The aim of the study was to compare morphometric characteristics of different types and grades of breast cancer. Morphometric analysis was performed using the SFORM software (Vamstec, Zagreb) on the May-Grünwald-Giemsa stained fine needle aspiration cytology (FNAC) breast tissue specimens. The study included 42 patients diagnosed with breast carcinoma by breast smear FNAC at Merkur University Hospital during the 2001–2005 period. Postoperative tumor histopathology and semi-quantitative tumor grading by the method of Elston and Ellis1 showed invasive ductal carcinoma grade I in 10, invasive ductal carcinoma grade II in 9, invasive ductal carcinoma grade III in 13, and invasive lobular carcinoma in 13 patients, the latter also including a subtype of invasive tubulolobular carcinoma. The following parameters were assessed by use of Statistica 7.1 and c 2-test: tumor area, circumference, maximal radius, minimal radius, convexity, length, width, elongation, nucleus/cytoplasm ratio, and shape factor. Morphometric analysis yielded statistically significant differences among all study groups (p<0.001). Morphometric parameters showed significant individual correlation with tumor type and grade, whereby the area, convexity and circumference were most significant at both nuclear and cellular level

Jakić-Razumović, Jasminka

Kardum-Skelin, Ika

Manojlović, Spomenka

Prvulović, Ivana

Susterčić, Dunja

English

University of Zagreb Medical School Repository

Coll. Antropol. 34 (2010) 1: 99–103Original scientific paperMorphometry of Tumor Cells in Different Gradesand Types of Breast CancerIvana Prvulovi}1, Ika Kardum-Skelin2,5, Dunja Suster~i}2, Jasminka Jaki}-Razumovi}3,5 andSpomenkaManojlovi}4,51 Department of Cytology, General Hospital »Dr. J. Ben~evi}«, Slavonski Brod, Croatia2 Laboratory for Cytology and Hematology, University Hospital »Merkur«, Zagreb, Croatia3 Department of Pathology, University Hospital Centre Zagreb, Zagreb, Croatia4 Department of Pathology, University Hospital Dubrava, Zagreb, Croatia5 University of Zagreb, School of Medicine, Zagreb, CroatiaA B S T R A C TThe aim of the study was to compare morphometric characteristics of different types and grades of breast cancer.Morphometric analysis was performed using the SFORM software (Vamstec, Zagreb) on the May-Grünwald-Giemsastained fine needle aspiration cytology (FNAC) breast tissue specimens. The study included 42 patients diagnosed withbreast carcinoma by breast smear FNAC at Merkur University Hospital during the 2001–2005 period. Postoperative tu-mor histopathology and semi-quantitative tumor grading by the method of Elston and Ellis1 showed invasive ductal car-cinoma grade I in 10, invasive ductal carcinoma grade II in 9, invasive ductal carcinoma grade III in 13, and invasivelobular carcinoma in 13 patients, the latter also including a subtype of invasive tubulolobular carcinoma. The followingparameters were assessed by use of Statistica 7.1 and c2-test: tumor area, circumference, maximal radius, minimal ra-dius, convexity, length, width, elongation, nucleus/cytoplasm ratio, and shape factor. Morphometric analysis yielded sta-tistically significant differences among all study groups (p<0.001). Morphometric parameters showed significant indi-vidual correlation with tumor type and grade, whereby the area, convexity and circumference were most significant atboth nuclear and cellular level.Key words: breast cancer, image analysis, morphometry, fine needle aspiration cytologyIntroductionInvasive breast cancer is the most common cancer inwomen. It accounts for 22% of all female cancers, whichis more than twice the occurrence of cancer in women atany other site. Invasive ductal carcinoma is the mostcommon type of invasive carcinoma of the breast, com-prising between 40–75%. This wide range is possibly dueto lack of application of strict criteria. Invasive lobularcarcinoma represents 5–15% of invasive breast tumorsthat also includes invasive tubulolobular carcinoma asits subtype2. Fine needle aspiration cytology (FNAC) issimple, quick and economic procedure that has impor-tant role in the early diagnosis of palpable and non-pal-pable breast lesions, and it is of great diagnostic value inthe preoperative evaluation of breast tumors3–5. Fre-quently, treatment and planning is preoperatively madelargely on the basis of the FNAC diagnosis. FNAC re-ports of breast malignancy should provide not only thediagnosis of malignancy but also the type and grade ofcarcinoma. Diagnosis of carcinoma may be made easily inthe majority of technically satisfactory aspirates, but de-termining the exact tumor type and grade may be trou-blesome. Standard morphologic analysis is subjective tosome extent and image analysis could be very useful sup-plement to FNAC. Image morphometry is quantitativemethod with two major goals in cytopathology: to add ob-jective measurement to diagnostic assessment and to im-prove diagnostic capabilities6. While the main aim of theFNAC is to differentiate between benign and malignantbreast disease, further classification could gain new ben-efits. Morphometric features which describe shape andsize could be used for diagnostic decisions for determina-tion of tumor type and grade7.99Received for publication June 28, 2009The aim of our study was to compare various morpho-metric features between different types and grades ofbreast cancer.Materials and MethodsMorphometric analysis was performed by means ofthe SFORM (Vamstec, Zagreb) software on the May--Grünwald-Giemsa (MGG) stained fine needle aspirationspecimens of breast cancer. Selected FNAC representedall breast smears from 2001 to 2005 that were malignanton cytological report and that was also postoperativelyhistologicaly confirmed in other Clinical Hospital in Za-greb. All of the cases were evaluated by computerized in-teractive morphometry (Figure 1). In the present studysystem was programmed to rapidly measure cell’s andnuclear features and to calculate their form factor, de-fined as physical size of object as measured by outside di-mensions, and nuclear/cytoplasmatic ratio. Nuclei andcell membrane of well preserved cells, without degenera-tive changes or artifacts and without overlapping be-tween them were outlined on the computer screen usinga computer mouse. At the same time a digital databasewas created in computer memory. Our investigated groupconsisted of 42 patients that were divided into 4 catego-ries regarding cancer type and grade, as follows:¿ 10 patients with invasive ductal carcinoma grade I(IDC I) (Figure 2)¿ 9 patients with invasive ductal carcinoma grade II(IDC II) (Figure 3¿ 13 patients with invasive ductal carcinoma gradeIII (IDC III) (Figure 4)¿ 10 patients with invasive lobular carcinoma (ILC)(Figure 5).From each case 100 cells and 100 nuclei were mea-sured, that totally made 4200 cells and 4200 nuclei. Thefollowing morphometric features were investigated: area,outline, maximum radius, minimum radius, convex area,breadth, length, nuclear/cytoplasmatic ratio, form factorand elongation. Statistical analysis was performed usingstatistic 7.1 and c2-test.ResultsFor all analyzed types and grades of breast cancer, thefollowing morphometric features showed increased meanvalues and were statistically significant for the cell andfor the nuclei: area, convex area and outline. No signifi-cant difference was noted between other investigatedfeatures: maximum radius, minimum radius, breadth,length, nuclear/cytoplasmatic ratio, form factor and elon-gation. In the group of patients with IDC malignant cellsshowed high variations in shape and size with increasingin mean cell and nuclear area following the progressionin tumor grade. Differences were highly statistically sig-nificant for mean nuclear and cell area and convex area(p<0.01) with increasing mean values of nuclear area inI. Prvulovi} et al.: Morphometry of Breast Cancer, Coll. Antropol. 34 (2010) 1: 99–103100Fig. 1. Computerized interactive morphometry.Fig. 2. FNAC smear of the breast. Invasive ductal carcinoma grade I.Fig. 3. FNAC smear of the breast. Invasive ductal carcinoma gradeII.tumors with grade progression from grade I to grade III.The same variables (area and convex area) were alsomost useful for distinction between ILC and all threegrades of IDC (p<0.001). Nuclear and cell features ofILC were greater then those of IDC I and much lowerthen those of IDC II and IDC III. The detailed resultswith mean values and standard deviation (SD) of themost useful morphometric features in our study (area,convex area and outline) for the cell and for the nucleiare shown in Tables 1 and 3 and p values are shown inTables 2 and 4.Discussion and ConclusionStandard morphologic analysis is to some extent sub-jective method and morphometric analysis, that is quan-titative method with objective and reproducible results,could be very useful supplement to FNAC. Most of ourtraditional knowledge of diagnostic cytology exists inconceptual form of descriptive terms and only a modestamount of such information is available in numeric form.Exact measurement could provide points of references,basic descriptive measures of value and variation, as abasis for checking reproducibility. Nowdays, demands forhigher productivity and higher accuracy in cytopatho-logy are increasing and it is clear, from abundant re-search on that subject, that, quantitative measurementof patterns and features of cells in cytological specimenscould be very helpful in routine cytopathology practice.Image morphometric analysis has already been appliedfor diagnosis and grading of IDC8–10 but only a smallnumber of studies compared different types and gradesof breast cancer using morphometric features as diagnos-tic variables. Yu et al. compared the morphometric fea-tures of IDC and ILC and found that 7 features, of 21 an-alyzed, were significant: area, defined as the number ofpoints within a nuclear image; kurtosis, a reflection ofthe »flatness« of the area histogram; long run length em-phasis, a measure of coarse texture; perimeter and bend-ing energy, a measure of object complexity and diameter1, a measure of nuclear width and diameter 2, a measureof nuclear length11. Compared to that study, Rajesh et al.investigated a large number of ILC (19 cases) and IDC(30 cases) and found that similar morphometric features(area, convex area, perimeter, convex perimeter, diame-ter and roundness) were helpful in distinguish ILC fromIDC12. However, in all studies mentioned above ILC wascompared to IDC regardless to its grade. It is well knownthat cytological differentiation between ILC and IDCgrade I could be very difficult. According to our results,I. Prvulovi} et al.: Morphometry of Breast Cancer, Coll. Antropol. 34 (2010) 1: 99–103101Fig. 4. FNAC smear of the breast. Invasive ductal carcinomagrade III.Fig. 5. FNAC smear of the breast. Invasive lobular carcinoma.TABLE 1CELL MORPHOMETRIC FINDINGS IN DIFFERENT TYPES AND GRADES OF BREAST CANCERType and grade of tumorArea (ìm2)X±SDOutline (ìm)X±SDConvex area (ìm2)X±SDIDC I 163.8397±25.29 51.2083±3.83 172.4295±25.48IDC II 184.2420±53.33 54.2363±8.64 194.5791±56.33IDC III 232.9815±66.61 60.4498±9.62 245.9972±69.76ILC 166.4137±37.46 53.2546±5.52 186.3856±31.20IDC I – invasive ductal carcinoma grade I, IDC II – invasive ductal carcinoma grade II, IDC III – invasive ductal carcinoma grade III,ILC – invasive lobular carcinomaapplied morphometrical analysis could be very helpful inthis area, especially with measuring of nuclear features.However, we analysed all subtypes of ILC as one groupbecause of small number of cases. ILC group in our study,including all subtypes of ILC, showed higher nuclear andcell pleomorphism, making the distinction of ILC andIDC I more successful.In our study, calculated values of mean cell and nu-clear area and convex area in IDC increased with pro-gression of grade, and differences were highly statisti-cally significant (p<0.01). Mean cell and nuclear outlinealso increased with grade progression (p<0.05).In our study out of all investigated features the fol-lowing three were the most useful ones: area, convexarea and outline, and they were significant for the celland for the nuclei comparing all three grades of IDC be-tween themselves.The differences in the observed values of differentmorphometric features among different publications maybe due to applications of different morphometric meth-ods. However, a strictly standardized and uniform mea-suring technique, with regular calibration of the compute-rized morphometric equipment could enhance reprodu-cibility.In addition to the previously mentioned studies, Elza-gheid and Collan investigated only nuclear morphometrybecause outlining of the nuclear borders was easily per-formed making nuclear measurements reliable and re-producible, while cell membrane outlining was more dif-ficult due to overlapping of cells in groups13. In our studyI. Prvulovi} et al.: Morphometry of Breast Cancer, Coll. Antropol. 34 (2010) 1: 99–103102TABLE 2STATISTICALLY SIGNIFICANT QUANTITATIVE CELL MORPHOMETRIC VARIABLES BETWEEN STUDIED GROUPSAREA CONVEX AREA OUTLINEIDC I / IDC II p<0.00000 p<0.00000 p<0.0431IDC I / IDC III p<0.00000 p<0.00000 p<0.00005IDC II / IDC III p<0.0253 p<0.00000 p<0.00000IDC I / ILC p<0.00000 p<0.00000 p<0.0191IDC II / ILC p<0.00000 p<0.00000 p<0.000015IDC III / ILC p<0.00000 p<0.00000 p<0.0243IDC I – invasive ductal carcinoma grade I, IDC II – invasive ductal carcinoma grade II, IDC III – invasive ductal carcinoma grade III,ILC – invasive lobular carcinomaTABLE 3NUCLEAR MORPHOMETRIC FINDINGS IN DIFFERENT TYPES AND GRADES OF BREAST CANCERType and grade of tumorArea (mm2)X±SDOutline (mm)X±SDConvex area (mm2)X±SDIDC I 85.4926±12.49 36.4892±3.20 88.4191±13.15IDC II 89.0447±34.24 36.0676±7.22 91.5290±34.93IDC III 116.9542±32.73 41.6016±6.16 120.3853±33.66ILC 87.2724±21.25 39.2048±6.15 90.6378±31.78IDC I – invasive ductal carcinoma grade I, IDC II – invasive ductal carcinoma grade II, IDC III – invasive ductal carcinoma grade III,ILC – invasive lobular carcinomaTABLE 4STATISTICALLY SIGNIFICANT QUANTITATIVE NUCLEAR MORPHOMETRIC VARIABLES BETWEEN STUDIED GROUPSAREA CONVEX AREA OUTLINEIDC I / IDC II p<0.00000 p<0.00000 p<0.0289IDC I / IDC III p<0.00000 p<0.00000 p<0.0206IDC II / IDC III p<0.00000 p<0.00000 p<0.0004IDC I / ILC p<0.00000 p<0.00000 p<0.256IDC II / ILC p<0.00000 p<0.00000 p<0.00088IDC III / ILC p<0.00000 p<0.00000 p<0.487IDC I – invasive ductal carcinoma grade I, IDC II – invasive ductal carcinoma grade II, IDC III – invasive ductal carcinoma grade III,ILC – invasive lobular carcinomawe analyzed only single cells, without overlapping, mak-ing both cytoplasmatic and nuclear features available forinvestigation.When performed as a part of routine everyday workmorphometric analysis is more laborious and time con-suming than the traditional cytological analysis. How-ever, if we improve the method, and investigate only themost valuable morphometric features, this method wouldnot be too time-consuming for cytopathologists, espe-cially if they were adequately trained. The morphometricanalysis could help in standardizing grading of breastcancer between different laboratories and an addition toquality control14.Development of a quantitative morphometrical me-thod requires continued efforts. Because of our encour-aging results, we started a larger, prospective study in-volving DNA image analysis with the aim to improvecytological grading and image morphometry.R E F E R E N C E S1. ELSTON CW, ELLIS IO, Histopathology, 19 (1991) 403. — 2.ELLIS IO, SCHNITT SJ, SASTRE-GARAU X, BUSSOLATI G,TAVASSOLI FA, EUSEBI V, PETERSE JL, MUKAI K, TABAR L,JACQUEMIER J, CORNELISSE CJ, SASCO AJ, KAAKS R, PISANI P,GOLDGAR DE, DEVILEE P, CLETON-JANSEN MJ, BØRRESEN--DALE AL, VAN’T VEER L, SAPINO A, Invasive breast carcinoma. In:JAFFE ES, HARRIS NL, STEIN H, VARDIMAN JW, (Eds) World HealthOrganization Classification of Tumors. Pathology and Genetics of Tu-mors of the Breast and Female Genital Organs (IARC Press, Lyon, 2003).— 3. TAYE AA, GEMECHU T, Ethiop Med J, 36 (1998) 219. — 4. SCOPACD, KOUKOURAS D, SPILIOTIS J, HARKOFTAKIS J, KOURELEAS S,KYRIAKOPOULOU D, TZORACOLEFTHERAKIS E, Cancer DetectPrev, 20 (1996) 620. — 5. SREENIVAS M, KUMAR GH, REDDY SJ,BHASKARAN CS, Indian J Pathol Microbiol, 32 (1989) 133. — 6.MARCHEVSKY AM, BARTELS PH, Image analysis a primer for patholo-gists (Raven Press, New York, 1994). — 7. CASTREN JP, KUOPIO T,NURMI MJ, COLLAN YU, J Urol, 154 (1995) 1302. — 8. DAWSON AE,AUSTIN RE JR, WEINBEREG DS, Am J Clin Pathol, 95 (1991) 29. — 9.THALAN A, NIJHAWAN R, JOSHI K, Analyt Quant Cytol Histol, 22(2001) 193. — 10. DEY P, GHOSHAL S, PATTARI SK, Analyt QuantCytol Histol, 22 (2000) 483. — 11. YU GH, SNEIGE N, KIDD LD,JOHNSTON DA, KATZ RL, Analyt Quant Cytol Histol, 17 (1995) 88. —12. RAJESH L, DEY P, JOSHI K, Analyt Quant Cytol Histol, 24 (2002)81. — 13. ELZAGHEID A, COLLAN Y, Analyt Quant Cytol Histol, 25(2003) 73. — 14. KRONQVIST P, KUOPIO T, JALAVA P, COLLAN Y, Br JCancer, 87 (2002) 1257.I. Prvulovi}Department of Cytology, General Hospital »Dr. Josip Ben~evi}«, Andrije [tampara 42, 35 000 Slavonski Brod, Croatiae-mail: ivanahodak@yahoo.comMORFOMETRIJA TUMORSKIH STANICA KOD RAZLI^ITIH GRADUSA I TIPOVA KARCINOMADOJKES A @ E T A KCilj istra`ivanja bio je usporediti morfometrijske karakteristike karcinoma dojke razli~itog subtipa i stupnja (gra-dusa) diferenciranosti. Morfometrijska analiza provedena je na citolo{kim razmazima punktata dojke obojenim po me-todi May-Grünwald-Giemsa (MGG) programom SFORM (Vamstec, Zagreb). Istra`ivanjem su obuhva}ene 42 bolesnicekod kojih je dijagnoza karcinoma dojke postavljena na uzorcima dobivenim citolo{kom punkcijom dojke u Klini~kojbolnici Merkur u razdoblju od 2001. do 2005. godine. Postoperativno je u~injena patohistolo{ka verifikacija te odre|i-vanje gradusa tumora semi-kvantitativnom metodom prema Elstonu i Ellisu1. Kod 10 bolesnica radilo se o invazivnomduktalnom karcinomu gradusa I (IDC I), kod 9 bolesnica o invazivnom duktalnom karcinomu gradusa II (IDC II), kod13 bolesnica o invazivnom duktalnom karcinomu gradusa III (IDC III), a kod 10 bolesnica postavljena je dijagnozainvazivnog lobularnog karcinoma (ILC) u koji je uklju~en i invazivni tubulo-lobularni karcinom (TLC) kao njegov pod-tip. Istra`ivani su slijede}i parametri: povr{ina, opseg, maksimalni radijus, minimalni radijus, konveksitet, du`ina,{irina, izdu`enost, omjer jezgra/citoplazma i faktor oblika. Kori{tena je statistika 7,1, c2-test. Morfometrijska je analizau ovom istra`ivanju pokazala statisti~ki zna~ajne razlike, (p<0,001), izme|u svih ispitivanih grupa. Morfometrijski suparametri, primjenjeni pojedina~no, bili zna~ajno povezani sa tipom i gradusom tumora, a najzna~ajniji su parametri, iza jezgru i za stanicu, bili: povr{ina, konveksitet i opseg.I. Prvulovi} et al.: Morphometry of Breast Cancer, Coll. Antropol. 34 (2010) 1: 99–103103

Morphometry of tumor cells in different grades and types of breast cancer [Morfometrija tumorskih stanica kod različitih gradusa i tipova karcinoma dojke]

Morphometry of tumor cells in different grades and types of breast cancer [Morfometrija tumorskih stanica kod različitih gradusa i tipova karcinoma dojke]

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