Clinicopathologic characteristics of invasive breast cancer
The clinical features of patients were shown in Table 1. Most cases (78.5%) were invasive carcinoma of no special type. Mean tumor size was 33 mm ranging from 6 to 150 mm. As for histologic grade, 65.9% was high. In addition, 143 cases (56.7%) were classified as T2. Results of ER, PR and HER2 expression were also summarized as follows: 58.7% were ER-positive, 53.2% were PR-positive and 46% were HER2-overexpression, respectively. Specially, 85% ER positive cases had PR expression.
Table 1
Clinicopathologic features of all patients suffering from breast carcinoma (N = 252)
Clinicopathologic parameters | Number of sample (%) |
Age (years) (mean 50, median 49, range 25–76) |
< 40 | 23 (9.1%) |
40 to 50 | 124 (49.2%) |
> 50 | 105 (41.7%) |
Tumor size (mm) (mean 33, median 30, range 6–150) |
< 20 | 41 (16.3%) |
20 to 50 | 174 (69.0%) |
> 50 | 19 (7.5%) |
Unknown | 18 (7.1%) |
Histologic grade |
1 | 6 (2.4%) |
2 | 73 (29.0%) |
3 | 166 (65.9%) |
Not assessable | 7 (2.8%) |
Expression of ER, PR and HER2 (Positive/Negative) |
ER (+/-) | 148/104(58.7%/41.3%) |
PR (+/-) | 134/118(53.2%/46.8%) |
HER2(+/-) | 116/136(46.0%/54.0%) |
Histologic subtype |
IDC (NOS) | 198 (78.5%) |
ILC | 24 (9.6%) |
Other invasive breast carcinomas | 25 (9.9%) |
Unknown | 5 (2.0%) |
Lymph node status |
Metastasis in 1 to 3 lymph nodes | 159 (70.6%) |
Metastasis in 4 or more lymph nodes | 60 (23.8%) |
Not available | 14 (5.6%) |
Stage | |
T1 | 35 (13.9%) |
T2 | 143 (56.7%) |
T3 | 41 (16.3%) |
T4 | 20 (8.4%) |
Unknown | 13 (5.2%) |
Cytomorphological features with ER+, PR + and HER2-overexpression tumors
The nuclei of ER + and/or PR + tumors (Fig. 1a) was smaller than HER2-overexpression tumors (Fig. 1b). In ER + and/or PR + tumor cells (Fig. 1c), the nucleoli accounted for about 1/5 of the nuclear diameter and was dispersed or clinging to the nuclear membrane. In HER2-overexpression tumor cells (Fig. 1d), the nucleoli accounted for about 1/3 of the nuclear diameter and was located in the center of the nuclei.
Correlation of cytomorphological features with status of ER, PR and HER2
To explore the relationship between cytological characteristics and prognostic factors, we analyzed 11 parameters. The results were summarized in Table 2. Among the eight standard parameters, dissociation and nuclear margin were from Robinson’s system, and nuclear/cytoplasmic ratio and density of chromatin were from Taniguchi’s system. And the three new parameters including nucleolar size, nucleolar location and nucleolar number were based on our observation. In 252 samples, ER + tumors had smaller cell than ER- tumors (P < 0.001). ER- tumors showed more marked pleomorphism (P < 0.001), nucleoli (P < 0.001) and folds, irregular nuclear margin (P < 0.001) than its competitor. In addition, significant difference was observed among PR + and PR- samples.
Table 2
Correlation between cytologic parameters (eight standard and three new parameters) and the status of ER, PR and HER2
| ER | | PR | | HER2 | |
| Positive | Negative | P-value | Positive | Negative | P-value | Positive | Negative | P-value |
Dissociation | | | 0.971 | | | 0.508 | | | 0.037 |
clusters | 34(23.0) | 28(26.9) | | 33(24.6) | 29(24.6) | | 26(22.4) | 36(26.5) | |
single and clusters | 76(51.4) | 45(43.3) | | 68(50.7) | 53(44.9) | | 49(42.2) | 72(52.9) | |
single | 38(25.7) | 31(29.8) | | 33(24.6) | 36(30.5) | | 41(35.3) | 28(20.6) | |
Cell size | | | < 0.001 | | | < 0.001 | | | < 0.001 |
1-2xRBC | 52(35.1) | 7(6.7) | | 48(35.8) | 11(9.3) | | 12(10.3) | 47(34.6) | |
2-4xRBC | 84(56.8) | 46(44.2) | | 71(53.0) | 59(50.0) | | 57(49.1) | 73(53.7) | |
≥ 5xRBC | 12(8.1) | 51(49) | | 15(11.2) | 48(40.7) | | 47(40.5) | 16(11.8) | |
Nuclear pleomorphism | | < 0.001 | | | < 0.001 | | | < 0.001 |
Uniform | 25(16.9) | 5(4.8) | | 23(17.2) | 7(5.9) | | 6(5.2) | 24(17.6) | |
Mild | 118(79.7) | 71(68.3) | | 104(77.6) | 85(72.0) | | 87(75.0) | 102(75.0) | |
Marked | 118(79.8) | 28(26.9) | | 7(5.2) | 26(22.0) | | 23(19.8) | 10(7.4) | |
Nucleoli | | | < 0.001 | | | < 0.001 | | | 0.09 |
Indistinct | 53(35.8) | 10(9.6) | | 46(34.3) | 17(14.4) | | 24(20.7) | 39(28.7) | |
Noticeable | 69(46.6) | 60(57.7) | | 65(48.5) | 64(54.2) | | 60(51.7) | 69(50.7) | |
Prominent | 26(17.6) | 34(32.7) | | 23(17.2) | 37(31.4) | | 32(27.6) | 28(20.6) | |
Nuclear margin | | | < 0.001 | | | 0.004 | | | 0.391 |
Smooth | 9(6.1) | 5(4.8) | | 9(6.7) | 5(4.2) | | 7(6.0) | 7(5.1) | |
Folds | 133(89.9) | 71(68.3) | | 115(85.8) | 89(75.4) | | 90(77.6) | 114(83.8) | |
Buds | 6(4.1) | 28(26.9) | | 10(7.5) | 24(20.3) | | 19(16.4) | 15(11.0) | |
Nuclear/cytoplasmic ratio | 0.507 | | | 0.844 | | | 0.517 |
< 50 | 8(5.4) | 15(14.4) | | 8(6.0) | 15(12.7) | | 13(8.9) | 10(9.4) | |
50–80 | 126(85.1) | 74(71.2) | | 114(85.1) | 86(72.9) | | 115(78.8) | 85(80.2) | |
> 80 | 14(9.5) | 15(14.4) | | 12(9.0) | 17(14.4) | | 18(12.3) | 11(10.4) | |
Density of chromatin | | 0.13 | | | 0.001 | | | 0.778 |
Not | 44(29.7) | 43(41.3) | | 33(24.6) | 54(45.8) | | 43(37.1) | 44(32.4) | |
Moderately | 97(65.5) | 54(51.9) | | 93(69.4) | 58(49.2) | | 64(55.2) | 87(64.0) | |
Markedly | 7(4.7) | 7(6.7) | | 8(6.0) | 6(5.1) | | 9(7.8) | 5(3.7) | |
Chromatin granularity | | 0.853 | | | 0.214 | | | 0.443 |
Fine | 80(54.1) | 58(55.8) | | 76(56.7) | 62(52.5) | | 62(53.4) | 76(55.9) | |
Moderately granular | 35(23.6) | 17(16.3) | | 32(23.9) | 20(16.9) | | 21(18.1) | 31(22.8) | |
Coarse | 33(22.3) | 29(27.9) | | 26(19.4) | 36(30.5) | | 33(28.4) | 29(21.3) | |
Nucleolar size# | | | | | | | | |
Micro | 115(77.7) | 17(16.3) | < 0.001a | 106(79.9) | 26(22.0) | < 0.001 | 39(33.6) | 94(69.1) | < 0.001 |
Medium | 31(21.0) | 63(60.6) | < 0.001b | 21(15.7) | 74(62.7) | | 62(53.4) | 33(24.3) | |
Macro | 2(0.01) | 24(23.1) | | 7(4.5) | 18(15.3) | | 15(12.9) | 9(6.6) | |
Nucleolar location# | | < 0.001c | | | < 0.001c | | | < 0.001c |
Peripheral | 106(71.6) | 6(5.8) | | 98(73.1) | 14(11.9) | | 24(20.7) | 88(64.7) | |
Centered | 42(28.4) | 98(94.2) | | 36(26.9) | 104(88.1) | | 92(79.3) | 48(35.3) | |
Nucleolar number# | | < 0.001 | | | < 0.001 | | | < 0.001 |
≤ 1 | 61(41.2) | 97(93.3) | | 54(40.3) | 104(88.1) | | 94(81.0) | 64(47.1) | |
2–3 | 32(21.6) | 5(4.8) | | 31(23.1) | 6(5.1) | | 9(7.8) | 28(20.6) | |
> 3 | 55(37.2) | 2(1.9) | | 49(36.6) | 8(6.8) | | 13(11.2) | 44(32.4) | |
a. Fisher’s exact test (mico vs medium and macro). b. Fisher’s exact test (medium vs macro). c. chi-square test (peripheral vs centered) and others were tested with Mann-Whitney U test. |
#: new parameters from our observation. |
There were two cytological parameters demonstrated statistically different between HER2-overexpression and HER2- samples. Larger cells and marked nuclear pleomorphism were detected in HER2 + samples (P < 0.001) (Fig. 1b). No significant difference was observed between positive and negative samples of the markers when we tested the cytological features, which included dissociation, nuclear/cytoplasmic ratio, density of chromatin and chromatin granularity.
Both ER + and PR + tumors showed smaller nucleolar size and less pleomorphism, compared to negative ones (P < 0.001). Additionally, in 106 ER + and 98 PR + tumors, the nucleolar location was scattered by contrast to 6 ER- samples and 14 PR- samples (P < 0.001). ER- or PR- tumors were more likely to show one or less nucleoli, compared with ER+ (P < 0.001) or PR+ (P < 0.001) tumors. In contrast, HER2 + samples had bigger nucleolar size (P < 0.001) but less cells than HER2- samples (P < 0.001). Besides, the nucleoli showed more centered in 92 (79.3%) HER2 + than HER2- samples (P < 0.001).
Multivariate analysis and cytological model for cytological grading and predicting status of the three factors
Based on our findings, we finally selected six parameters, which were significantly associated with three markers, to form a new system. Detailed standards were shown in Table 3.
Table 3
The new scoring system for predicting the status of ER, PR and HER2
| Score 1 | | Score 2 | | Score 3 |
Cell size | < 3×RBC size | | 3–4×RBC size | | > 4×RBC size |
Nucleoli | Indistinct | | Noticeable | | Prominent |
Nucleolar size | Micro | | Medium | | Macro |
Nucleolar location | Scattered | | | | Centered |
Nucleolar number | > 3 | | 2–3 | | ≤ 1 |
Nucleolar margin | Round, smooth | | Smooth | | Irregular |
We next performed a multivariate analysis to identify the cytological features which were independently associated with the expression of ER, PR and HER2 (Table 4). The results indicated that the cell size (P < 0.001), nucleolar size (P = 0.030), nucleolar location (P = 0.005), nucleolar number (P = 0.043) and nuclear margin (P = 0.043) were the independently associated with the expression of ER. In addition, only the cell size and nucleolar location were correlated with the status of PR and HER2.
Table 4
Multivariate logistic regression analysis for determination of the likelihood of ER, PR and HER2 status from new system's cytologic features
| ER+ | PR+ | HER2+ |
| OR (95% CI) | P- value | OR (95% CI) | P- value | OR (95% CI) | P- value |
Cell size | 0.237 (0.122–0.460) | < 0.001 | 0.469 (0.277–0.791) | 0.005 | 2.548 (1.583–4.104) | < 0.001 |
Nucleoli | 0.565 (0.303–1.052) | 0.072 | 0.688 (0.414–1.145) | 0.15 | 0.992 (0.638–1.543) | 0.992 |
Nucleolar size | 0.425 (0.196–0.921) | 0.03 | 0.678 (0.337–1.3255) | 0.271 | 0.861 (0.457–1.624) | 0.65 |
Nucleolar location | 6.201 (1.758–21.878) | 0.005 | 6.889 (2.546–18.638) | < 0.001 | 0.224 (0.090–0.563) | 0.001 |
Nucleolar number | 0.363 (0.136–0.969) | 0.043 | 0.697 (0.377–1.289) | 0.25 | 1.253 (0.746–2.104) | 0.394 |
Nuclear margin | 0.367 (0.139–0.967) | 0.043 | 0.890 (0.402–1.969) | 0.773 | 0.761(0.378–1.533) | 0.445 |
OR, odds radio |
The cutoff value was set as 12.5, according to the multivariate analysis. The score of 12.5 or lower was also regarded as a highly possibility with the expression of ER or PR upon the Receiver Operating Characteristic (ROC) analysis. At this cutoff (Table 5), the sensitivity and specificity were 94% and 77% for detecting the ER-expression. We also verified the scoring system for the expression of PR, the sensitivity was 86% and the specificity was 78%, respectively. With regard of HER2 status, the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) was 76%, 68%, 67% and 77%, respectively.
Table 5
Sensitivity, specificity, PPV and NPV from the three system for assessment the status of ER, PR and HER2
| Sensitivity | | Specificity | | PPV | | NPV |
| ER | PR | HER2 | | ER | PR | HER2 | | ER | PR | HER2 | | ER | PR | HER2 |
New system | 0.94 | 0.86 | 0.76 | | 0.77 | 0.78 | 0.68 | | 0.74 | 0.77 | 0.67 | | 0.95 | 0.87 | 0.77 |
Robinson system | 0.55 | 0.57 | 0.72 | | 0.76 | 0.75 | 0.55 | | 0.77 | 0.72 | 0.58 | | 0.54 | 0.61 | 0.7 |
Taniguchi system | 0.61 | 0.58 | 0.63 | | 0.62 | 0.56 | 0.64 | | 0.69 | 0.6 | 0.6 | | 0.52 | 0.54 | 0.67 |
PPV, positive predictive value; NPV, negative predictive value |