Chinese-German Journal of Clinical Oncology May 2010, Vol. 9, No. 5, P249–P252 DOI 10.1007/s10330-010-0048-0

Breast cancer has become the leading cause of death for women around the world, which could be classified into five phenotypes on the basis of recent gene- expres-sion profiling analytical techniques: the normal breast type, luminal A type, luminal B type, Her-2 type, and the basal type [1, 2]. In recent years, the mortality rate is be-ing effectively decreased and the prognosis increased due to advanced integrated therapy. However, a number of studies have confirmed that the basal-like breast cancer shows a high grade of malignancy and a poor prognosis [1, 3, 4]. This subtype generally expresses basal markers, and in most cases they are estrogen receptor (ER)-negative, progesterone receptor (PR)-negative, and Her2-negative; this subtype is called triple-negative (TN) breast cancer [5]. As this subtype shows a high grade of malignancy and lacks effective integrated treatment, it is rated as the most vicious and adverse prognosis of all breast cancer sub-types.

Sasa et al [6] showed that it is necessary to classify the

lesions as basal phenotype (BP) or non-basal phenotype (NBP) by performed immunohistochemical (IHC) staining for the CK5/6, CK14, and CK17 basal markers. For normal cases, clinicopathological test only involves screening for ER, PR, and Her-2, which could not differentiate the two subtypes; therefore the therapeutic approach based on the diagnosis result can’t effectively target the two subtypes [1,

7, 8]. The differences and contacts between these two kinds of subtypes in clinicopathology and prognosis would be-come a new vision for treating triple-negative breast can-cer. It has been reported that approximately 50%–60% of TN breast cancers are BP [3], and its prognosis is worse than the NBP subgroup [9]. So it is necessary to classify the lesions for the TN breast cancer, which can make the therapeutic approach more appropriately [3, 10]. In our study, we performed immuno-histochemical staining for the CK5/6, CK17 and EGFR markers to classify the can-cer as basal-marker-positive BP or basal-marker-negative NBP; then the clinicopathological and related prognostic factors investigations were conducted.

Clinicopathologic features and related prognosis factors analysis of the basal and non-basal phenotype of triple negative breast cancer*Lin Sun, Lin Zhang, Shasha Ren, Deding Tao, Yaqun Wu

Department of Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China

Received: 8 April 2010 / Revised: 11 April 2010 / Accepted: 25 April 2010© Springer-Verlag Berlin Heidelberg 2010

Abstract Objective: Triple-negative breast cancer (estrogen receptor-negative, progesterone receptor-negative and Her2-negative) can be classified into two subtypes: basal and non-basal phenotype. And the basal phenotype is associated with poor outcome. The purpose of this study was to figure out the differences of clinicopathological characters and related factors of prognosis between these two subtypes. Methods: Immunohistochemical staining was performed for the CK5/6, CK17 basal markers and EGFR on biopsy samples from 40 triple-negative patients and the clinicopathology features of these samples were investigated. Results: Seventy percent of the patients were diagnosed as the basal phenotype. Compared with the non-basal phenotype, the basal phenotype lesions were significantly larger in diameter with a high nuclear grade. In the node-negative group the basal phenotype clearly showed the same clinicopathological differences. There was statistically significant concordance among all three antibodies. Conclusion: Expression of basal markers identifies a biologically and clinically distinct subgroup of TN tumors, justifying the use of basal markers to define the basal or the non-basal phenotype. It is important to help the doctor deciding the therapeutic strategy for patient with triple-negative breast cancer.

Key words breast cancer; triple-negative; basal phenotype; non-basal phenotype; clinicopathology; prognosis

Correspondence to: Lin Zhang. Email: zhanglin3@medmail.com.cn* Supported by a grant from the National Natural Science Foundation of Hubei Province (No. 2009CDB063).

250 www. springerlink. com/content/1613-9089

Materials and methods

Sample collectionForty patients with triple-negative breast cancer were

involved in this study who were treated at Department of Surgery of Tongji Hospital between January 2007 and June 2009. All cases were characterized by surgical exci-sion (modified radical mastectomy of breast cancer), and the surgery specimens were confirmed that the ER, PR and Her-2 by immunohistochemical methods. Her-2 was determined as negative on the basis of a staining score of 0 or 1 and as positive when the score was 3, the score 2 cases are usually subjected to FISH or CISH analysis and rated as positive (high amplification) or negative (no or low amplification). The clinicopathological data were collected to establish a database.

IHC staining and judge methodThe Paraffin-embedded tumor specimens were first

sectioned to 4 μm thickness. IHC reaction tests were performed using EnVision method to detect the CK5/6, CK17, EGFR and P53 markers (CK5/6 1:50, CK17 1:500, EGFR 1:200 and P53 1:50). CK5/6 and CK17 stain the tumor cytomembrane and/or cytoplasmic, EGFR in cy-tomembrane, and P53 in cytoblast. The known positive sections were used as the positive control, and PBS was used to replace the first antibody as the negative control. The standard to judge the positive sections referred to the method of Xu et al [11].

SubgroupOn the basis of the three markers, the biopsy sample

was judged to be BP when the specimen had been found to be positive for any of CK5/6, CK14, or CK17. Disease found to be negative for all three of these basal markers was diagnosed as NBP.

Statistical methodsThe Chi-square test was used to test the correlations

between BP and NBP cases with the clinicopathological parameter based on the staining results for the three basal markers. A P-value of < 0.05 was considered to indicate statistical significance.

Results

Clinicopathological findings of TNBCAll of the patients were women aging from 28 to 71

(median, 52.5 years). There were 2 patients (5%) younger than 36 years, 16 patients (40%) age ranged from 36 to 50 years, and 22 patients (55%) older than 50 years. The number of premenopausal cases was 16 (40%), and the postmenopausal was 24 (60%) (Table 1).

Comparison of clinicopathological findings for BP and NBP

Of the total 40 cases, 26 patients were CK5/6-posi-tive, 14 patients were CK17- positive, and 16 patients are EGFR-positive. Based on the results, 28 of the 40 pa-tients (70%) were judged to be basal phenotype (BP) and 12 (30%) were judged to be non-basal phenotype (NBP). Twenty-six patients (65%) were P53-positive. Fig. 1 showed a P53-positive in BP.

The tumor diameter (T1: ≤ 2 cm; T2: 2.1–5 cm; T3: > 5 cm) was significantly larger in the BP group, and a high nuclear grade was also significantly higher in the BP group than in the NBP group. No differences were found between the BP and NBP groups in relation to the age, menopause, pathological classification, and status of lymph node metastasis, and expression of P53 (Table 1).

In the node-negative group, the BP subgroup had a significantly high nuclear grade (II + III, 14/15) than the NBP subgroup (2/6). But in the node-positive group there were no differences.

Basal marker expression of each three antibodies

The basal marker expression was analyzed among three antibodies. The concordance rates for staining were 65% for CK5/6 and CK17, 70% for EGFR and CK17, and 65% for CK5/6 and EGFR, and there was statistically signifi-cant concordance among all of the antibodies.

Table 1 Comparison of clinic pathological findings for BP and NBPBP (n) NBP (n) P

Age (years)≤ 35 2 036–50 8 8> 50 18 4

MenopausePostmenopausal 17 7Premenopausal 11 5

TT1 4 7 < 0.05T2 + T3 24 5

Pathological classificationInvasive ductal carcinoma 20 9Others 8 3

Nuclear gradeI 1 5 < 0.05II + III 27 7

Lymph node metastasis+ 13 6– 15 6

P53+ 18 8– 10 4

251Chinese-German J Clin Oncol, May 2010, Vol. 9, No. 5

Discussion

Most of the patients in this study were senior age (> 50 years, 55%) and postmenopausal (60%) women, which were opposite with many literatures [12, 13]. A clinical analysis in China [14] for the TN breast cancer contains 69 patients, whose average age was 47.5 years old and the premenopausal was 71%. As many studies reported [3, 12,

13, 15–17], there were many clinical and biological features which were positive associated with the TN breast can-cers: larger tumor size, more lymph node metastasis, and higher nuclear grade (most cases were II–III), invasive ductal carcinoma, more P53 variation, etc. These entire conclusions coincided with the findings in this study: the average diameter of the TN breast cancer tumor was 3 cm, lymph node metastasis was 47.5%, more than 85% was II–II grade, the invasive ductal carcinoma accounts 72.5%, and the rate of P53-positive was 65%.

Biomarkers in the subgroup of TNAlthough more attention has been attracted to the sub-

types of the TN breast cancer, the standard of determina-tion is still unclear. Sasa et al [6] showed that the lesions could be classified as basal phenotype (BP) or non-basal phenotype (NBP) by performed immunohistochemical (IHC) staining for the basal markers (CK5/6 and CK17). Nielsen [5] and Rodriquez-Pinilla [18] consider using ER, PR, Her-2 and CK5/6, and other researchers add CK14 for more information. Liu [9] presumes that adding the detec-tion of EGFR on the base of CK5/6 and CK17 can elevate the detection rate for BP of the TN breast cancer. In this study, we chose CK5/6, CK17 and EGFR as the judge-markers. On the basis of the three markers, the disease was judged to be BP when the specimen had been found to be positive. Therefore, the IHC difference of these two subtypes of the TN breast cancer on the clinicopathologic and prognosis will be a new field for research and diag-nosis.

The CKs-positive was more than 60% of the TN breast

cancer in Nielsen [5] and Livasy [19], 61% (40/66) in Sasa [6] and 70% (28/40) in this study. The higher rate in this study may be due to the difference in selecting the ob-jects.

There is a statistically significant concordance among all of the three antibodies (CK5/6, CK17, and EGFR). Similar result can also be found in Sasa et al [6]. As very few studies detect all of these three markers, the result was very important for clinicopathologic analysis. We can also choose one or two markers, especially CK5/6, to detect and decide the BP type based on the expression.

Subgroups in TN and prognosisWe found in this study that the BP type has higher

nuclear grade and larger tumor size than the NBP type, and has statistical significance (P < 0.05), which coincided with other studies [1, 3, 4, 6]. Many reports [20] have shown that CK5/6, CK17, and EGFR were correlated with poorer prognosis and lower survival rate, and the nuclear grade and tumor size are also valid independent factors to judge the prognosis of breast cancer. As a result, we can pre-sume that the NBP type has better prognosis, which can be found in other’s work. We also can presume that the nuclear grade and tumor size are better factors to esti-mate the prognosis of the worse breast cancer type. All of these results can prove that the therapeutic approach that is decided for TN disease lacking the information for the subtype is clearly inappropriate.

We classify the TN breast cancer into two subtypes based the lymph node metastasis condition, then inves-tigate the differences between the BP type and the NBP type in this two subtypes separately and found the same results in the lymph node metastasis negative subtype as before. However similar result could not be found in the lymph node metastasis positive subtype. All of these find-ings can demonstrate that the BP type is a poor prognosis factors for the lymph node metastasis negative TN breast cancer. We can classify the TN breast cancer based on the lymph node metastasis and the basal-cell keratins expres-sion; the lymph node metastasis negative and NBP type has a better prognostic result. These results were the same as Rakha [3] and Sasa [6]. In the study of vande Rijn [20], who choose all types breast cancer as the object, can not get the same result. This difference can be due to the popula-tion selection differences.

Because of the correlation of the BP type and NBP type on clinicopathologic and prognosis factors, we should classic the TN breast cancer when choosing treatment ap-proach. Maybe we can use high dose chemotherapy to treat the BP type in the future. As the EGFR expresses in the BP type frequently, we can choose EGFR targeted therapy probably. Study of Nielsen et al [5] also shows that this is a feasible way to treat this breast cancer. All of these may be the new way for the treat of breast cancer

Fig. 1 P53-positive in BP subgroup (Envision × 400)

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[21].This study also has some limitations. For example, we

only have 40 patients in this study, and all of these are from 2007 to 2009, which lacks the follow-up information of the relapse, metastasis, and live time. Therefore com-parison of these two subtypes could not be made directly. Further investigation is needed to verify the results.

References

Cleator S, Heller W, Coombes RC. Triple-negative breast cancer: Therapeutic options. Lancet Oncol, 2007, 8: 235–244.van’t Veer LJ, Dai H, van de Vijver MJ. Gene expression profiling pre-dicts clinical outcome of breast cancer. Nature, 2002, 415: 530–536.Rakha EA, EI-Sayed ME, Green AR, et al. Prognostic markers in triple negative breast cancer. Cancer, 2006, 109: 25–32.Rakha EA, Putti TC, EI-Rehim DMA, et al. Morphological and immu-nophenotypic analysis of breast carcinomas with basal and myoepi-thelial differentiation. J Pathol, 2006, 208: 495–506.Nielsen TO, Hsu FD, Jensen K, et al. Immunohistochemical and clini-cal characterization of the basal-like subtype of invasive breast carci-noma. Clin Cancer Res, 2004, 10: 5367–5374.Sasa M, Bando Y, Takahashi M, et al. Screening for basal marker expression is necessary for decision of therapeutic strategy for triple-negative breast cancer. J Surg Oncol, 2008, 97: 30–34.Kim MJ, Ro JY, Ahn SH, et al. Clinicopathologic significance of the basal-like subtype of breast cancer: a comparison with hormone re-ceptor and Her2/neu-overexpressing phenotypes. Hum Pathol, 2006, 37: 1217–1226.Haffty BG, Yang QF, Reiss M, et al. Locoregional relapse and dis-tant metastasis in conservatively managed triple negative early-stage breast cancer. J Clin Oncol, 2006, 24: 5652–5657.Liu ZB, Wu J, Ping B, et al. Expression of CK5/6 and CK17 and it cor-relation with prognosis of triple-negative breast cancer patients. Chin J Oncol (Chinese), 2008, 30: 610–614.

1.

2.

3.

4.

5.

6.

7.

8.

9.

Siziopikou KP, Cobleigh M. The basal subtype of breast carcinomas may represent the group of breast tumors that could benefit from EGFR-targeted therapies. Breast, 2007, 16: 104–107.Xu LZ. The biology markers of prognosis. Breast Pathology. Shang-hai: Shanghai University Publishing Company, 1999. 359.Bauer KR, Brown M, Cress RD, et al. Descriptive analysis of estrogen receptor (ER)-negative, progesterone receptor (PR)-negative, and HER2-negative invasive breast cancer, the so-called triple-negative phenotype: a population-based study from the California cancer Reg-istry. Cancer, 2007, 109: 1721–1728.Dent R, Trudeau M, Pritchard KI, et al. Triple-negative breast cancer: clinical features and patterns of recurrence. Clin Cancer Res, 2007, 13: 4429–4434.Zhou X, Yang JX, Lv G, et al. Clinical analysis of 69 cases of triple negative breast cancer. J Clin Surg (Chinese), 2008, 16: 815–816.Yuan ZY, Wang SS, Gao Y, et al. Clinical characteristics and prog-nosis of triple-negative breast cancer: a report of 305 cases. Chin J Cancer (Chinese), 2008, 27: 561–565.Sun B, Wu SK, Song ST, et al. Research prognosis in triple-negative breast cancer. Oncol Prog (Chinese), 2007, 5: 549–552.Tan DS, Marchio C, Jones RL, et al. Triple negative breast cancer: molecular profiling and prognostic impact in adjuvant anthracycline-treated patients. Breast Cancer Res Treat, 2008, 111: 27–44.Rodriquez-Pinilla SM, SarrióD, Honrado E. Vimentin and laminin expression is associated with basal-like phenotype in both sporadic and BRCA1-associated breast carcinomas. J Clin Pathol, 2007, 60: 1006–1012.Livasy CA, Karaca G, Nanda R, et al. Phenotypic evaluation of the basal-like subtype of invasive breast carcinoma. Mod Pathol, 2006, 19: 264–271．van de Rijn M, Perou CM, Tibshirani R, et al. Expression of cytokera-tins 17 and 5 identifies a group of breast carcinomas with poor clinical outcome. Am J Pathol, 2002, 161: 1991–1996.Dabbs DJ, Chivukula M, Carter G, et al. Basal phenotype of duc-tal carcinoma in situ: recognition and immunohistologic profile. Mod Pathol, 2006, 19: 1506–1511.

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.