Short CommunicationCorrelation of E-Cadherin Expression withDifferentiation Grade and Histological Type inBreast Carcinoma
Carlos Gamallo,* Jose Palacios,*Asuncion Suarez,* Angel Pizarro,*Pilar Navarro,t Miguel Quintanilla,t andAmparo CanotFrom the Departamento de Anatomia Patol6gica,* HospitalLa Paz, and Instituto de Investigaciones Biomedicas,Consejo Superior de Investigaciones Cientificas; and theDepartamento de Bioquimica,t Facultad de MedicinaUniversidad Aut6noma, Madrid, Spain
Recently, a correlation has been suggested be-tween a loss ofE-cadherin (E-CD) and increasedinvasiveness of neoplastic ceUs. In this study,E-CD expression in breast cancer was investi-gated using an affinity-purifled antibody(ECCD-2) in an immunoenzymatic (avidin-biotin-alkaline phosphatase) test. Intensity and exten-sion ofE-CD immunoreactivity were evaluated in61 breast carcinomas and correlated with theirhistological type and grade, nodal involvement,and hormonal receptor status. Histological typeswere infiltrating ductal carcinoma of no specialtype (n = 54) and inflltrating lobular carcinoma(n = 7). AU infiltrating ductal carcinomas of nospecial type except two grade 3 carcinomasshowedpositive immunoreactivity that was vari-able among different cases. Grade 1 breast car-cinomas (n = 10) showed greater immunoreac-tivity than grade 2 (n =25) andgrade 3 (n = 19)carcinomas. E-CD immunoreactivity correlatedpositively with the degree of tubularformationand inversely with the mitoses number. None ofthe inflltrating lobular carcinomas expressedE-CD in their infiltrating ceUs, whereas theyshowed only weak immunostains in areas ofatyp-ical lobular hyperplasia and lobular carcinomain situ These results indicate that E-CD expres-
sion correlates with histological type and gradein breast carcinomas. (Am J Pathol 1993, 142:987-993)
Cadherins are a family of Ca2+-dependent cell-celladhesion receptors that play a major role during em-bryonic development and in the maintenance of adulttissue architecture.1 Several types of cadherins havebeen identified, such as E-cadherin (E-CD) (uvo-morulin, cell-CAM 120/180, L-CAM), P-CD, and N-CD(A-CAM). Although all subclasses share similar prop-erties such as molecular weight, Ca2+ dependence,and protease sensitivity, each homophilic subclass ischaracterized by a unique tissue pattern distributionand selective interactions.2 In adult organisms, E-CDis expressed in most normal epithelial tissues but isabsent in lymphoid and connective tissues. Recently,E-CD expression has been demonstrated in a par-ticular subset of sensory neurons.3 E-CD immunore-activity has been identified in carcinomas of differentorigins but not in mesenchymal or lymphoid neopla-sias.45 It has been suggested that the selective lossof E-CD can generate dedifferentiation and invasive-ness in human carcinomas, supporting a role forE-CD as an invasion suppressor molecule.67
Breast carcinoma is an important cause of mor-bidity and mortality among women. A major researchfield is the identification of molecular events under-lying invasiveness and metastatic progression of thisneoplasia to develop prognostic indicators. In this re-
Supported by research grants 92/0505 and BAE 92/5704 (AP) ofthe Fondo de Investigaciones Sanitarias de la Seguridad Social,Spain.Accepted for publication December 28, 1992.
Address reprint requests to Dr. Carlos Gamallo, Departamentode Anatomia Patol6gica, Hospital La Paz, Paseo de la Castellana,261, 28046 Madrid, Spain.
987
988 Gamallo et alAJP April 1993, Vol. 142, No. 4
port, we describe the results of our studies concern-ing the E-CD expression in breast carcinoma in anattempt to correlate these data with the usual clini-copathological features useful in the assessment ofbreast cancer prognosis.
Materials and MethodsSpecimens
Breast tissue was obtained from 61 unselectedmastectomy specimens submitted to the Depart-ment of Pathology, La Paz Hospital, Madrid. Neo-plastic and nonneoplastic breast tissue sampleswere embedded in ornithine carbamoyltransferasecompound (Miles Laboratory, Naperville, IL), snap-frozen in liquid nitrogen-cooled isopentane, andstored at -70 C. The remaining breast tissue andaxillary lymph nodes were routinely fixed in 10% for-malin for 24 hours and embedded in paraffin.
Immunohistochemical Staining
Immunostaining was performed as previously re-ported.8 Briefly, 5 to 6-p cryostat sections were im-munostained by the avidin-biotin-alkaline phos-phatase method. Nonspecific antibody binding wasblocked with Tris buffer containing 2% goat serum.Tissue sections were incubated with the monoclonalantibody ECCD-29 (1/250), biotinylated sheep anti-rat IgG (1/200, Amersham International, Bucking-hamshire, UK), and extravidin-alkaline-phosphatasecomplex (1/250, BioMakor, Rehovot, Israel). The al-kaline phosphatase activity was developed usingnaphtyl ASMX phosphate as substrate and Fast-Red as the chromogen group (Sigma Chemical Co.,St. Louis, MO). The sections were finally counter-stained with Meyer hematoxylin.The monoclonal antibody ECCD-2 was a gener-
ous gift of M. Takeichi (University of Kyoto, Kyoto,Japan). This rat monoclonal antibody againstmouse cadherin also recognizes E-CD in the humanmammary tumor cell line MCF-7.10 Dilutions ofECCD-2 were carried out in 150 mmol/L NaCI, 10mmol/L Hepes, pH 7.4, 10 mmol/L CaCI2 (HMF-Cabuffer), containing 1% (wt/vol) bovine serum albu-min. The secondary antibody was diluted in Trisbuffer containing 1 % bovine serum albumin. Normalmouse and human skin were used as positivecontrol. These samples showed intense E-CD im-munoreactivity along the cell membrane of kera-tinocytes. As negative control, we used rat anti-mouse IgG as the primary antibody. To evaluatetissue sample preservation, in all E-CD-negative
specimens, serial sections were immunostained bymonoclonal antibody class major histocompatibil-ity complex antigen (Dako, Copenhagen, Denmark).All these specimens showed a positive immunore-activity of variable intensity for class major histo-compatibility complex in the epithelial and stromalcells.
Evaluation of ImmunohistochemicalStaining
A semi-quantitative estimation based on the stain-ing intensity and relative abundance of E-CD immu-noreactive cells was performed independently bytwo pathologists. The intensity was graded from 0(equivalent to background staining of the acellularstroma) to +3 (intense stain equivalent to normalbreast epithelium). The abundance of E-CD positivecells was graded from 0 to 4 by counting at least100 tumor cells in areas of heterogeneous E-CD ex-pression (0 = less than 5% of positive cells; 1 = 5to 25%; 2 = 26 to 50%; 3 = 51 to 75%; 4 = 76 to100%). With these data, a composite score was ob-tained by adding the values of the immunoreactionintensity and relative abundance. Preserved E-CDexpression was estimated when the compositescore was 6 or 7. Score 5 indicated moderately re-duced E-CD expression, and scores 0 to 4 severelyreduced E-CD expression. In six instances, therewas a disagreement in the score assigned by thetwo pathologists, and the slides were reviewed by athird observer who did not know the nature of theantigen being tested and the hypothesis under in-vestigation. A consensus based in the two most co-incidental opinions was obtained.Carcinoma histological typing was performed on
formalin-fixed and paraffin-embedded samples. Thecombined histological grade (1, 2, and 3) of infiltrat-ing ductal carcinomas was obtained according toElston,11 using the scale assigned to three features:tubular formation (1 to 3), nuclear atypia (1 to 3),and mitoses (1 to 3). The tumor size, lymph nodestatus (0 versus any positive axillary lymph nodes),and the amount of estrogen and progesterone re-ceptors of the tumor defined by the steroid bindingassay (Dextran-coated charcoal analysis) were alsoevaluated. The x2 test was used to analyze the sta-tistical significance between E-CD expression andthe histological grade. The Kendall's test was usedto evaluate the statistical correlations between E-CDexpression and tubular formation, nuclear atypia,mitoses, tumoral size, nodal status, and estrogenand progesterone receptors.
E-Cadherin in Breast Carcinoma 989AJP April 1993, Vol. 142, No. 4
Results
E-CD Expression in Normal Breast Tissueand Benign Breast Lesions
Lobular and ductal epithelium of normal breast tis-sue expressed E-CD in a regular array on lateralcell borders. A similar immunoreactivity pattern wasobserved in ductal hyperplasia.
E-CD Expression and Histological Type ofBreast Carcinoma
Of the 61 samples analyzed, 54 cases were infiltrat-ing ductal carcinomas of no special type (DCNST)and the remaining seven cases were infiltrating lob-ular carcinomas (ILC). The immunoreactivity scoresestimated in the 54 DCNSTs are shown in Table 1.Twenty-seven DCNSTs had preserved E-CD expres-sion (Figure 1, A and B, and Figure 2A), whereas in27 cases it was reduced (Figure 1, C and D, andFigure 2B), including two cases with complete lossof E-CD (Figure 2C). The seven ILCs showed amarked alteration of E-CD expression. The areas ofatypical lobular hyperplasia and lobular carcinomain situ had decreased and heterogeneous E-CD im-munoreactivity, whereas it was absent in both thepagetoid ductal spread and infiltrating cells in thestroma (Figure 3, A and B). In all evaluated ILCs,normal breast epithelium was present and showedpreserved E-CD expression.
Correlation between E-CD Expression andPathological Features in DCNSTs
Table 1 shows E-CD expression level in DCNSTs inrelation to their histological grade. The frequency of
Table 1. E-CD Expression Compared with HistologicalGrade in Infiltrating Ductal Carcinoma of theBreast
Histological gradeE-CD expression* 1 2 3
0-4 1 4 65 0 11 56 4 5 77 5 5 1
Total (54) 10 25 19
X2 test. P = 0.0233. Histological grade was obtained by assess-ing tubular formation, nuclear atypia, and mitoses according to El-ston.10
* Composite score obtained by adding the intensity of immu-noreaction (1 to 3) and relative abundance of positive cells (1 to 4).Normal breast epithelium had expression score 6 to 7. E-CD -E-cadherin.
tumors with reduced E-CD expression was signifi-cantly higher (P = 0.0233) in histological grade 2and 3 breast carcinomas than in grade 1 tumors. Asignificant positive correlation was obtained whencomparing E-CD expression with the tubular forma-tion pattern, but a negative correlation resulted be-tween the E-CD expression and mitoses. No corre-lation was observed with nuclear atypia, hormonalreceptor levels (estrogen and progesterone), lymphnode status, or tumor size.
DiscussionThe E-CD expression in breast carcinoma has beenmainly studied in cell lines,5 and only two previousstudies have analyzed the expression of this mole-cule in a reduced number of surgical specimens.4,12 In these latter works, only a few examples of in-filtrating ductal carcinomas have been evaluated. Inthe present study, we have analyzed for the firsttime the E-CD expression among different histologi-cal types of breast carcinomas. An interesting find-ing is the observation that lobular neoplasias arecharacterized by an important alteration in the E-CDexpression. Decreased and heterogeneous expres-sion was observed in lobular hyperplasia and inlobular carcinoma in situ, whereas all ILC infiltratingcells showed a complete loss of E-CD immunoreac-tivity. Similar results have been recently reported inILC.13 ILC is characterized by a diffuse infiltratingpattern of small and round, regular cells in singlelines between collagen bundles.14 Further studiesanalyzing a higher number of ILC and other adhe-sion molecules would be of interest to evaluatewhether or not lack of E-CD expression could par-ticipate in the lack of cell adherence characteristicof ILC.
The present study confirms the previous observa-tion in infiltrating ductal carcinomas that most tu-mors express this adhesion molecule, but some ofthem show quantitative abnormalities in their E-CDexpression. Eidelman et a14 studied five tumors andobserved that all cases expressed E-CD. Shiozakiet al12 have reported that nine of 20 cases (45%)displayed reduced E-CD expression in infiltratingductal carcinomas. In concordance with these latterresults, we have observed reduced E-CD expres-sion in 27 of 54 cases studied (50%). However,these authors did not find any correlation betweenE-CD expression and the differentiation grade ofbreast carcinoma nor other clinicopathological fea-tures derived from the TNM classification. In con-trast, we have found that E-CD expression corre-lates with the differentiation grade, the degree of
990 Gamallo et alAJP Apil 1993, Vol. 142, No. 4
Figure 1. Different patterns ofE-CD expressionin several infiltrating ductal carcinomas grade1 and 2. E-CD immunoreactivity is preservedin a breast carcinoma grade I with prominenttubularformation (A) and in a grade 2 breastcarcinoma with cohesive solid nests (B). Re-duced E-CD expression in two grade 2 breastcarcinomas with heterogeneous immunoreac-tivity among trabecular neoplastic cells (C andD). Note the weak immunostaining in (D)(Original magnification X40 in A, X 63 in B,and x 60 in C and D).
tubule formation, and mitoses. The discrepancywith both studies may be due in part to the differ-ence in the number of cases analyzed. Our resultsin breast carcinomas are similar to those reported ingastric carcinoma.12 In both types of neoplasias,highly differentiated tumors generally maintainstrong and homogeneous E-CD expression,whereas in poorly differentiated carcinomas, E-CD-positive and negative cells can be mixed or, in ex-treme cases, expression can be virtually sup-pressed throughout the tumor, mainly in areascomposed by poorly cohesive cells.
From studies with a variety of human cell lines, ithas been suggested that E-CD represents an addi-tional differentiation marker of human breast carci-noma cells. In addition, E-CD expression inverselycorrelates with in vitro invasiveness.6 Cell lines de-rived from well-differentiated human carcinomas(which kept the epithelial phenotype in tissue cul-tures) generally express E-CD, whereas the mole-
cule is not detected in most cell lines from poorlydifferentiated carcinomas. These cells show a fibro-blastoid phenotype in tissue culture. Moreover,E-CD-negative cell lines were found to be invasivein collagen gels, whereas E-CD-positive lines gen-erally did not.6 Other experimental studies havesuggested a reversible down-regulation of E-CD ex-pression. Thus, ras-transformed Madin Darby Ca-nine Kidney cells lose E-CD during tumor formationin the nude mouse, but re-expression of this adhe-sion molecule is observed in derived cell lines.15
It has been reported that in vivo E-CD expressioninversely correlates with lymph node metastasis insquamous cell carcinomas of the head and neck16but not in gastric carcinomas.17 Although we havefound an inverse correlation between the differentia-tion grade of breast carcinomas and E-CD expres-sion, numerous invasive high-grade tumors largelyretain normal E-CD immunoreactivity, and no corre-lation was observed between E-CD expression and
E-Cadherin in Breast Carcinoma 991AJP Apil 1993, Vol. 142, No. 4
Figure 2. Different patterns ofE-CD expressionin several infiltrating ductal carcinomas grade3. A: E-CD immunoreactivity is preserved inthis breast carcinoma with cohesive cells. B:Partial loss ofE-CD expression. C: E-CD immu-noreactivity is completely absent in a poorlydifferentiated breast carcinoma with noncohe-sive cells. Note E-CD expression in normal epi-thelium duct. (Original magnification X40 inA and x 63 in B and C).
nodal status. These observations could be ex-plained by a temporary loss and posterior re-expression of E-CD or by biochemical suppressionof the action of cadherins or their associated pro-teins. Thus, it has been reported that cadherin-
mediated cell-cell adhesion is perturbed by v-srctyrosine phosphorylation of the cadherin-cateninsystem in metastatic fibroblast.18 According to ourresults, we consider it unlikely that any single cell-surface alteration alone, such as loss of E-CD ex-
Figure 3. E-CD expression in lobular carcino-ma. E-CD immunoreactivity is negative in twoexamples of infiltrating lobular carcinoma withan Indian file pattern (A) and a targetoid pat-tern (B) of infiltration; note preserved immu-noreactivity in normal ducts (arrows in A).(Original magnification X 16).
992 Gamallo et alAJP Apil 1993, Vol. 142, No. 4
pression, is responsible for the invasive and meta-static potential of neoplastic cells. Althoughinvasiveness and metastasis can be enhanced bythe down-regulation of E-CD expression, the alter-ation of other adhesion processes, such as cell-substrate adhesion,19 or other genetic events suchas low nm23 expression20'21 might be necessary forthe invasiveness and metastases development. Fur-ther studies are necessary to evaluate survivalamong breast carcinoma patients with differentE-CD expression and to establish if this might be anindependent predictor of survival in patients withthis type of neoplasia, mainly in histological grade 2and 3 carcinomas.The estrogens seem to modulate the adhesive
properties of breast carcinoma cells. For example,the expression of integrin adhesive receptors inbreast carcinomas closely parallels the expressionof estrogen receptors as judged immunohistochem-ically.18 Moreover, it has been demonstrated thatthe breast carcinoma cell line MCF-7, which ex-presses estrogen receptors, also expressesE-CD,6'10 whereas three dedifferentiated hormonalindependent MDA-MB lines are E-CD-negative.6However, we could not find any correlation betweenthe E-CD expression in ductal breast carcinomaand the estrogen and progesterone receptors ex-pression assessed biochemically. Because this ap-proach does not allow a morphological evaluationof both E-CD and hormonal receptor expression inthe same cells, immunohistochemical analysis ofestrogen and progesterone receptors is currentlybeing performed in this DCNST series.
In short, results in the present series suggest thatE-CD expression in breast carcinoma is more re-lated to histological type and differentiation gradethan with invasiveness and metastatic potential.
AcknowledgmentsWe thank Josefina Duran and Pilar Ocania for tech-nical assistance with the immunohistochemicalstudy. We are also grateful to Dr. M. Takeichi for hisgenerous gift of the monoclonal antibody ECCD-2and to Rosario Madero for the statistical survey.
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15. Mareel MM, Behrens J, Birchmeier, De Bruyne GK,Vleminckx, Hoogewijs A, Fiers W, Van Roy FM: Down-regulation of E-cadherin expression in Madin Darbycanine kidney (MDCK) cells inside tumors in nudemice. Int J Cancer 1991, 47:922-928
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