三陰乳腺癌與基底細(xì)胞樣乳腺癌病理形態(tài)和生物學(xué)特征探討

王成鑫，黃萱，封華

（江大病理診斷所；江漢大學(xué)醫(yī)學(xué)院病理學(xué)與病理生理學(xué)教研室，湖北武漢430056）

李培林

（加州大學(xué)舊金山分校醫(yī)學(xué)院醫(yī)學(xué)系，舊金山CA94121)

三陰乳腺癌與基底細(xì)胞樣乳腺癌病理形態(tài)和生物學(xué)特征探討

王成鑫，黃萱，封華

（江大病理診斷所；江漢大學(xué)醫(yī)學(xué)院病理學(xué)與病理生理學(xué)教研室，湖北武漢430056）

目的探討三陰乳腺癌（triple-negative breast cancer，TNBC）與基底細(xì)胞樣乳腺癌（basal-like breast cancer，BLBC）及非三陰乳腺癌（non triple-negative breast cancer，NTNBC）的關(guān)系及其形態(tài)、生物學(xué)特征。方法應(yīng)用免疫組織化學(xué)（immunohistochemistry，IHC）方法對(duì)96例乳腺癌標(biāo)本進(jìn)行HER2、ER、PR蛋白的檢查；應(yīng)用FISH（fluorescent in situ hybridization）方法對(duì)3例HER2 IHC 3+及5例HER2 IHC 2+標(biāo)本進(jìn)行HER2基因擴(kuò)增的檢查；對(duì)22例TNBC進(jìn)行了CK5/6或EGFR的檢查。按照檢查結(jié)果，將其分為TNBC、NTN?BC和BLBC，比較三者的病理形態(tài)及生物學(xué)特征。結(jié)果NTNBC占72.92%（70/96），其組織學(xué)Ⅰ、Ⅱ及Ⅲ級(jí)的病例分別為28.57%（20/70）、57.14%（40/70）和14.29%（10/70），淋巴結(jié)轉(zhuǎn)移率為44.44%（16/36）；TN?BC占乳腺癌的27.08%（26/96），無(wú)組織學(xué)Ⅰ級(jí)病例，組織學(xué)Ⅱ及Ⅲ級(jí)的病例分別為53.85%，（14/26）及46.15%，（12/26）例，淋巴結(jié)轉(zhuǎn)移率為61.11%（11/18）；BLBC占TNBC的63.64%（14/22），同TNBC一樣，無(wú)組織學(xué)Ⅰ級(jí)病例，Ⅱ、Ⅲ級(jí)的病例分別為57.14%（8/14）及42.86%（6/14），淋巴結(jié)轉(zhuǎn)移率為55.56%（5/9）。TN?BC與BLBC低分化病例的比例以及淋巴結(jié)的轉(zhuǎn)移率均高于NTNBC。3例HER2 IHC 3+的病例FISH檢查結(jié)果同IHC，5例HER 2 IHC 2+標(biāo)本FISH檢查1例基因擴(kuò)增，3例陰性，1例結(jié)果不確定。結(jié)論TNBC與BLBC占乳腺癌1/4的比例，組織學(xué)多為中、低分化，浸潤(rùn)性生長(zhǎng)，易發(fā)生淋巴結(jié)轉(zhuǎn)移和復(fù)發(fā)，臨床預(yù)后較差。雖然BLBC與TNBC大部分有重疊，但其具有獨(dú)自的特異性，應(yīng)成為獨(dú)立的組織病理學(xué)類型。CK5/6或EGFR可用作從TNBC中篩選BLBC的指標(biāo)。IHC 2+病例要做FISH檢查，以正確指導(dǎo)治療。

三陰乳腺癌；基底細(xì)胞樣乳腺癌；病理形態(tài)學(xué)；熒光原位雜交

乳腺癌是異質(zhì)性明顯的惡性腫瘤，為女性最常見(jiàn)的惡性腫瘤之一，占女性死因的首位。在傳統(tǒng)分類上，將乳腺癌分為激素受體（ER和/或PR）陽(yáng)性或陰性腫瘤［1-2］。近年來(lái)，根據(jù)基因表達(dá)譜及基因分型，將乳腺癌分為4種亞型［2-3］：①基底細(xì)胞樣型（basal-like subtype，ER、PR、HER2均陰性）；②HER2高表達(dá)型（ER、PR、陰性，HER2陽(yáng)性）；③管腔型（1uminal subtype），此型又按照有無(wú)HER2的過(guò)表達(dá)分為：Luminal A（ER和/或PR陽(yáng)性，HER2陰性）；④Luminal B（ER和/或PR陽(yáng)性，HER2陽(yáng)性）。也有分為5型及其他亞型的［1，4］，即多一型正常乳腺樣腫瘤（Normal breast-like）。管腔型以ER及其相關(guān)基因高表達(dá)為特征，預(yù)后較好；基底細(xì)胞樣型乳腺癌（BLBC）是指具有基底細(xì)胞基因表型并不同程度地表達(dá)基底細(xì)胞角蛋白和肌上皮標(biāo)記物的乳腺癌，占全部乳腺癌的15%，與早已發(fā)現(xiàn)的免疫組化三聯(lián)陰乳腺癌（TNBC）在免疫表型及臨床預(yù)后等方面存在相似之處，特別是在組織學(xué)上多為高級(jí)別腫瘤。更因?yàn)锽LBC占TNBC的85%［5］，有人將BLBC與TNBC相提并論，并視為完全相同的乳腺癌亞型［6］，但目前諸多研究顯示，BLBC與TNBC是不同范疇的兩類乳腺癌［2，5，7］，BLBC與TNBC兩型之間至少有30%以上存在不一致性，基因分型、免疫表型及組織學(xué)類型也不能完全對(duì)應(yīng)。

TNBC指雌激素受體（ER）、孕激素受體（PR）和HER2均無(wú)表達(dá)的乳腺癌，被認(rèn)為是一個(gè)單獨(dú)的組織學(xué)類型，占全部乳腺癌的10%～17%，其中任何一項(xiàng)陽(yáng)性者即定義為NTNBC［8-11］。本研究目的在于分析TNBC、BLBC及NTNBC間的關(guān)系及其病理形態(tài)特點(diǎn)及生物學(xué)特征。

1 資料和方法

1.1 臨床資料

選取2008年1月至2012年12月間江大病理診斷所收檢的乳腺癌手術(shù)標(biāo)本96例，進(jìn)行臨床病理資料分析，包括年齡、淋巴結(jié)轉(zhuǎn)移、病理類型、組織學(xué)分級(jí)等。所有患者均為女性，年齡23～86歲，平均年齡51.12歲。

1.2 病理資料

所有標(biāo)本均為浸潤(rùn)性乳腺癌，90%以上為浸潤(rùn)性導(dǎo)管癌，少數(shù)為黏液癌、小葉癌及混合性乳腺癌。按照癌組織的分化程度的不同，將其分為三級(jí)：GⅠ、GⅡ、GⅢ。有淋巴結(jié)清掃的病例，每例檢查淋巴結(jié)均在12枚以上。

1.3 方法與試劑

標(biāo)本經(jīng)40 g/L緩沖甲醛固定，常規(guī)石蠟包埋切片，切片厚3～4 μm，分別作常規(guī)HE及應(yīng)用免疫組織化學(xué)S-P法檢查HER2、ER、PR蛋白在乳腺癌組織中的表達(dá)，對(duì)22例TNBC進(jìn)行了CK5/6或EGFR的檢查，二者中任意一項(xiàng)陽(yáng)性即為BLBC。應(yīng)用FISH方法對(duì)3例HER2 IHC 3+及5例IHC 2+HER2標(biāo)本進(jìn)行HER2基因擴(kuò)增的檢查。試劑購(gòu)自福州邁新公司（產(chǎn)品編號(hào)：ER：MAB-0349；PR：MAB-0236；C-erbB-2（HER2）：MAB-0198；CK5/6：MAB-0276、EGFR：RMA-0554；HER2雙色探針試劑盒：Z2020-5）。染色步驟按試劑盒說(shuō)明進(jìn)行。已知陽(yáng)性組織切片作陽(yáng)性對(duì)照，PBS液代替一抗作陰性對(duì)照。

1.4 IHC及FISH結(jié)果判定

HER2免疫組織化學(xué)及FISH檢測(cè)結(jié)果判定按照ASCO/CAP指南［7］推薦標(biāo)準(zhǔn)判定；ER、PR結(jié)果判定：采用文獻(xiàn)［12］介紹的評(píng)分方法，即將腫瘤細(xì)胞核著色數(shù)量、著色程度分別評(píng)分，兩者相加，3分及以上為陽(yáng)性。根據(jù)IHC及FISH檢查結(jié)果，將其分為3組：TNBC、NTNBC和BLBC組。TNBC最大年齡85歲，最小年齡23歲，平均年齡48.32歲；NTNBC最大年齡86歲，最小年齡26歲，平均年齡52.68歲；BLBC最大年齡77歲，最小年齡39歲，平均年齡51.5歲。

1.5 統(tǒng)計(jì)學(xué)方法

應(yīng)用SPSS 13.0軟件進(jìn)行統(tǒng)計(jì)學(xué)處理，比較用非參數(shù)檢驗(yàn)。P＜0.05為差異有統(tǒng)計(jì)學(xué)意義。

2 結(jié)果

2.1 組織形態(tài)學(xué)

在TNBC中非特殊型導(dǎo)管癌占了絕大部分，多為浸潤(rùn)性導(dǎo)管癌Ⅱ～Ⅲ級(jí)，少數(shù)為黏液及化生性癌。它們與NTNBC不同的形態(tài)學(xué)特點(diǎn)為：較低的組織學(xué)分級(jí)，無(wú)Ⅰ級(jí)病變，細(xì)胞具有明顯的多形性，高的核漿比、較少腺管樣結(jié)構(gòu)、核分裂多見(jiàn)（圖1，見(jiàn)封三），細(xì)胞凋亡現(xiàn)象明顯，間質(zhì)稀少以及推擠性邊緣和中央地圖樣壞死（圖2，見(jiàn)封三）或粉刺樣壞死等，有時(shí)還可見(jiàn)梭形細(xì)胞及鱗狀分化（圖3，見(jiàn)封三），有中央?yún)^(qū)微血管增生和炎性細(xì)胞浸潤(rùn)。

2.2 ER、PR、HER2蛋白、CK5/6和EGFR及HER2基因的表達(dá)

ER、PR、HER2蛋白表達(dá)的情況如表1所示，96例患者中，ER：58例陽(yáng)性；PR：41例陽(yáng)性；HER2：31例陽(yáng)性。TNBC 26例，占全部乳腺癌的27.08%（26/96），NTNBC 70例，占72.92%（70/96）。CK5/6和EGFR表達(dá)見(jiàn)圖4（見(jiàn)封三）和圖5（見(jiàn)封三）FISH結(jié)果：3例HER2 IHC 3+的病例有基因擴(kuò)增（圖6，見(jiàn)封三）；5例HER2 IHC2+標(biāo)本2例基因擴(kuò)增，其中1例為導(dǎo)管內(nèi)癌擴(kuò)增，無(wú)實(shí)際意義，2例陰性（圖7，見(jiàn)封三），1例結(jié)果不確定。22例TNBC中，14例CK5/6或EGFR陽(yáng)性表達(dá)，為BLBC，占TNBC的63.64%（14/22），低于文獻(xiàn)［5］中占85%的報(bào)道。

表1 ER、PR、HER-2蛋白表達(dá)的情況/例（%）Tab.1 Expression of ER,PR and HER2 in breast cancer

2.3 各組乳腺癌組織學(xué)分級(jí)及淋巴結(jié)轉(zhuǎn)移率

乳腺癌組織學(xué)分級(jí)及淋巴結(jié)轉(zhuǎn)移率如表2和表3所示，TNBC（包括BLBC）無(wú)組織學(xué)Ⅰ級(jí)病例，Ⅱ～Ⅲ級(jí)的病例分別為53.85%（14/26）及46.15%（12/26），本組有完整淋巴結(jié)資料的病例18例，11例有淋巴結(jié)轉(zhuǎn)移，轉(zhuǎn)移率為61.11%；NTNBC組70例，組織學(xué)Ⅰ、Ⅱ及Ⅲ級(jí)的病例分別為28.57%（20/70）、57.14%（40/70）和14.29%（10/70），有完整淋巴結(jié)資料的病例36例，16例有淋巴結(jié)轉(zhuǎn)移，轉(zhuǎn)移率為44.44%（16/36）。TNBC組織學(xué)高級(jí)別的比例遠(yuǎn)高于非三陰型組，差異有顯著的統(tǒng)計(jì)學(xué)意義（P＜0.001），淋巴結(jié)轉(zhuǎn)移率三陰型組61.11%也高于非三陰型組的44.44%；BLBC組Ⅱ～Ⅲ級(jí)的病例分別為57.14%（8/14）及42.86%（6/14），本組有完整淋巴結(jié)檢查資料的病例9例，5例有淋巴結(jié)轉(zhuǎn)移，轉(zhuǎn)移率為55.56%（5/9），同TNBC相比，在組織學(xué)分級(jí)上差別不大，淋巴結(jié)轉(zhuǎn)移率高于NTNBC，而低于TNBC，可能和標(biāo)本基數(shù)太少有關(guān)。

表2 三陰與非三陰型乳腺癌組織學(xué)分級(jí)/例Tab.2 Histological grade of triple-negative and non-triple-negative breast cancer

表3 三陰與非三陰型乳腺癌淋巴結(jié)轉(zhuǎn)移率/例Tab.3 Rate of lymph node metastasis of triple-negative and non-triple-negative breast cancer

3 討論

TNBC是早已廣泛認(rèn)識(shí)、具有特殊免疫表型的一類乳腺癌。激素受體陽(yáng)性者往往對(duì)內(nèi)分泌治療有效，而具有HER2基因過(guò)表達(dá)的患者則對(duì)靶向治療敏感，因此目前對(duì)乳腺癌患者常規(guī)進(jìn)行激素受體和HER2基因表達(dá)的檢測(cè)。TNBC則不適用于內(nèi)分泌治療及靶向治療。TNBC占乳腺癌的10%～17%［2，9-11］，但也有TNBC占乳腺癌比例較高的報(bào)道。袁中玉等［11］的研究TNBC占23.8%，也有TNBC占24.8%的報(bào)道，這和本研究TNBC占乳腺癌的27.08%比較接近，究其原因，可能是本研究嚴(yán)格按照ASCO/CAP指南推薦標(biāo)準(zhǔn)進(jìn)行判定，27.08%應(yīng)該是最實(shí)際的數(shù)據(jù)。實(shí)際工作中，TNBC是按照免疫組化結(jié)果判定的，不同的實(shí)驗(yàn)室對(duì)同一病例的判斷也會(huì)必然不同。

BLBC由PEROU等在2000年時(shí)首次提出，對(duì)內(nèi)分泌及靶向治療均不敏感，容易復(fù)發(fā)及發(fā)生轉(zhuǎn)移。ER、PR和HER2陰性與CK5/6和（或）EGFR陽(yáng)性目前已經(jīng)成為經(jīng)典的BLBC診斷標(biāo)準(zhǔn)。本研究發(fā)現(xiàn)，BLBC與TNBC在形態(tài)、免疫表型及生物學(xué)特征上互有重疊。本研究結(jié)果BLBC占TNBC的63.64%（14/ 22），低于文獻(xiàn)［5］中占85%的報(bào)道，這也可能是乳腺癌國(guó)內(nèi)外發(fā)病與分型的差異。但須注意的是EGFR單獨(dú)使用時(shí)的陽(yáng)性率很低，CK5/6與EGFR聯(lián)合應(yīng)用優(yōu)于單獨(dú)使用，可以從TNBC中篩選出大部分的BLBC。

TNBC及BLBC其惡性度高，侵襲性強(qiáng)，容易復(fù)發(fā)及發(fā)生淋巴結(jié)轉(zhuǎn)移，更傾向于經(jīng)血道轉(zhuǎn)移至腦或肺，其無(wú)病生存期及總體生存率均較NTNBC為低，是生物學(xué)行為及臨床預(yù)后較差的乳腺癌亞型，大部分患者5年內(nèi)死亡。本研究的三陰乳腺癌組無(wú)組織學(xué)Ⅰ級(jí)病例，其組織學(xué)Ⅱ及Ⅲ級(jí)的病例分別為14例（53.85%，14/26）及12例（46.15%，12/26），淋巴結(jié)轉(zhuǎn)移率為61.11%（11/18）；BLBC在組織學(xué)分級(jí)上基本同TNBC，淋巴結(jié)轉(zhuǎn)移率為55.56%（5/9），高于非三陰乳腺癌，而低于TNBC；TNBC低分化病例的比例以及淋巴結(jié)的轉(zhuǎn)移率均高于NTNBC，差異有統(tǒng)計(jì)學(xué)意義（P＜0.001）。免疫組化HER2 IHC2+以下的病例，是否給予HER2靶向治療，需要進(jìn)行FISH或CISH（色素原位雜交）的檢測(cè)，以確定該基因有無(wú)擴(kuò)增。TNBC與BLBC發(fā)病均傾向于中、老年女性，臨床表現(xiàn)、生物學(xué)行為相似，兩者的預(yù)后與各自組織學(xué)類型的關(guān)系尚待進(jìn)一步研究。

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（責(zé)任編輯：范建鳳）

Abstract:ObjectiveTo study whether exogenous HA can reduce macrophage-tropic(CCR5-dependent,R5) HIV infectivity.MethodsIn this study，first using TZM-bl cell and un-stimulated CD4+T cells assays to assess whether exogenous HA can reduce R5-HIV infectivity.Next treat the un-stimulated CD4+T cells with hyaluroni?dase to study endogenous HA impact on R5-HIV infectivity.At last measures both exogenous HA,and endoge?nous HA effect on R5-HIV binding on CD4+T cells.Results(1)100 μg of exogenous HA treatment can reduce R5-HIV infectivity on TZM-bl cells and un-stimualted CD4+T cells(P＜0.001).(2)Hyaluronidase treatment can enhance HIV infectivity,by adding 100 μg of exogenous HA can reverse the function of hyaluronidase treatment(P＜0.001).(3)100 μg of exogenous HA can reduce R5-HIV binding to CD4+T cells,while hyaluronidase treatment can enhance R5-HIV binding to CD4+T cells(P＜0.001).ConclusionExogenous HA can reduce R5-HIV in?fectivity on un-stimulated CD4+T cells,while hyaluronidase treatment can enhance R5-HIV binding to CD4+T cells,and R5-HIV infectivity.

Keywords：hyaluronic acid(HA)；R5-HIV；infectivity；CD4+T cell

李培林

（加州大學(xué)舊金山分校醫(yī)學(xué)院醫(yī)學(xué)系，舊金山CA94121)

摘要：目的探討外源性透明質(zhì)酸（HA)是否可以降低巨噬細(xì)胞嗜性（CCR5依賴，R5）人類免疫缺陷病毒（HIV）對(duì)CD4+T細(xì)胞的感染性。方法首先用TZM-bl細(xì)胞和未受刺激的CD4+T細(xì)胞檢測(cè)，以評(píng)估外源性透明質(zhì)酸（HA）能否降低R5-HIV的感染性。用透明質(zhì)酸酶去處理未受刺激的CD4+T細(xì)胞，研究?jī)?nèi)源性HA對(duì)R5-HIV感染性的影響。最后，同時(shí)測(cè)量外源性和內(nèi)源性透明質(zhì)酸（HA）對(duì)R5-HIV對(duì)CD4+T細(xì)胞粘和力的影響。結(jié)果（1）100 μg外源性HA處理能夠顯著降低R5-HIV感染TZM-bl細(xì)胞和未受刺激的CD4+T細(xì)胞（P＜0.001）。（2）透明質(zhì)酸酶處理可增強(qiáng)HIV的感染性，但是如果加上100 μg外源性HA可以逆轉(zhuǎn)透明質(zhì)酸酶的處理（P＜0.001）。（3）100 μg外源性HA可以減少R5-HIV對(duì)CD4+T細(xì)胞的粘和力，透明質(zhì)酸酶處理可以提高R5-HIV對(duì)CD4+T細(xì)胞的粘和力（P＜0.001）。結(jié)論外源性HA減少R5-HIV對(duì)未受刺激的CD4+T細(xì)胞的感染性，而透明質(zhì)酸酶處理可以提高R5-HIV對(duì)CD4+T細(xì)胞的粘和力，能增強(qiáng)R5-HIV對(duì)CD4+T細(xì)胞的感染性。

關(guān)鍵詞：透明質(zhì)酸；R5-HIV；病毒感染；CD4+T細(xì)胞

0 Introduction

Prevention of HIV transmission is still the most direct way to stem the HIV/AIDS epidemic[1].However,to date,large-scale clinical trials of vaccines to produce an HIV-specific antibody or a T-cell response to pre?vent HIV infection have been disappointing[2-3].Since 80%of HIV infection occurs through sexual contact[4], there is intense interest in the prevention of HIV mucosal transmission,and CCR5-tropic viruses(R5 viruses) are preferentially transmitted.To design a better strategy to prevent mucosal transmission of HIV,it needs more fully understand the mechanism of HIV mucosal transmission[5].

Mucosal tissues are the front-line defense against pathogen invasion and greatly impede HIV transmis?sion.Studies using the simian immunodeficiency virus(SIV)rhesus macaque model demonstrate that the geni?tal tract mucosal barrier limits exposure of CD4+T cells,dendritic cells(DCs),and macrophages to the majority of the viral inoculum,and only a small number of infectious virions pass through the mucosal barrier to estab?lish the infected founder population[6-7].These findings are confirmed by clinical studies showing that a small number of infectious virions breach the mucosal barrier to infect resting CD4+T cells,generating a clonal or oli?goclonal founder population[5,8-9].

Mucosal integrity plays an important role in HIV transmission,and mucosal inflammation can increase HIV transmission[10-12].The mucosal tissues are composed of epithelial cells,extracellular matrix,interstitial cells and surface mucus.In addition to providing a full complement of host immune cells that variably facili?tate or impede HIV infection,the mucosal surface also serves as a physical barrier to mucosal HIV invasion. Mucosal mucus can trap HIV virions[13]and reduce virion movement[14].An acidic vaginal mucosal environment can decrease the rate of HIV sexual transmission[15].How these effects on mucosal HIV transmission are mediat?ed remains largely unknown[5,9].

The surface of the mucosal layer is a scaffold with extracellular matrix，a major component of the extracel?lular matrix is hyaluronic acid(HA,or hyaluronan).HA is a large glycosaminoglycan(GAG)that can be remod?eled and degraded by hyaluronidase.On the surface of the cells,HA polymers extend up to 25 μm in length, forming pericellular coats.HA interaction with its receptors can induce cellular signaling and is involved in mu?cosal tissue homeostasis and maintenance of tissue integrity[16-18].HA is also a regulator of immunity.HA inter?action with its main receptor,CD44,regulates recruitment and extravasation of T-cells into sites of inflamma?tion[19-20]and participates in the inflammatory process［16，21］.HA interaction with CD44 can reduce cytokine pro?duction from macrophages in the setting of inflammation[22]and lowers PKCa activity to decrease histamine re?lease from leukemic cell lines[23].

In our lab，recently we observed that exogenous HA reduced HIV infectivity when both virions and CD4+T cells expressed CD44.Effects were seen on both early infection events like viral binding and probably later events through reduction of PKCa activation,while treatment with hyaluronidase reduced endogenous HA thick?ness and enhanced susceptibility of CD4+T cells to infection,but in that study the HIV was generated from NL4-3 plasmid,which is a T-cell-tropic(CXCR4-dependent,X4)[24].Clinical studies have found that CCR5-tropic viruses(R5 viruses)are preferentially transmitted[5].The main aim of this study was to assess the role of HA in CCR5-tropic HIV infection on un-stimulated CD4+T cells.We observed that exogenous HA re?duced both X4,and R5-HIV infectivity on un-stimulated CD4+T cells.Effects were seen on viral binding, while treatment with hyaluronidase reduced endogenous HA thickness and enhanced susceptibility of CD4+T cells to infection.

1 Material and Methods

1.1 Material

Hyaluronic acid(HA)(Sigma,St.Louis,MO),hyaluronidase(Hase)(Sigma,St.Louis,MO),Phytomeagglu?tinin-M(PHA-M)(Roche,Indianapolis IN)，human-Interleukin 2(hIL-2)(Roche,Indianapolis,IN),MDEM and PRMI(Life Technologies,Carlsbad,CA),anti-HIV-1-p24 antibody(Santa Cruz Biotechnology,Dallas, Texas),Alexa Fluor647(Life Technologies,Carlsbad,CA),cell membrane staining(Life Technologies,Carls?bad,CA),stained nuclei with DAPI(Life Technologies,Carlsbad,CA),anit-CD4-PE(R&D System),EasySep Human CD4+T cell Enrichment kit(STEMCell,Vancouver,BC),HIV-1p24 ELISA kit(PerkinElmer, Waltham,MA).Beta-Glo?assay system(Promega,Madison WI).

1.2 Methods

1.2.1 Isolation of CD4+T cells from healthy donorsCD4+T cells were isolated from healthy donors PBMCs using the EasySep Human CD4+T cell Enrichment kit(STEMCell,Vancouver,BC)according to the manufac?turer′s instructions.

1.2.2 Generation of X4(NL4-3)and R5(YK-JRCSF)HIV virus stock from PBMCIsolated PBMCs were stimulated with 3 μg/mL of phytomeagglutinin-M(PHA-M)(Roche,Indianapolis IN);and 20 unit/mL of hu?man-Interleukin 2(hIL-2)(Roche,Indianapolis,IN)for 3 days,X4 and R5 HIV viral supernatant from trans?fection of 293T cells was used to infect pooled mixed stimulated PBMCs prepared from 2 healthy donors.The supernatant from HIV infected PBMCs was harvested at day 7 and day 11.Viral titers were determined by HIV-1p24 ELISA(PerkinElmer,Waltham,MA).

1.2.3 Reduction of HA from cell surface1x106/mL of unstimulated CD4+T cells were washed 3 times in RPMI without FCS and incubated in RPMI with 12 unit/mL of hyaluronidase(Hase)(Sigma,St.Louis,MO)for 60 min,followed by removal of supernatant and three washes with RPMI without FCS.

1.2.4 Staining HIV-p24 on unstimulated CD4+T cellsHIV staining:Unstimulated CD4+T cells were incu?bated with R5-HIV for 5 hours,fixed with 2%formaldehyde,and incubated with mouse anti-HIV-1 p24 anti?body at 4℃overnight,then stained with donkey anti-mouse IgG antibody conjugated with Alexa Fluor647 (Life Technologies,Carlsbad,CA)for 2 hours.HIV-p24 staining was measured by 2 methods:laser scanning microscopy(LSM),and flow cytometry(FACS).For LSM analysis:the cells were further stained with anit-CD4-PE(R&D System),cell membranes with cell membrane staining(Life Technologies,Carlsbad,CA), and stained nuclei with DAPI(Life Technologies,Carlsbad,CA),and LSM510 META laser scanning micro?scope(Carl Zeiss Microimaging Inc.Thornwood,NY,USA)was used to scan the cells with Z-stack projections. For FACS analysis:50 000 HIV-p24 stained cells were analyzed by EasyCyte6HT-2L(Millipore,MA).

1.2.5 Assays for HIV Infection(1)TZM-bl cells(NIH AIDS Research and Reference Reagent Program):3× 104cells plated into 96 well plate for overnight,then removed supernatant and cells were washed with complete DMEM without FCS,then treated with study reagents,inoculated with 100pg HIV-p24 of HIV/well for 5 hours, washed 3 times with complete DMEM,and cultured in complete DMEM.HIV infectivity was measured by us?ing Beta-Glo?assay system(Promega,Madison WI)and measured with a plate reader(VICTOR3)(PerkinEl?mer)at 3 days post-infection[25].The luciferase activity is presented as relative light units(RLU).(2)Unstimulat?ed CD4+T cells:1×106healthy donor unstimulated cells were washed with RPMI without FCS,treated with dif?ferent reagents,inoculated with 1 000 pg HIV-p24 of HIV for 5 hours,washed 3 times with complete RPMI,re?suspended in complete RPMI with 5 μg of PHA-M and 20 unit of hIL-2 for 24 hours,washed 3 times with com?pleted RPMI,and finally resuspended in completed RPMI with 20 unit of hIL-2.HIV infectivity was measured by HIV-1 p24 production in supernatant at day 7 post-infection by using HIV-1p24 ELISA kit(PerkinElmer, Waltham,MA).

1.3 Statistical analysis

TZM cell assay was run in triplicate and repeated at least 3 times.Experiments with primary CD4+T cells were run in duplicate and repeated with at least 3 different donors.Data averages and standard deviations(SD)were calculated in Microsoft Excel 2008 and presented as means±standard deviation(x±s).Significance was assessed by a t-test,with P＜0.05 considered significant.

2 Results

2.1 Exogenous HA reduces both X4 and R5-HIV infectivity on TZM-bl cells line,and unstimulated CD4+T cells

Earlier studies reported that exogenous HA could interrupt T cell rolling and impede extravasation of T cells into an inflamed site through interactions with CD44[19].To study whether exogenous HA can impact HIV infectivity,TZM-bl cells[25]were treated with exogenous HA for 1 hour,then inoculated with X4(NL4-3)or R5 (YK-JRCSF)produced from PBMCs cells and normalized by p24.HIV infectivity was measured at day 3 post infection by luciferase activity[25]as relative light units(RLU).Exogenous HA reduced infectivity of both X4 (NL4-3)and R5(YK-JRCSF)HIV significantly(Fig.1A).

In vivo studies of acute HIV and SIV infection demonstrate that virus initially targets resting CD4+T cells and establishes an infected founder population during mucosal transmission[5,8-9].To study whether exogenous HA treatment can reduce infectivity of both R5(YK-JRCSF)and X4(NL4-3)HIV on CD4+T cells,primary unstimulated CD4+T cells from healthy donors were treated with exogenous HA,and then inoculated with vi?ral stocks of both X4(NL4-3)or R5(YK-JRCSF)HIV normalized by p24,then stimulated with PHA and IL-2 for 24 hours.HIV infectivity was assessed by measurement of supernatant HIV-1 p24 7 days post-infection. Exogenous HA treatment significantly reduced HIV-1 p24 production during both X4(NL4-3)and R5 (YK-JRCSF)infection(Fig.1B).

Fig.1ATZM-bl cells assay:Exogenous HA(Sigma,St. Louis,MO)reduced infectivity of both NL4-3(X4)(open bar)and YK-JRCSF(R5)HIV(filled bar).(Data are mean±SEM of triplicate samples and represent 3 independent experiments)

Fig.1BUn-stimulated CD4+T cells assay:Exogenous HA decreased infectivity of both NL4-3(X4)(open bar) and YK-JRCSF(R5)on primary un-stimulated CD4+T cells.(Data are mean±SEM of duplicate samples and representative of 3 donors）

2.2 Hyaluronidase treatment can enhance both X4 and R5-HIV infectivity on unstimulated CD4+T cells

Endogenous HA has been observed to form a cushion on the cell surface,to play an anti-adhesive role[18]and to impede ligand access to cell-surface receptors[26].Hyaluronidase treatment can reduce endogenous HA from the cell surface,thus promoting cell-cell contact[17].Hyaluronidase is highly activated during sexual inter?course as well as inflammation[16-18].Our initial experiments showed that exogenous HA reduced both X4 (NL4-3)and R5(YK-JRCSF)HIV infection of unstimulated CD4+T cells.Thus,we studied the impact of hyal?uronidase treatment on HIV infection of primary unstimulated CD4+T cells.Healthy donor,unstimulated primaryCD4+T cells were treated with hyaluronidase prior to inoculation with an equivalent input(by p24)of either X4 (NL4-3)or R5(YK-JRCSF),then stimulated with PHA and IL-2 for 24 hours.At day 7 post-infection,infec?tivity was assessed by measurement of HIV-1 p24 in culture supernatants.Consistent with the ability of endog?enous HA to inhibit HIV infection,hyaluronidase treatment significantly increased both X4(NL4-3)and R5( YK-JRCSF)infection of unstimulated CD4+T cells,but this enhancement could be completely reversed by ad?dition of exogenous HA(Fig.2).

These results indicate that hyaluronidase treatment renders unstimulated CD4+T cells more susceptible to HIVCD44infection,possibly through reduction of cell surface HA.

Fig.2Hyaluronidase treatment enhanced HIV infection of unstimulated CD4+T cells.Hase(Hyaluronidase treatment) (Sigma,St.Louis,MO);medium(medium treatment);Hase+HA:(Hyaluronidase treatment with 100 μg of exogenous HA(Sigma,St.Louis,MO));Medium+HA:(medium treatment with100 μg of exogenous HA).Treatment of healthy donor unstimulated CD4+T cells by hyaluronidase(Hase)boosted both X4(open bar)and R5(filled bar)HIV infection, and exogenous HA reduced this enhancement(Hase+HA).(All data are mean±SEM from duplicate samples and representative of at least 3 donors)

2.3 Hyaluronidase treatment enhances R5-HIV binding on unstimulated CD4+T cells

Next,we investigated how hyaluronidase treatment impacts HIV binding HA on the surface of unstimulated CD4+T cells.Our early results shown that hyaluronidase treatment can reduce endogenous HA thickness on surface of the un-stimulated CD4+T cells[24].Because the thickness of endogenous HA has been postulated to prevent ligand binding to cognate receptors on the surface of T-cells[27]and hyaluronidase treatment enhanced infectivity of both X4(NL4-3)and R5(YK-JRCSF),we hypothesized that hyaluronidase-mediated reduction of HA on the cell surface may enhance HIV binding.To test this hypothesis,unstimulated,primary CD4+T cells were treated by hyaluronidase and then incubated with R5(YK-JRCSF)in the presence 10 μg/mL of the peptide fusion inhibitor T-20[25]for 5 hours.Cells were washed 5 times to remove unbound HIV,stained with anti-HIV-1 p24 antibody,and studied by laser scanning microscopy.Hyaluronidase treatment significantly in?creases HIV binding on surface of the unstimulated,primary CD4+T cells(Fig.3A,B,C,D，E).

3 Discussion

Devising more effective interventions to prevent mucosal HIV transmission will require a deeper under?standing of the interplay between virus and target cells in the mucosal environment.Studies of both human and rhesus macaque models indicate that mucosal tissues provide multiple mechanisms to prevent HIV and SIV in?fection,including physical barriers and innate and adaptive immune responses[5,9].However,a small amount of HIV-1 and SIV can penetrate the mucosal barrier,gaining access to the sub-mucosa and the initial recipient cell targets,which appear to be predominantly resting CD4+T cells[8].Details of the interaction of HIV and SIVwith resting CD4+T cells in the sub-mucosa are not fully understood,but CCR5-tropic viruses(R5-HIV)are preferentially transmitted[5,9].In this study,we show that endogenous HA on unstimulated CD4+T cells is an ob?stacle for HIV infection and that hyaluronidase treatment can increase binding of HIV particles,and enhance HIV infectivity.Furthermore,addition of exogenous HA at 100 μg/mL,the concentration of HA in human epi?dermis[28],reduces HIV infectivity on unstimulated CD4+T cells.These data collectively argue that HA inter?feres with HIV infection during early stage,perhaps by directly impacting the interactions between CD44 andother molecules,or altering the distribution and/or activation status of local CD4+T cells,thus reducing targets which are needed for efficient transmission.

Fig.3(A,B,C)LSM510 META laser scanning microscope(Carl Zeiss Microimaging Inc.Thornwood,NY,USA)was used to scan the cells.Images are 40x;CD4+T cells were stained with mouse anti-HIV-1p24(Santa Cruz Biotech)and donkey anti-mouse IgG antibody with AlexaFluor@680(Life Technologies,Carlsbad,CA).CD4-PE(R&D Sytem),cell membrane staining(Life Technologies,Carlsbad,CA);and nuclear staining-DAPI(Life Technologies,Carlsbad,CA). (A)CD4+T cells treated with 100 μg of exogenous HA；(B)CD4+T cells treated with medium；(C)CD4+T cells treated with hyaluronidase

Fig.3 DFACS results:cells with isotype control,cells treated with 100 μg of HA,cells treated with medium and cells treated with hyaluronidase

Fig.3EPercentage of HIV-1-p24 positive cells treated with hyaluronidase(Hase)or with medium(Medium)or with 100 μg of HA(100 μg HA).(All data are mean± SEM from duplicate experiments representative of at least 3 donors)

HA is the main component of the extra cellular matrix(ECM)in the mucosal tissue.The main receptor for HA,CD44,is highly expressed in all cells,including CD4+T cells,in mucosal tissues,especially those along the reproductive tract[18,29].HA interactions with CD44 are involved in mucosal immunity and participate in mu?cosal inflammation[16,21].It has been reported that exogenous HA can interfere with interaction of endogenous HA with CD44,limiting recruitment of T cells by reducing T cell adhesion[30],preventing T cell rolling,and in?hibiting T cell extravasation into an inflamed site[19-20].Other studies identify the recruitment of T cells into sub?mucosa as important and necessary for mucosal HIV transmission[5,9].

Our study demonstrates that exogenous HA reduces both X4 and R5 HIV infection of unstimulated CD4+T cells.Others have reported that the endogenous HA coat on the surface of cells can prevent extraneous or in?discriminant ligand binding[18]and can prevent infection with newcastle disease virus(NDV),vesicular stomati?tis virus(VSV),and rubella virus(RV).Reducing the thickness of HA on the cell surface by hyaluronidase treatment can make target cells more susceptible to NDV,VSV,and RV infection[31].We also observed that treatment of unstimulated CD4+T cells with hyaluronidase reduced the thickness of endogenous HA on the cell surface[24],allowing more HIV particles to bind(Fig.3),an effect that could be reversed by the addition of exoge?nous HA(Fig.2).While these experimental results suggest that the inhibitory effects of HA on HIV infection occur at least in part at an early stage of the viral life cycle,including effects localizable to viral binding, further work is needed.

It is notable that HA,and hyaluronidase are all highly expressed and play important roles in maintaining the integrity and function of the mucosal tissue along the reproductive tract.Hyaluronidase is also a major con?stituent of semen[29].Both HA and hyaluronidase are highly up-regulated during sexual intercourse as well as in?flammation[18].The processes of reproductive fertilization and HIV transmission intersect at many levels anatom?ically and functionally.Both involve the reproductive tract and permissiveness of mucosal tissue and surface barriers.Clinical studies have demonstrated that inflammation in the mucosal tissue can facilitate HIV trans?mission[32],and that transmission rates are higher for HIV in semen compared to blood[33].The identification of topical agents that can be applied to mucosal surfaces to prevent mucosal HIV transmission has been identified as a priority area for research and development in the effort to control the HIV/AIDS epidemic[34].

In summary,HA present on the surface of unstimulated CD4+T cells can influence both X4 and R5-HIV infection.Both HA and hyaluronidase have well described roles in reproductive and inflammatory processes. Our data now show that the status of the HA coat may play an important role in the initial interaction of X4 and R5-HIV virions with resting CD4+T cells,a feature that may be particularly relevant for mucosal HIV transmis?sion.Intriguingly,exogenous HA appears to block HIV engagement and infection of target cells.HA is a non-immunogenic natural biopolymer that has been used in a variety of clinical applications.These findings should be further explored in vivo and,if confirmed,could lead to the development of novel interventions to re?duce HIV mucosal transmission.

Conflict of Interest

The authors declare that they have no conflict of interest.

Acknowledgments

This work was supported by the National Institutes of Health[grants 1R21AI104445-01A1(PL)].

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（責(zé)任編輯：陳曠）

Investigation of Pathomorphology and Biologic Characteristics of Triple-Negative Breast Cancer and Basal-Like Breast Cancer

WANG Chengxin，HUANG Xuan，F(xiàn)ENG Hua
（Jiangda Pathologic Diaglostic Institute；Department of Pathology&Pathophysiology,School of Medicine，Jianghan University, Wuhan 430056,Hubei,China）

ObjectiveTo investigate the pathomorphology，biologic characteristics and the relation?ships oftriple-negative breast cancer(TNBC)，non triple-negative breast cancer(NTNBC)andbas?al-like breast cancer（BLBC）.MethodsHER2，ER，PR，CK5/6 or EGFR protein were deter?mined with immunohistochemistry（IHC）in breast cancer,3 cases of HER2 IHC 3+and 5 cases of HER2 IHC 2+were analysed for HER2 gene amplification with fluorescent in situ hybridization（FISH）.CK5/6 or EGFR were checked in 22 cases of TNBC.The cases were divided into the TNBC，NTNBC and BLBC groups according to the examination results.The pathomorphology and bi?ologic characteristics of the three groups were compared.ResultsOf the 96 cases，27.08%（26/96）were confirmed to be TNBC in which gradeⅡandⅢcases were 53.85%（14/26）and 46.15%（12/ 26）respectively，lymph nodes metastasis rate was 61.11%（11/18）；BLBC accounted for 63.64%（14/ 22）of TNBC，in which gradeⅡandⅢcases were 57.14%（8/14）and 42.86%（6/14）respective?ly，lymph nodes metastasis rate was 55.56%（5/9）；There were no gradeⅠcases in TNBC and BLBC.72.92%（70/96）were confirmed to be NTNBC in which gradeⅠ，ⅡandⅢcases were 28.57%（20/70），57.14%（40/70）and14.29%（10/70）respectively，lymph nodes metastasis rate was 44.44%（16/ 36）.The percentage of poorly differentiated cases and lymph nodes metastasis rate were higher in the TNBC and BLBC group than those in NTNBC，besides there was no gradeⅠcases in TNBC and BLBC group.Of the 5 HER2 IHC 2+cases，one case there was gene amplification，one equivocal and three negative and of 3 HER2 IHC 3+cases,the FISH result was consistent with IHC.Conclu?sionMore than 1/4 of breast cancer are TNBC or BLBC in our study.Compared with NTNBC，even though TNBC and BLBC share a great deal of overlap and have a poor differentiation with aggressive behavior and increased local recurrence or distant metastasis，indicating a poor prognosis，they may be independent histopathologic entity.CK5/6 or EGFR can be used to differentiate the BLBC from TNBC.IHC 2+cases should be examined with FISH in order to avoid misleading treatment.

triple-negative breast cancer；basal-like breast cancer；pathomorphology；FISH

Exogenous Hyaluronic Acid Reduces R5-HIV Infection of CD4+T Cells

LI Peilin
（Department of Medicine,University of California San Francisco(UCSF),San Francisco CA 94121,USA)

外源性透明質(zhì)酸減少R5-HIV對(duì)CD4+T細(xì)胞感染的研究

2014-08-13

1R21AI104445-01A1(PL)

R737.902

A文章編號(hào)：1673-0143（2014）06-0057-05

R512.91文獻(xiàn)標(biāo)志碼：A

1673-0143（2014）06-0062-09

2014-09-29

王成鑫（1954—），男，教授，主任醫(yī)師，碩士生導(dǎo)師，研究方向：腫瘤病理。

Biography:LI Peilin(1958—)，male，associate professor，majors in molecular immunology.