Differences in the Immune Response of the Nonmetastatic Axillary Lymph Nodes between Triple-Negative and Luminal A Breast Cancer Surrogate Subtypes

Breast cancer (BC) comprises four immunohistochemical surrogate subtypes of which triple-negative breast (TNBC) has the highest risk mortality. Axillary lymph nodes (ALNs) are regions where BC cells first establish before distant metastasis, and presence tumor in ALN causes an immune tolerance profile that contrasts with nonmetastatic (ALN?). However, few studies have compared components ALNs? subtypes. The present study aimed to determine whether differences between populations primary were associated luminal A or TNBC subtype. We evaluated a retrospective cohort 144 patients using paraffin-embedded biopsies. samples tended higher histologic grade proliferation index had levels markers tumors ALNs?. Two methods for validating multivariate analysis found grade, intratumoral S100 dendritic cells, CD8 T lymphocytes CD57 natural killer factors TNBC, whereas CD83 In conclusion, we contained concentrations related than A. This finding partially explains worse prognosis TNBC. 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Ethics Committee Research approved (reference number 24p/2012). provided written informed consent participate data used. Strengthening Reporting Observational Studies Epidemiology (STROBE) guidelines followed. Pathologists selected representative areas obtain 2-mm cylinders: (intratumoral region) central region ALN? specimens. gave total 576 cylinders (352 224 TNBC), incorporated microarrays (TMAs) previously described.25Callau Garcia Lopez Evaluation cytokeratin-19 samples: automatic manual evaluations scanned microarray cylinders.Biomed Eng Online. 14: S2Crossref (12) 12 TMAs examind each TMA block 50 cylinders. Eleven slides sectioned TMA, studied, producing 132 slides. allow evaluation large cases, drawback they closely correlated whole-tissue sections permit diagnosis. Even so, use purposes considered adequate,26Pinder S.E. Brown J.P. Gillett Purdie C.A. Speirs Thompson A.M. 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Green Ki67 whole sections.Breast 164: 341-348Crossref (32) detection studied here, following antibodies (Figure 1) used: helper (anti-CD4, clone 4B12, Dako, Santa Clara, CA), cytotoxic (anti-CD8, C8/144B, Dako), (NK) (anti-CD57, NK1, Zymed, Thermo Fisher Scientific, Waltham, MA), regulatory [anti-forkhead box protein P3 (FOXP3), 236A/E7, CNIO, Madrid, Spain], macrophages (anti-CD68, KP1, follicular (DCs) (anti-CD21, 1F8, Langerhans DCs (anti-CD1a, 010, plasmacytoid (anti-CD123, 6H6, eBioscience, San Diego, interdigitant (anti-S100, polyclonal, Leica Microsystems GmbH, Wetzlar, Germany), lysosome-associated membrane glycoprotein (LAMP3) (anti-CD208, Proteintech, Rosemont, IL), mature (anti-CD83, clone1H4b, GmbH). final staining was ENDVISION FLEX method (Dako), chromogen diaminobenzidine (Dako) substrate hematoxylin counterstain, accordance manufacturer's protocol. Slides at ×40 magnification Aperio ScanScope XT scanner (Leica Nussloch, Germany). 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Springer, Berlin, Heidelberg2010: 213-221Google 34Korzynska Zak METINUS Plus nuclei quantification section.Biomed Signal Process Control. 32: 1-9Crossref pathologic compiled: age, diameter, nodes, axillar lymphovascular invasion, perineural (Ki-67), menopausal status. variables subytpes. mean median quantitative subtypes, independent-samples t-test U-test Categorical ?2 exact test. assess immunologic, clinical, (with reference), univariate logistic regression models fitted variable estimate odds ratio (OR) 95% CI. allowed us identify differentially attaining level significance P < 0.10 analyses develop model. Model goodness fit concluded if Hosmer-Lemeshow test nonsignificant variables. Any significant contributed model dichotomized according distribution: than/equal (normally distributed variable) (non-normally variable). As rule thumb, should based minimum 10 events included model.35Peduzzi Concato Kemper E. Holford T.R. Feinstein simulation analysis.J Epidemiol. 1996; 49: 1373-1379Abstract (4468) Scholar,36Vittinghoff McCulloch C.E. Relaxing ten Cox regression.Am 2007; 165: 710-718Crossref (2011) Luminal used reference analyses, event. With 4 variables, sample 57 therefore sufficient. ability correctly assign Nagelkerke's R2, sensitivity specificity, under receiver operating characteristic curve (AUC). validated statistical techniques. validation bootstrapping technique IBM SPSS Statistics version 23.0 (IBM, Armonk, NY), assuming CI 10,000 samples. second Multiple Imputation Stata software 14.0 (StataCorp LLC, College Station, TX). Ten imputed sets handle missing data, predictive outcomes imputation Rubin's rules combine estimates SEs. AUC calculated both methods. 0.05 statistically analyses. Table 1 summarizes characteristics expected, significantly proportion grades those group. Similarly, also percentage high Ki-67 expression.Table 1Differences Clinical Pathologic Variables Patients Triple-Negative ProfilesVariableLuminal (n = 88)Triple-negative 56)PAge, years61.1 ± 11.758.7 12.00.234?Independent-samples t-test.Tumor mm18.0 12.018.5 19.50.959†U-test.Metastatic n1.0 (2.0)0.0 (2.0)0.625†U-test.Axillar Positive46 (52.3)26 (46.4)0.608‡The Negative42 (47.7)30 (53.6)LVI Present42 (47.7)8 (50.0)1.000‡The Absent46 (52.3)8 (50.0)PNI Present28 (31.8)5 (31.3)1.000‡The Absent60 (68.2)11 (68.8)Histologic 124 (27.3)3 (5.4)<0.001‡The 245 (51.1)10 (17.9) 319 (21.6)43 (76.8)Ki-67 Low35 (39.8)26 (47.3)0.037‡The Medium35 (39.8)11 (20.0) High18 (20.5)18 (32.7)Menopausal status Premenopausal13 (15.9)2 (15.4)1.000‡The Menopausal69 (84.1)11 (84.6)LVI, invasion; PNI, invasion.Data expressed means SD age (interquartile range) n (%) rest data.? Independent-samples t-test.† U-test.‡ Open table tab LVI, invasion. Data data. concentration CD4, CD8, FOXP3, CD21, CD1a, CD123, S100, CD208 (Table 2). Nine differed respect area. similar results region, CD57, CD123 Conversely, group.Table 2Differences Concentration Populations Intratumoral Regions SubtypesVariableLuminal A(n 88)TNBC(n 56)PIntratumoral CD40.61 (1.11)3.34 (7.15)<0.001?U-test. CD81.06 (1.53)1.54 (3.31)0.050?U-test. CD570.10 (0.33)0.12 (0.24)0.993?U-test. FOXP30.06 (0.13)0.17 (0.31)<0.001?U-test. CD210.001 (0.027)0.006 (0.017)0.021?U-test. CD682.63 (2.16)3.69 (4.46)<0.001?U-test. CD1a0.09 (0.20)0.22 (0.68)0.009?U-test. CD1230.000 (0.000)0.035 (0.099)<0.001?U-test. S1000.12 (0.29)0.32 (0.62)0.002?U-test. CD2080.02 (0.08)0.07 (0.15)0.015?U-test. CD830.11 (0.18)0.11 (0.26)0.650?U-test.ALN? CD456.25 13.30)63.32 14.24)0.004†Independent-samples t-test. CD813.07 (8.47)19.45 (8.35)<0.001?U-test. CD570.18 (0.23)0.76 (0.82)<0.001?U-test. FOXP31.99 (1.42)2.12 (1.64)0.927?U-test. CD210.70 (1.76)1.01 (1.17)0.539?U-test. CD689.32 (4.93)9.91 (6.82)0.225?U-test. CD1a1.64 (3.49)1.76 (3.19)0.797?U-test. CD1231.32 (1.84)2.35 (3.13)0.001?U-test. S1004.16 (5.40)3.95 (4.48)0.621?U-test. CD2080.24 (0.41)0.18 (0.27)0.112?U-test. CD831.07 (1.26)0.19 (0.59)<0.001?U-test.Data stained marker.ALN?, node; forkhead P3; cancer.? U-test.† ALN?, cancer. associations estimated regressions presented 3. independently 3) indicated none when analysis. On hand, regarding DCs, lymphocytes, NK protective (P 0.839), indicating good fit.Table 3Univariate Multivariate Analysis Differences Compared SubtypeVariableUnivariate OR(95% CI)PMultivariate CI)PAge, years0.98 (0.95 1.01)0.233Tumor mm1.00 (0.98 1.02)0.761Metastatic n1.01 (0.94 1.08)0.837Axillar Positive0.79 (0.40 1.55)0.494 Negative1.0LVI Present1.10 (0.38 3.18)0.867 Absent1.0PNI Present0.97 (0.31 3.07)0.964 Absent1.0Histologic 318.11 (4.86 67.51)<0.001 21.78 (0.45 7.08)0.415 11.0PI (Ki-67) High1.35 (0.59 3.08)0.481 Medium0.42 (0.18 0.99)0.046 Low1.0Menopausal Postmenopausal1.04 (0.21 5.23)0.966 Premenopausal1.0Intratumoral CD41.26 (1.12 1.41)<0.001 CD81.07 (0.97 1.18)0.181 CD571.00 1.06)0.886 FOXP335.60 (4.79 264.68)0.001 CD21cGreater median2.50 (1.24 5.05)0.011Median less1.0 CD681.14 (1.01 1.30)0.034 CD1a1.82 (1.06 3.14)0.030 CD123c<0.001Greater median11.55 (5.12 26.06)Median S1001.93 3.70)0.0462.03 (1.09 3.77)0.026 CD2083.45 (1.65 7.19)0.001Greater medianMedian CD833.50 12.63)0.056ALN? CD41.04 1.07)0.005 CD81.09 (1.03 1.15)0.0011.15 1.28)0.011 CD57c<0.001<0.001Greater median10.82 (4.69 24.92)22.65 (4.63 110.73)Median less1.01.0 FOXP31.06 (0.79 1.43)0.685 CD210.96 (0.80 1.15)0.642 CD681.06 1.14)0.151 CD1a1.03 (0.93 1.14)0.541