Arch Gynecol Obstet (2014) 290:1221–1229 DOI 10.1007/s00404-014-3331-4
GYNECOLOGIC ONCOLOGY
Identification of prognostic different subgroups in triple negative breast cancer by Her2-neu protein expression Gilda Schmidt • Gabriele Meyberg-Solomayer • Christoph Gerlinger Ingolf Juhasz-Bo¨ss • Daniel Herr • Achim Rody • Cornelia Liedtke • Erich-Franz Solomayer
•
Received: 27 January 2014 / Accepted: 17 June 2014 / Published online: 11 July 2014 Ó Springer-Verlag Berlin Heidelberg 2014
Abstract Purpose Many patients with triple negative breast cancer (TNBC) have a poor outcome, but not all of them. This study has the aim to analyse the prognostic impact of tumour size, nodal status, grading, Her2-neu (human epithelial growth factor receptor 2) score and Ki-67 index. The main goal of this analysis is to find out if there are any differences in survival between patients with TNBC and a Her2-neu score 0 versus 1?2. Experimental design Retrospectively, we studied a cohort of 121 patients with TNBC, diagnosed at the Saarland University Medical Center between December 2004 and June 2013. We compared the disease-free survival (DFS) and overall survival (OS) in those women on the basis of the different Her2-neu scores (0 versus 1 or 2 with negative FISH). Results One hundred and twenty one patients were included in this study. 58.68 % of them had a T2–4 tumour. 39.67 % were nodal positive and 67.77 % had high-grade tumours. The Her2-neu score was determined in 119 patients. 54.62 % of them had a score 0. In the 103 patients with a Ki-67 determination, the mean index was 44.5 %. We found that tumour size, nodal status and Her2-neu score are important prognostic factors. Patients with a Her2-neu score 0 had a significantly poorer outcome regarding DFS and OS. In contrast, the expression level of
G. Schmidt (&) G. Meyberg-Solomayer C. Gerlinger I. Juhasz-Bo¨ss D. Herr E.-F. Solomayer Department of Obstetrics and Gynaecology, Homburg University Medical Centre, University of Homburg, 66421 Homburg, Germany e-mail: [email protected] A. Rody C. Liedtke Department of Obstetrics and Gynaecology, Kiel University Medical Centre, Kiel, Germany
Ki-67 and the grading do not seem to have any prognostic value in TNBC. Conclusion Besides tumour stage, grading and nodal status, the Her2-neu score 0 is able to function as a prognostic factor in patients with TNBC. Keywords TNBC (triple negative breast cancer) Her2 neu score Ki-67
Introduction Breast cancer is the most common cancer in women. Approximately, 15–20 % of the breast cancers are triple negative and associated with a poor prognosis [1–4]. These tumours do not express oestrogen and progesterone receptors and show negativity for Her2-neu protein. Triple negative breast cancers (TNBCs) are very aggressive and more prevalent in younger women [5] and they are characterized by early relapse, early metastasis and early death [6–11]. Previous studies showed that the peak risk of recurrence is within 3 years [12, 13]. In addition, patients with TNBC also have an increased risk for cerebral metastasis compared to patients bearing the ER?/Her2neu-phenotype [14]. In TNBC, there are different intrinsic subtypes known with different molecular features [15]. Besides, triple negative tumours can be divided into six molecular subgroups: basal-like 1, basal-like 2, immunomodulatory, mesenchymal, mesenchymal-stem-like and luminal/AR tumours [16, 17]. It is known that basal-like tumours usually express cytokeratins like epidermal growth factor receptor (EGFR) which is also known as Her1-receptor [18–20]. Previous studies revealed that increased intratumoural expression level of the Her1-receptor in TNBC is associated with nodal
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and distant metastases in the basal-like subtype and show furthermore that the Her1-receptor has a significant prognostic value [21–23]. Except the Her1-receptor, these days there are further three members of the Her family known: Her2-neu, Her3 and Her4. While the importance of Her1 as prognostic marker could be already shown, the question arises if the Her2-neu-receptor is also able to function as a prognostic factor in TNBC. Therefore, in this study, we focused on Her2-neu. Like the other members of the Her family, the Her2-neu is a tyrosinkinase receptor located on the surface of tumour cells and c-erbB2-gen (cerbB2 = cellular avian erythroblastosis homologue B2) coded for Her2-neu. In TNBC, Her2-neu can show three different scores: 0, 1?, 2? (with negative FISH test). Among the determination of the Her2-neu score in breast cancer, the determination of the proliferation marker Ki-67 is also clinical routine. Ki-67 is a nuclear antigen which is present in the nuclei of cells in all phases of the cell cycle as well as in mitosis, but quiescent cells in the G0 phase do not express it [24–26]. The protein Ki-67 is widely used in several cancers as an efficient immunohistochemical marker for determining the proliferation of malignant cells. Patients with TNBC show a significantly higher expression of Ki-67 compared to patients with non TNBCs [21, 22]. In hormone receptor positive breast cancer, the biomarker Ki-67 is useful as a prognostic and likely predictive marker [27, 28]. In the St. Gallen Consensus 2013, its use is recommended as additional factor to divide the large group of receptor positive breast cancers in luminal A and B [29]. Generally, in breast cancer, high Ki-67 is associated not only with a better response to chemotherapy [30–33] but also with a poorer prognosis [23, 34–36]. Hence, the role of Ki-67 in TNBC needs to be further analysed. Our study retrospectively investigates the distribution of Her2-neu score and Ki-67 in TNBC to answer the question concerning the importance of those two markers with regard to disease-free survival (DFS) and overall survival (OS). We thereby wanted to clarify the role of Her2-neu and Ki-67 as prognostic markers arising the questions, if patients with a low Ki-67 index have a better outcome than patients with a high Ki-67 level index and if there are any differences in survival (OS and DFS) between patients with Her2-neu score 0 and Her2-neu score 1?2 in TNBC?.
Methods Between December 2004 and June 2013, a total of 144 patients were diagnosed with TNBC at the Saarland University Medical Center. Patients with breast cancer in their medical history were excluded (n = 11) and patients who have already an event at timepoint 0 were also excluded (n = 12).
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A total of 121 patients were included in this study. The follow-up was until September 2013. Triple negative breast cancer was defined as oestrogen receptor negative (ER 0/12), progesterone receptor negative (PR 0/12) and without overexpression of Her2-neu. These tests were carried out with standard kits by IHC. For each patient in the database, antibody staining of a set of paraffin embedded slides for ER and PR was carried out. Any positivity was taken as positive. The overexpression of Her2-neu status was evaluated using the HercepTestTM. The score is determined by immunohistochemical analyses carried out by a pathologist (HercepTestTM). Score 0 means that the pathologist observes no staining or membrane staining in\10 % of the tumour cells. Score 1? is also negative and indicates that a faint/barely perceptible membrane staining is detected in [10 % of tumour cells. The cells exhibit incomplete membrane staining. A score 2? is also negative in case the pathologist observes a weak to moderate complete membrane staining in [10 % of the tumour cells and no Her2neu amplification in fluorescence in situ hybridization (FISH) test. Her2-neu report of 3? by IHC and 2? with a Her2-neu amplification in FISH test were considered as positive [37]. High expression of Ki-67 was defined as C14 %, with 14 % as cutoff based on a retrospective study realized by Cheang [38]. Disease-free survival was calculated as the interval (in months) between the date of breast cancer diagnosis to first recurrence event: locoregional recurrence or distant metastasis or death of any cause or the date of last followup, if no recurrence event was recorded. The date of diagnosis was defined as the date of first histology of breast cancer. Survival times were censored to the date of last contact for subjects who were lost to follow-up. The OS was defined as the time (in months) between the date of diagnosis and the date of death (breast cancer- and non-breast cancer related) or last follow-up. Following the intent to treat principle, all patients who provided data were included in the statistical analyses. Missing data were not imputed. All continuous variables were analysed during the descriptive statistics number of non-missing observations, mean, standard deviation, minimum, 1st quartile, median, 3rd quartile, and maximum. All categorical variables were analysed by absolute and relative frequency counts. Time to event data was analysed using the Kaplan–Meier estimator. The null hypothesis of no difference between strata was tested using the logrank test. We used a comparison wise significance level a of 5 %, two sided. The statistical analyses were performed using SAS software version 9.2. (SAS Inc. Cary, NC, USA, www.sas.com).
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Results Patients’ characteristics A total of 121 patients with TNBC were included in the study. The characteristics of the study population are presented in Table 1. The age of patients ranged from 28 to 91 years, with a mean age of 55.58 years (Table 1). T2 was the most common (47.94 %) and T4 (3.31 %) the least common presentation. Node negative patients were the largest group with 60.33 %. Positive lymph nodes had 39.67 % of them. 67.77 % of the patients with TNBC had a high-grade tumour and almost 30 % of the patients had an invasion into lymphatic vessels. The Her2-neu score was numerally determined in 119 of the 121 patients. The distribution of the Her2-neu score among the 119 patients with TNBC is also represented in Table 1. 65 patients had a Her2-neu score 0 and 54 patients had a score 1 or 2. The biomarker Ki-67 was determined in 103 of the 121 patients. The mean Ki-67-index was 44.5 % Table 1 Patients’ characteristics
Characteristicsa
N
(Table 1). 92 of the 103 patients (89.32 %) showed a high expression of Ki-67. One hundred and one of the 121 patients were treated with chemotherapy (Table 1). 30 of them obtained a neoadjuvant chemotherapy, the rest of the patients received an adjuvant or primary systemic chemotherapy. The majority of the patients (78.22 %) obtained a taxane-containing Table 2 Incidence of locoregional relapse, distant metastases and death N
Percent (%)
Her2-neu 0, N (%)
Her2-neu 1 or 2, N (%)
Locoregional relapse
16
13.22
11 (100)
5 (100)
Metastasis (multiple incidence possible)
24
19.83
19 (100)
5 (100)
Pulmonary
18
14.88
18 (100)
4 (100)
Osseous
9
7.44
Hepatic
7
5.79
Cerebral Death
6
4.96
22
18.18
Percent (%)
Her2-neu 0, N (%)
Her2-neu 1 or 2, N (%)
Age at diagnosis \40
13
10.74
10 (15.38)
3 (5.56)
40–55
52
42.97
27 (41.54)
23 (42.59)
[55
56
46.28
28 (43.08)
28 (51.85)
T1
50
41.32
23 (35.38)
27 (50.00)
T2
58
47.94
33 (50.77)
24 (44.44)
T3
9
7.44
6 (9.23)
3 (5.56)
T4
4
3.31
3 (4.62)
0 (0.00)
Size of the tumour (T)
Lymph nodes stages (N) N0
73
60.33
36 (55.38)
36 (66.67)
N?
48
39.67
29 (44.62)
18 (33.33)
0.83
1 (1.54)
0 (0.00)
38 82
31.40 67.77
17 (26.15) 47 (72.31)
21 (38.89) 33 (61.11)
Grading (G) G1 G2 G3
Invasion into lymphatic vessels (L) L0
82
67.77
43 (66.15)
37 (68.52)
L1
33
27.27
19 (29.23)
14 (25.96)
Lx
6
4.96
3 (4.61)
3 (5.56)
11
10.67
4 (8.00)
7 (13.73)
92
89.32
46 (92.00)
44 (86.27)
101
83.47
51 (100 %)
48 (100 %)
Ki-67 (%)b \14 C14 Chemotherapy a
Two patients without Her2neu determination
b
Twenty-one patients with unknown Ki-67 (15.79 %)
Taxane-containing
79
78.22
40 (100)
37 (100)
Neoadjuvant
30
29.70
15 (100)
13 (100)
-yT0, yN0
6
20.00
2 (100)
3 (100)
20
16.52
14 (100)
6 (100)
No chemotherapy
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Fig. 1 Her2-neu score as prognostic factor in TNBC. Kaplan–Meier curve of (a) DFS and (b) OS dependent on Her2neu score in triple negative breast cancer. The observed differences between Her2-neu score 0 and Her2-neu score 1?2 (FISH test negative) are significant for both DFS and OS (p = 0.0021 and 0.0.0105, respectively)
regime. Six patients (20.00 %) who obtained a neoadjuvant chemotherapy had a complete remission. 20 patients did not obtain a chemotherapy (7 patients were multimorbid and the resting 13 patients refused chemotherapy). Sixteen of the 121 patients fell into locoregional relapse during the follow-up time, and 2 of them (1.65 %) had breast cancer contralateral. The median time to relapse was approximately 2 years and 4 months. Distant metastasis
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was seen in 24 patients (Table 2). Most of the patients with distant metastasis had visceral metastasis. 15 of the 24 patients with metastasis had only visceral and/or cerebral metastasis, further 8 patients showed both visceral/cerebral and osseous metastasis and 1 patient had only osseous metastasis. The incidence of cerebral metastasis was in 4.96 % of our patients. Patients with locoregional relapse or distant metastasis had three times more frequently a
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Fig. 2 Distribution of nodal status by Her2-neu score. No significant differences between the Her2-neu score 0 group and Her2-neu score 1 and 2 group regarding lymph node status (p = 0.2100; Chi-square test)
Her2-neu score 0 compared to patients with a score Her2neu 1 or 2 (Table 2: 30 patients versus 10 patients). A total of 22 of the 121 patients died until September 2013. This corresponds to 18.18 % (Table 2). 18 of the 22 deaths (82 %) had a Her2-neu score 0. Prognostic factors in TNBC
score 0 had a significantly worse DFS (p = 0.0021) and also a worse OS (p = 0.0105) compared to patients with a Her2-neu score of 1 and 2. In the Her2-neu score 0 group, there was a larger proportion of patients with positive lymph nodes. An additional analysis showed no significant difference from the Her2-neu score 1 and 2 group (p = 0.2100; Chi-square test) (Fig. 2).
Tumour size Ki-67 Patients with a small tumour (T1) had a significantly better outcome (DFS p = 0.0114; OS p = 0.0020) compared to patients with a T2–4 tumour (data not shown). Nodal status Patients with negative nodal status (N0) showed a significantly better DFS (p \ 0.0001) and a significantly better OS (p = 0.0005) compared to patients with lymph node positivity (N1–N3) (data not shown).
The DFS and OS dependent on Ki-67 index are displayed in Fig. 3a, b. It becomes clear that there is no significant difference between patients with Ki-67 C14 and \14 % in DFS (p = 0.5156) and OS (p = 0.6467). A Ki-67 cutoff of 50 % was also calculated, and it confirms the same results (Figure not shown). We did not detect any correlation between expression of Ki-67 and overall, respectively, DFS.
Grading
Discussion
The DFS and OS were not significant better for patients with G1 or G2 tumours compared to patients with G3 tumours (DFS p = 0.1026; OS p = 0.2235) (data not shown).
In this retrospective study, 121 patients with TNBC were analysed regarding DFS and OS. During the observation time, 16 of the 121 patients developed a locoregional relapse, 24 patients had distant metastasis and 22 patients died. In view of tumour size and nodal status, we could show that patients with small tumours and negative nodal status had a significant better outcome compared to patients with a large tumour size and/or a nodal positive status,
Her2-status Figure 1a, b shows the patients outcome to be dependent on the Her2-neu score. Patients with TNBC and a Her2-neu
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Fig. 3 Ki-67 as prognostic factor in TNBC. Kaplan–Meier curve of (a) DFS and (b) OS dependent on Ki-67 index in triple negative breast cancer. The observed differences between Ki-67 \ 14 and Ki67 C 14 % are not significant neither in DFS nor in OS (p = 0.5156 and 0.6467, respectively)
whereas the DFS and OS were not significant worse for patients with high-grade tumours. Furthermore, regarding the Her2-neu distribution in these patients, we could show that patients with a Her2-neu score 0 had a poorer outcome regarding DFS and OS compared to patients with a Her2neu score 1 or 2. Hence our study demonstrates that the Her2-neu score is able to function as a prognostic factor in
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patients with TNBC. On the other hand, Ki-67 failed to predict the DFS and OS. Neither DFS nor OS was significantly better in patients with a low Ki-67 level (\14 %) compared to patients with a high Ki-67 level (C14 %). TNBC is associated with aggressive behaviour and characterized by early relapse, early metastasis and death. The mean time to the local relapse was in our study 2 years
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and 4 months. These results are in line with the published data [39]. Especially between the first and third year after diagnosis, patients with TNBC fell into local relapse. It is known that visceral metastases like hepatic and pulmonary were common in TNBC. In our study, 95.83 % of the patients with distant metastasis had visceral and/or cerebral metastases. 4.16 % of the patients had osseous metastases only. This explains the aggressive behaviour of these tumours. Our results are in line with published data. Ko¨nigsberg et al., for example, demonstrated that patients with TNBC develop distant visceral metastases more frequently than bone metastases [7, 14, 40]. The incidence of cerebral metastasis in 4.96 % of our patients is also in line with the published data [14]. Heitz et al. showed that 6.7 % of all patients with TNBC developed cerebral metastases. We found that patients with a small tumour size showed a better outcome, so that the tumour size acts as a prognostic factor in TNBC. This observation is in agreement with a previous report where the tumour size was the most important prognostic factor in TNBC patients [41]. Similar results show a further study published 2013 [41, 42]. Other studies also revealed that even small tumours (T1N0) seem to have a higher recurrence rate [43, 44]. Our results showed furthermore that not only the tumour size but also the nodal status is able to function as a prognostic factor. This is in agreement with previous published studies [42, 45, 46]. Hernandez-Aya et al. [47] found also that patients with N0 had a significant better OS and RFS (relapse-free survival) (p \ 0.001 all comparisons), but in contrast there was no difference in survival among patients with N1, N2, N3. Histological grading is also known as an important independent prognosis factor [48]. Surprisingly, our study shows no difference regarding outcome (DFS and OS) in patients with low- or intermediate-grade tumours compared to patients with high-grade tumours. Considering the distribution of the histological grading, 82 of our patients had a G3 tumour, 38 patients had a G2 tumour and only 1 patient had a G1 tumour. Published reports showed that the determination of the histologic grading is not always precise and reproducible and, furthermore, the prognosis prediction for the intermediate-grade tumours (G2) is limited [49]. Approximately, 67.77 % of the included patients had G3 tumours which accentuate the aggressivity of the TNBC. In our study, patients with TNBC and a Her2-neu score 0 have a poorer outcome compared to patients with a score of 1 or 2. Patients with locoregional relapse or distant metastasis had three times more frequently a Her2-neu score 0 compared to patients with a score Her2-neu 1 or 2 and furthermore 18 of the 22 deaths had a score 0. These facts explain furthermore the aggressivity of the TNBC and
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especially the aggressivity of TNBC with Her2-neu score 0. Tumour cells with a Her2-neu score 0 are more basallike and more immature than tumour cells with a Her2-neu score 1 or 2. The existence of more immature cells is a possible explanation for the aggressivity of these tumours. It is also possible that the prognosis is also dependent on the different stages of development of the cancer cells. Our data show completely new results. Up to now, tumour cells with a score 0 were not enough investigated. Yet there are no published data yet and further research is necessary. The subgroup with the Her2-neu score 0 may easily be identified, because the determination of the Her2-neu score is routine in the investigation of breast cancer. But the determination of the Her2-neu score depends on the pathologist. A meta-analysis on concordance between immunohistochemistry (IHC) and FISH to detect Her2-neu gene overexpression in breast cancer, published 2014, shows very reliable results. The analysis with over 6,600 patients shows that the overall concordance and discordance rates between IHC staining with score 0 and 1? and FISH for detection failure of Her2-neu expression were 96 and 4 %, respectively [50]. Cases of Her2-neu FISH positive but IHC negative tumours have been reported in the past, making up about 2 % of all breast cancer patients [51, 52]. Much of this discrepancy has been attributed to pre-analytic and analytic issues associated with IHC of paraffin preserved sections. Furthermore, Luoh et al.’s [53] finding of frozen breast tumour samples that score positive for Her2-neu gene amplification by FISH but do not overexpress Her2-neu protein by Western analysis nor Her2-neu mRNA by RT-PCR indicates that some of these tumours may indeed be true Her2-neu FISH false positives, i.e. Her2-neu gene amplification without concomitant protein overexpression. Of note, we did not perform Her2-neu FISH analysis of all patient samples in our cohort, but we estimate that only 2–4 % of our triple negative tumours with an IHC score 0 or 1 have Her2-neu FISH amplification, so that our findings cannot be changed by this bias. Despite standardization, deviations are possible, and both intern controls and extern quality assurance programmes are recommended. Furthermore, our results must be analysed in further studies also with FISH test. In summary, Her2-neu score 0 appears to be a prognostic marker, yet further studies with a higher patient number may be necessary to validate this finding. High risk patients may require closer surveillance in the follow-up, and it is essential to improve the therapy by developing novel regimes. Our study shows furthermore that the proliferation marker Ki-67 failed to predict the survival (DFS and OS). There was no difference in survival between the patients with a low Ki-67 level (\14 %) to patients with a high Ki-
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67 level (C14 %). The results are in line with Aleskandarany et al’s [54] published data. The study group showed that the Ki-67 expression did not correlate with the OS and the distant metastasis-free survival in patients with TNBC. In contrast, Munzone et al. [55] demonstrated in 2012 that the Ki-67 expression could be used like a prognostic factor in TNBC. The importance of Ki-67 in TNBC has not been finally defined yet. Ki-67 is regular topic of discussion because of the cutoff values and the large inter- and intraobserver variation. In 2013, Polley et al. [56] compared the Ki-67 levels in eight of the world’s most experienced laboratories and observed a large variation among those laboratories. It has to be assumed that these days a standardization of Ki-67 pathological assessment is not existent [28]. This might be one of the reasons of the limited value of Ki-67 [57]. Finally, further studies may be required to validate the importance of Ki-67. Although patients with TNBC have a poorer outcome compared to patients with an ER?/Her2-neu- breast cancer, there are a lot of patients with a good survival and without relapse. These days proper prognostic factors for the TNBC are missing. It needs further study to investigate prognostic markers. In conclusion, our data show that tumour size, nodal status and Her2-neu score 0 are very important prognostic markers in patients with TNBC. Patients with large tumours, nodal positive and/or Her2-neu score 0 had a poorer outcome. In contrast, we observed the Ki-67 and grading failed to predict DFS and OS. Conflict of interest
We declare that we have no conflict of interest.
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Identification of prognostic different subgroups in triple negative breast cancer by Her2-neu protein expression Gilda Schmidt • Gabriele Meyberg-Solomayer • Christoph Gerlinger Ingolf Juhasz-Bo¨ss • Daniel Herr • Achim Rody • Cornelia Liedtke • Erich-Franz Solomayer
•
Received: 27 January 2014 / Accepted: 17 June 2014 / Published online: 11 July 2014 Ó Springer-Verlag Berlin Heidelberg 2014
Abstract Purpose Many patients with triple negative breast cancer (TNBC) have a poor outcome, but not all of them. This study has the aim to analyse the prognostic impact of tumour size, nodal status, grading, Her2-neu (human epithelial growth factor receptor 2) score and Ki-67 index. The main goal of this analysis is to find out if there are any differences in survival between patients with TNBC and a Her2-neu score 0 versus 1?2. Experimental design Retrospectively, we studied a cohort of 121 patients with TNBC, diagnosed at the Saarland University Medical Center between December 2004 and June 2013. We compared the disease-free survival (DFS) and overall survival (OS) in those women on the basis of the different Her2-neu scores (0 versus 1 or 2 with negative FISH). Results One hundred and twenty one patients were included in this study. 58.68 % of them had a T2–4 tumour. 39.67 % were nodal positive and 67.77 % had high-grade tumours. The Her2-neu score was determined in 119 patients. 54.62 % of them had a score 0. In the 103 patients with a Ki-67 determination, the mean index was 44.5 %. We found that tumour size, nodal status and Her2-neu score are important prognostic factors. Patients with a Her2-neu score 0 had a significantly poorer outcome regarding DFS and OS. In contrast, the expression level of
G. Schmidt (&) G. Meyberg-Solomayer C. Gerlinger I. Juhasz-Bo¨ss D. Herr E.-F. Solomayer Department of Obstetrics and Gynaecology, Homburg University Medical Centre, University of Homburg, 66421 Homburg, Germany e-mail: [email protected] A. Rody C. Liedtke Department of Obstetrics and Gynaecology, Kiel University Medical Centre, Kiel, Germany
Ki-67 and the grading do not seem to have any prognostic value in TNBC. Conclusion Besides tumour stage, grading and nodal status, the Her2-neu score 0 is able to function as a prognostic factor in patients with TNBC. Keywords TNBC (triple negative breast cancer) Her2 neu score Ki-67
Introduction Breast cancer is the most common cancer in women. Approximately, 15–20 % of the breast cancers are triple negative and associated with a poor prognosis [1–4]. These tumours do not express oestrogen and progesterone receptors and show negativity for Her2-neu protein. Triple negative breast cancers (TNBCs) are very aggressive and more prevalent in younger women [5] and they are characterized by early relapse, early metastasis and early death [6–11]. Previous studies showed that the peak risk of recurrence is within 3 years [12, 13]. In addition, patients with TNBC also have an increased risk for cerebral metastasis compared to patients bearing the ER?/Her2neu-phenotype [14]. In TNBC, there are different intrinsic subtypes known with different molecular features [15]. Besides, triple negative tumours can be divided into six molecular subgroups: basal-like 1, basal-like 2, immunomodulatory, mesenchymal, mesenchymal-stem-like and luminal/AR tumours [16, 17]. It is known that basal-like tumours usually express cytokeratins like epidermal growth factor receptor (EGFR) which is also known as Her1-receptor [18–20]. Previous studies revealed that increased intratumoural expression level of the Her1-receptor in TNBC is associated with nodal
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and distant metastases in the basal-like subtype and show furthermore that the Her1-receptor has a significant prognostic value [21–23]. Except the Her1-receptor, these days there are further three members of the Her family known: Her2-neu, Her3 and Her4. While the importance of Her1 as prognostic marker could be already shown, the question arises if the Her2-neu-receptor is also able to function as a prognostic factor in TNBC. Therefore, in this study, we focused on Her2-neu. Like the other members of the Her family, the Her2-neu is a tyrosinkinase receptor located on the surface of tumour cells and c-erbB2-gen (cerbB2 = cellular avian erythroblastosis homologue B2) coded for Her2-neu. In TNBC, Her2-neu can show three different scores: 0, 1?, 2? (with negative FISH test). Among the determination of the Her2-neu score in breast cancer, the determination of the proliferation marker Ki-67 is also clinical routine. Ki-67 is a nuclear antigen which is present in the nuclei of cells in all phases of the cell cycle as well as in mitosis, but quiescent cells in the G0 phase do not express it [24–26]. The protein Ki-67 is widely used in several cancers as an efficient immunohistochemical marker for determining the proliferation of malignant cells. Patients with TNBC show a significantly higher expression of Ki-67 compared to patients with non TNBCs [21, 22]. In hormone receptor positive breast cancer, the biomarker Ki-67 is useful as a prognostic and likely predictive marker [27, 28]. In the St. Gallen Consensus 2013, its use is recommended as additional factor to divide the large group of receptor positive breast cancers in luminal A and B [29]. Generally, in breast cancer, high Ki-67 is associated not only with a better response to chemotherapy [30–33] but also with a poorer prognosis [23, 34–36]. Hence, the role of Ki-67 in TNBC needs to be further analysed. Our study retrospectively investigates the distribution of Her2-neu score and Ki-67 in TNBC to answer the question concerning the importance of those two markers with regard to disease-free survival (DFS) and overall survival (OS). We thereby wanted to clarify the role of Her2-neu and Ki-67 as prognostic markers arising the questions, if patients with a low Ki-67 index have a better outcome than patients with a high Ki-67 level index and if there are any differences in survival (OS and DFS) between patients with Her2-neu score 0 and Her2-neu score 1?2 in TNBC?.
Methods Between December 2004 and June 2013, a total of 144 patients were diagnosed with TNBC at the Saarland University Medical Center. Patients with breast cancer in their medical history were excluded (n = 11) and patients who have already an event at timepoint 0 were also excluded (n = 12).
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A total of 121 patients were included in this study. The follow-up was until September 2013. Triple negative breast cancer was defined as oestrogen receptor negative (ER 0/12), progesterone receptor negative (PR 0/12) and without overexpression of Her2-neu. These tests were carried out with standard kits by IHC. For each patient in the database, antibody staining of a set of paraffin embedded slides for ER and PR was carried out. Any positivity was taken as positive. The overexpression of Her2-neu status was evaluated using the HercepTestTM. The score is determined by immunohistochemical analyses carried out by a pathologist (HercepTestTM). Score 0 means that the pathologist observes no staining or membrane staining in\10 % of the tumour cells. Score 1? is also negative and indicates that a faint/barely perceptible membrane staining is detected in [10 % of tumour cells. The cells exhibit incomplete membrane staining. A score 2? is also negative in case the pathologist observes a weak to moderate complete membrane staining in [10 % of the tumour cells and no Her2neu amplification in fluorescence in situ hybridization (FISH) test. Her2-neu report of 3? by IHC and 2? with a Her2-neu amplification in FISH test were considered as positive [37]. High expression of Ki-67 was defined as C14 %, with 14 % as cutoff based on a retrospective study realized by Cheang [38]. Disease-free survival was calculated as the interval (in months) between the date of breast cancer diagnosis to first recurrence event: locoregional recurrence or distant metastasis or death of any cause or the date of last followup, if no recurrence event was recorded. The date of diagnosis was defined as the date of first histology of breast cancer. Survival times were censored to the date of last contact for subjects who were lost to follow-up. The OS was defined as the time (in months) between the date of diagnosis and the date of death (breast cancer- and non-breast cancer related) or last follow-up. Following the intent to treat principle, all patients who provided data were included in the statistical analyses. Missing data were not imputed. All continuous variables were analysed during the descriptive statistics number of non-missing observations, mean, standard deviation, minimum, 1st quartile, median, 3rd quartile, and maximum. All categorical variables were analysed by absolute and relative frequency counts. Time to event data was analysed using the Kaplan–Meier estimator. The null hypothesis of no difference between strata was tested using the logrank test. We used a comparison wise significance level a of 5 %, two sided. The statistical analyses were performed using SAS software version 9.2. (SAS Inc. Cary, NC, USA, www.sas.com).
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Results Patients’ characteristics A total of 121 patients with TNBC were included in the study. The characteristics of the study population are presented in Table 1. The age of patients ranged from 28 to 91 years, with a mean age of 55.58 years (Table 1). T2 was the most common (47.94 %) and T4 (3.31 %) the least common presentation. Node negative patients were the largest group with 60.33 %. Positive lymph nodes had 39.67 % of them. 67.77 % of the patients with TNBC had a high-grade tumour and almost 30 % of the patients had an invasion into lymphatic vessels. The Her2-neu score was numerally determined in 119 of the 121 patients. The distribution of the Her2-neu score among the 119 patients with TNBC is also represented in Table 1. 65 patients had a Her2-neu score 0 and 54 patients had a score 1 or 2. The biomarker Ki-67 was determined in 103 of the 121 patients. The mean Ki-67-index was 44.5 % Table 1 Patients’ characteristics
Characteristicsa
N
(Table 1). 92 of the 103 patients (89.32 %) showed a high expression of Ki-67. One hundred and one of the 121 patients were treated with chemotherapy (Table 1). 30 of them obtained a neoadjuvant chemotherapy, the rest of the patients received an adjuvant or primary systemic chemotherapy. The majority of the patients (78.22 %) obtained a taxane-containing Table 2 Incidence of locoregional relapse, distant metastases and death N
Percent (%)
Her2-neu 0, N (%)
Her2-neu 1 or 2, N (%)
Locoregional relapse
16
13.22
11 (100)
5 (100)
Metastasis (multiple incidence possible)
24
19.83
19 (100)
5 (100)
Pulmonary
18
14.88
18 (100)
4 (100)
Osseous
9
7.44
Hepatic
7
5.79
Cerebral Death
6
4.96
22
18.18
Percent (%)
Her2-neu 0, N (%)
Her2-neu 1 or 2, N (%)
Age at diagnosis \40
13
10.74
10 (15.38)
3 (5.56)
40–55
52
42.97
27 (41.54)
23 (42.59)
[55
56
46.28
28 (43.08)
28 (51.85)
T1
50
41.32
23 (35.38)
27 (50.00)
T2
58
47.94
33 (50.77)
24 (44.44)
T3
9
7.44
6 (9.23)
3 (5.56)
T4
4
3.31
3 (4.62)
0 (0.00)
Size of the tumour (T)
Lymph nodes stages (N) N0
73
60.33
36 (55.38)
36 (66.67)
N?
48
39.67
29 (44.62)
18 (33.33)
0.83
1 (1.54)
0 (0.00)
38 82
31.40 67.77
17 (26.15) 47 (72.31)
21 (38.89) 33 (61.11)
Grading (G) G1 G2 G3
Invasion into lymphatic vessels (L) L0
82
67.77
43 (66.15)
37 (68.52)
L1
33
27.27
19 (29.23)
14 (25.96)
Lx
6
4.96
3 (4.61)
3 (5.56)
11
10.67
4 (8.00)
7 (13.73)
92
89.32
46 (92.00)
44 (86.27)
101
83.47
51 (100 %)
48 (100 %)
Ki-67 (%)b \14 C14 Chemotherapy a
Two patients without Her2neu determination
b
Twenty-one patients with unknown Ki-67 (15.79 %)
Taxane-containing
79
78.22
40 (100)
37 (100)
Neoadjuvant
30
29.70
15 (100)
13 (100)
-yT0, yN0
6
20.00
2 (100)
3 (100)
20
16.52
14 (100)
6 (100)
No chemotherapy
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Fig. 1 Her2-neu score as prognostic factor in TNBC. Kaplan–Meier curve of (a) DFS and (b) OS dependent on Her2neu score in triple negative breast cancer. The observed differences between Her2-neu score 0 and Her2-neu score 1?2 (FISH test negative) are significant for both DFS and OS (p = 0.0021 and 0.0.0105, respectively)
regime. Six patients (20.00 %) who obtained a neoadjuvant chemotherapy had a complete remission. 20 patients did not obtain a chemotherapy (7 patients were multimorbid and the resting 13 patients refused chemotherapy). Sixteen of the 121 patients fell into locoregional relapse during the follow-up time, and 2 of them (1.65 %) had breast cancer contralateral. The median time to relapse was approximately 2 years and 4 months. Distant metastasis
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was seen in 24 patients (Table 2). Most of the patients with distant metastasis had visceral metastasis. 15 of the 24 patients with metastasis had only visceral and/or cerebral metastasis, further 8 patients showed both visceral/cerebral and osseous metastasis and 1 patient had only osseous metastasis. The incidence of cerebral metastasis was in 4.96 % of our patients. Patients with locoregional relapse or distant metastasis had three times more frequently a
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Fig. 2 Distribution of nodal status by Her2-neu score. No significant differences between the Her2-neu score 0 group and Her2-neu score 1 and 2 group regarding lymph node status (p = 0.2100; Chi-square test)
Her2-neu score 0 compared to patients with a score Her2neu 1 or 2 (Table 2: 30 patients versus 10 patients). A total of 22 of the 121 patients died until September 2013. This corresponds to 18.18 % (Table 2). 18 of the 22 deaths (82 %) had a Her2-neu score 0. Prognostic factors in TNBC
score 0 had a significantly worse DFS (p = 0.0021) and also a worse OS (p = 0.0105) compared to patients with a Her2-neu score of 1 and 2. In the Her2-neu score 0 group, there was a larger proportion of patients with positive lymph nodes. An additional analysis showed no significant difference from the Her2-neu score 1 and 2 group (p = 0.2100; Chi-square test) (Fig. 2).
Tumour size Ki-67 Patients with a small tumour (T1) had a significantly better outcome (DFS p = 0.0114; OS p = 0.0020) compared to patients with a T2–4 tumour (data not shown). Nodal status Patients with negative nodal status (N0) showed a significantly better DFS (p \ 0.0001) and a significantly better OS (p = 0.0005) compared to patients with lymph node positivity (N1–N3) (data not shown).
The DFS and OS dependent on Ki-67 index are displayed in Fig. 3a, b. It becomes clear that there is no significant difference between patients with Ki-67 C14 and \14 % in DFS (p = 0.5156) and OS (p = 0.6467). A Ki-67 cutoff of 50 % was also calculated, and it confirms the same results (Figure not shown). We did not detect any correlation between expression of Ki-67 and overall, respectively, DFS.
Grading
Discussion
The DFS and OS were not significant better for patients with G1 or G2 tumours compared to patients with G3 tumours (DFS p = 0.1026; OS p = 0.2235) (data not shown).
In this retrospective study, 121 patients with TNBC were analysed regarding DFS and OS. During the observation time, 16 of the 121 patients developed a locoregional relapse, 24 patients had distant metastasis and 22 patients died. In view of tumour size and nodal status, we could show that patients with small tumours and negative nodal status had a significant better outcome compared to patients with a large tumour size and/or a nodal positive status,
Her2-status Figure 1a, b shows the patients outcome to be dependent on the Her2-neu score. Patients with TNBC and a Her2-neu
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Fig. 3 Ki-67 as prognostic factor in TNBC. Kaplan–Meier curve of (a) DFS and (b) OS dependent on Ki-67 index in triple negative breast cancer. The observed differences between Ki-67 \ 14 and Ki67 C 14 % are not significant neither in DFS nor in OS (p = 0.5156 and 0.6467, respectively)
whereas the DFS and OS were not significant worse for patients with high-grade tumours. Furthermore, regarding the Her2-neu distribution in these patients, we could show that patients with a Her2-neu score 0 had a poorer outcome regarding DFS and OS compared to patients with a Her2neu score 1 or 2. Hence our study demonstrates that the Her2-neu score is able to function as a prognostic factor in
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patients with TNBC. On the other hand, Ki-67 failed to predict the DFS and OS. Neither DFS nor OS was significantly better in patients with a low Ki-67 level (\14 %) compared to patients with a high Ki-67 level (C14 %). TNBC is associated with aggressive behaviour and characterized by early relapse, early metastasis and death. The mean time to the local relapse was in our study 2 years
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and 4 months. These results are in line with the published data [39]. Especially between the first and third year after diagnosis, patients with TNBC fell into local relapse. It is known that visceral metastases like hepatic and pulmonary were common in TNBC. In our study, 95.83 % of the patients with distant metastasis had visceral and/or cerebral metastases. 4.16 % of the patients had osseous metastases only. This explains the aggressive behaviour of these tumours. Our results are in line with published data. Ko¨nigsberg et al., for example, demonstrated that patients with TNBC develop distant visceral metastases more frequently than bone metastases [7, 14, 40]. The incidence of cerebral metastasis in 4.96 % of our patients is also in line with the published data [14]. Heitz et al. showed that 6.7 % of all patients with TNBC developed cerebral metastases. We found that patients with a small tumour size showed a better outcome, so that the tumour size acts as a prognostic factor in TNBC. This observation is in agreement with a previous report where the tumour size was the most important prognostic factor in TNBC patients [41]. Similar results show a further study published 2013 [41, 42]. Other studies also revealed that even small tumours (T1N0) seem to have a higher recurrence rate [43, 44]. Our results showed furthermore that not only the tumour size but also the nodal status is able to function as a prognostic factor. This is in agreement with previous published studies [42, 45, 46]. Hernandez-Aya et al. [47] found also that patients with N0 had a significant better OS and RFS (relapse-free survival) (p \ 0.001 all comparisons), but in contrast there was no difference in survival among patients with N1, N2, N3. Histological grading is also known as an important independent prognosis factor [48]. Surprisingly, our study shows no difference regarding outcome (DFS and OS) in patients with low- or intermediate-grade tumours compared to patients with high-grade tumours. Considering the distribution of the histological grading, 82 of our patients had a G3 tumour, 38 patients had a G2 tumour and only 1 patient had a G1 tumour. Published reports showed that the determination of the histologic grading is not always precise and reproducible and, furthermore, the prognosis prediction for the intermediate-grade tumours (G2) is limited [49]. Approximately, 67.77 % of the included patients had G3 tumours which accentuate the aggressivity of the TNBC. In our study, patients with TNBC and a Her2-neu score 0 have a poorer outcome compared to patients with a score of 1 or 2. Patients with locoregional relapse or distant metastasis had three times more frequently a Her2-neu score 0 compared to patients with a score Her2-neu 1 or 2 and furthermore 18 of the 22 deaths had a score 0. These facts explain furthermore the aggressivity of the TNBC and
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especially the aggressivity of TNBC with Her2-neu score 0. Tumour cells with a Her2-neu score 0 are more basallike and more immature than tumour cells with a Her2-neu score 1 or 2. The existence of more immature cells is a possible explanation for the aggressivity of these tumours. It is also possible that the prognosis is also dependent on the different stages of development of the cancer cells. Our data show completely new results. Up to now, tumour cells with a score 0 were not enough investigated. Yet there are no published data yet and further research is necessary. The subgroup with the Her2-neu score 0 may easily be identified, because the determination of the Her2-neu score is routine in the investigation of breast cancer. But the determination of the Her2-neu score depends on the pathologist. A meta-analysis on concordance between immunohistochemistry (IHC) and FISH to detect Her2-neu gene overexpression in breast cancer, published 2014, shows very reliable results. The analysis with over 6,600 patients shows that the overall concordance and discordance rates between IHC staining with score 0 and 1? and FISH for detection failure of Her2-neu expression were 96 and 4 %, respectively [50]. Cases of Her2-neu FISH positive but IHC negative tumours have been reported in the past, making up about 2 % of all breast cancer patients [51, 52]. Much of this discrepancy has been attributed to pre-analytic and analytic issues associated with IHC of paraffin preserved sections. Furthermore, Luoh et al.’s [53] finding of frozen breast tumour samples that score positive for Her2-neu gene amplification by FISH but do not overexpress Her2-neu protein by Western analysis nor Her2-neu mRNA by RT-PCR indicates that some of these tumours may indeed be true Her2-neu FISH false positives, i.e. Her2-neu gene amplification without concomitant protein overexpression. Of note, we did not perform Her2-neu FISH analysis of all patient samples in our cohort, but we estimate that only 2–4 % of our triple negative tumours with an IHC score 0 or 1 have Her2-neu FISH amplification, so that our findings cannot be changed by this bias. Despite standardization, deviations are possible, and both intern controls and extern quality assurance programmes are recommended. Furthermore, our results must be analysed in further studies also with FISH test. In summary, Her2-neu score 0 appears to be a prognostic marker, yet further studies with a higher patient number may be necessary to validate this finding. High risk patients may require closer surveillance in the follow-up, and it is essential to improve the therapy by developing novel regimes. Our study shows furthermore that the proliferation marker Ki-67 failed to predict the survival (DFS and OS). There was no difference in survival between the patients with a low Ki-67 level (\14 %) to patients with a high Ki-
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67 level (C14 %). The results are in line with Aleskandarany et al’s [54] published data. The study group showed that the Ki-67 expression did not correlate with the OS and the distant metastasis-free survival in patients with TNBC. In contrast, Munzone et al. [55] demonstrated in 2012 that the Ki-67 expression could be used like a prognostic factor in TNBC. The importance of Ki-67 in TNBC has not been finally defined yet. Ki-67 is regular topic of discussion because of the cutoff values and the large inter- and intraobserver variation. In 2013, Polley et al. [56] compared the Ki-67 levels in eight of the world’s most experienced laboratories and observed a large variation among those laboratories. It has to be assumed that these days a standardization of Ki-67 pathological assessment is not existent [28]. This might be one of the reasons of the limited value of Ki-67 [57]. Finally, further studies may be required to validate the importance of Ki-67. Although patients with TNBC have a poorer outcome compared to patients with an ER?/Her2-neu- breast cancer, there are a lot of patients with a good survival and without relapse. These days proper prognostic factors for the TNBC are missing. It needs further study to investigate prognostic markers. In conclusion, our data show that tumour size, nodal status and Her2-neu score 0 are very important prognostic markers in patients with TNBC. Patients with large tumours, nodal positive and/or Her2-neu score 0 had a poorer outcome. In contrast, we observed the Ki-67 and grading failed to predict DFS and OS. Conflict of interest
We declare that we have no conflict of interest.
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