTAGCTGAGGTAATC-3′. nested listed, (5′-AAGGGTAAGTAACACAGGCAGATGTA-3′). Both PCRs performed 50 μL standard [1.5 mm μm unlabeled (MWG Biotech, Ebersberg, Germany), 6-FAM–labeled (Applied Biosystems, Milan, Italy), Gold]. Input template 500-ng genomic first-round 2 product second round. profile 40 (first-round) (second-round) 58°C 60 minutes. exclude water controls every five run. Fluorescent capillary appeared peaks electropherogram. amplicon size extrapolated molecular ladder resuspended run parallel. Briefly, 3 products both rounds mixed ROX-labeled (Gene Scan 400 HD ROX; Biosystems) 11.5 formamide (Hi-Di Formamide; Biosystems). After denaturation 95°C minutes, loaded onto ABI PRISM 3100 genetic analyzer GENESCAN software, version 3.1 Foster City, CA). fragments CISH probe purified gels QIAquick Gel Extraction kit (Qiagen, Valencia, CA) sequenced 3130XL instrument Each aligned BLAST search function (Basic Local Alignment Tool, http://blast.ncbi.nlm.nih.gov, last accessed March 8, 2010) deposited GenBank. Extracted qPCR. two patients, NEC, DCIS, available, seven available. 25-μL prepared 2× Master Mix (MesaGreeen qPCR; Eurogentec, San Diego, (0.3 μmol/L) (nested 202 bp). determined mean triplicate sample values. glyceraldehyde-3-phosphate dehydrogenase (GAPDH), single-copy gene, internal reference Four 10-fold dilutions (1 × 101 1 104 copies/mL) (GAPDH) curves plate. corresponding GAPDH measurements plate minimize errors. An MMTV-positive template-negative (PCR mix only) Rotor-Gene Q (Qiagen). plotted software detector system printing CT values against concentration standards (MMTV sequence), copy number per calculated detector. in-house 2.7-kb described.27Liu Najfeld V. Identification proviral structure 2001; 61: 1754-1759PubMed 50-μL volume polymerase enzyme (Takara Bio Inc., Shiga, Japan), 5 10× buffer, 8 (5L LTR3),27Liu μg virus. followed seconds 68°C amplified labeled DIG-NICK translation (Roche, Basel, Switzerland). experiments sections (4-μm thick), mounted positively charged microscope slides, left dry 37°C. then deparaffinized rehydrated. Antigen retrieval achieved heat bath (Dako, Glostrup, Denmark). slides placed Coplin jars (Sigma-Aldrich, St. Louis, MO) enough 0.01 mol/L sodium citrate solution 6.0) cover sections, incubated 80°C expose nuclear components, K applied procedures applying 500 ng 16 hours 37°C, respectively, ThermoBrite hybridizer (ABBOTT, Park, IL). anti-digoxigenin-AP conjugate produced characteristic black stain NBT-BCIP substrate. staining, prepared. study. block but instead probe, sequences, tissue. one-way analysis variance test statistically evaluate numbers later stages patient. Our use shows studies, when comparing representative before (Figure 1A) after 1B). frequency phases reported Table Figure 2. NECs, (5 26) mammoplasty negative. hyperplasia, MMTVels, (6 22) ADH. (40 49) samples; (7 20) positive. As controls, adenocarcinoma, donors Regarding structures 13 (case 1), three positive; four (cases 6 9), positive, negative; 4), 12 17), DCISs 41 46) whom and/or IDCs latter 11 27, respectively). eight 18 25) 5) 28 40) 47 negative.Table 1Presence Frequency Concomitant c-NECs IDCsSample no.c-NECsDCISsIDCsSample no.c-NECsDCISsIDCs1+++26NA+−2++−27NA+−3++−28NA+NA4++−29NA+NA5++NA30NA+NA6−++31NA+NA7−++32NA+NA8−++33NA+NA9−++34NA+NA10NA++35NA+NA11NA++36NA+NA12−+−37NA+NA13−+−38NA+NA14−+−39NA+NA15NA+−40NA+NA16NA+−41−−−17−+−42−−NA18−+NA43−−NA19−+NA44−−NA20−+NA45−−NA21−+NA46NA−−22−+NA47NA−NA23−+NA48NA−NA24−+NA49NA−NA25−+NAPositive c-NECs, (19.0%) 26; (82.0%) 49; IDCs, (35.0%) 20; + (8.0%) 13; (24%) 21.NA, assessed. Open table tab Positive 21. NA, determine whether homologous prototype (GenBank accession AY152 721 AF243039, Multiple alignment showed 97% homology sequences. significant hits compared genome GenBank, indicating retrovirus origin. patients. control; therefore, bar MMTV/GAPDH ratio 3). average 3A) shown. There dramatic decrease (P = 0.01). result increase epithelium complete loss stage. shown 4, successfully None revealed any signal. inside 4B), 4A multiple coming nucleus varies among cells, 3.5; addition, fewer than (data shown). sporadic unknown, exception rare radiation-induced neoplasms.3Ronckers establishe