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Background: The aim of this study was to investigate the clinicopathological characteristics of GATA binding protein 3
(GATA3)-positive breast cancers as well as the association of GATA3 expression with response to chemotherapy.
Patients and methods: Tumor specimens obtained before neoadjuvant chemotherapy [ paclitaxel followed by
5-uorouracil/epirubicin/cyclophosphamide)] from breast cancer patients (n = 130) were subjected to
immunohistochemical and mutational analysis of GATA3 and DNA microarray gene expression analysis for intrinsic
subtyping.
Results: Seventy-four tumors (57%) were immunohistochemically positive for GATA3. GATA3-positive tumors were
signicantly more likely to be lobular cancer, estrogen receptor (ER)-positive, progesterone receptor (PgR)-positive, Ki67-
negative, and luminal A tumors. Somatic mutations were found in only three tumors. Pathological complete response (pCR)
was observed in 8 (11%) GATA3-positive tumors and in 22 (39%) GATA3-negative tumors. Multivariate analysis showed that
tumor size, human epidermal growth factor receptor 2 (HER2), and GATA3 were independent predictors of pCR.
Conclusions: GATA3-positive breast cancers showed luminal differentiation characterized by high ER expression and
were mostly classied as luminal-type tumors following intrinsic subtyping. Interestingly, GATA3 was an independent
predictor of response to chemotherapy, suggesting that GATA3 might be clinically useful as a predictor of a poor
response to chemotherapy.
Key words: breast cancer, GATA3, intrinsic subtype, microarray, neoadjuvant chemotherapy, pathological complete
response
The Author 2012. Published by Oxford University Press on behalf of the European Society for Medical Oncology.
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original articles Annals of Oncology
GATA3 is critical for the normal development of the Each patient underwent a vacuum-assisted core tumor biopsy
mammary gland. Introduction of GATA3 into a puried (Mammotome 8G; Ethicon Endosurgery; Johnson & Johnson, Cincinnati,
mammary progenitor-enriched population has been shown to OH) under ultrasonographic guidance before neoadjuvant chemotherapy.
induce luminal cell differentiation [3]. In human breast tissue, Tumor biopsy samples were xed in 10% buffered formaldehyde for
GATA3 is expressed in the luminal epithelial cells, and its histological examination and also snap-frozen in liquid nitrogen and kept
expression is also seen in breast cancer [4, 5]. The expression at 80C until use for DNA and RNA extraction. Every patient underwent
breast-conserving surgery or mastectomy after neoadjuvant chemotherapy.
of GATA3 is closely associated with estrogen receptor (ER) and
Patient characteristics entered in this study are shown in Table 1. Informed
forkhead box A1 (FOXA1) expression in breast cancer [4, 6].
consent for the study was obtained from each patient before tumor biopsy.
FOXA1 plays an important role in the transcriptional function
of ER [7]. It has also been suggested that GATA3 may be a
mediator for the transcriptional up-regulation of mucin 1 Table 1. Associations between GATA3 expression and clinicopathological
(MUC1) in breast cancer [8]. These results clearly indicate that parameters
GATA3 plays a pivotal role in the development and
differentiation of luminal epithelial cells, which is consistent Characteristic Number GATA3 P value
with the preferential expression of GATA3 in luminal-type of patients, Negative, n Positive, n
breast cancer [9]. Somatic mutations of GATA3 are reported to n (%)
occur infrequently (4%5%) in breast tumors. Interestingly, Total, n (%) 130 56 (43) 74 (57)
these mutations are mostly seen in ER-positive tumors, Median age, year (range) 52 (2773)
suggesting involvement of GATA3 mutation in the Histological type 0.002a
pathogenesis of luminal-type breast cancer [10, 11]. IDC 119 (92) 56 63
Because of this crucial function of GATA3, GATA3-positive ILC 11 (9) 0 11
tumors are thought to be derived from luminal progenitors and Stage 0.894a
to maintain the luminal epithelial cell phenotype. In fact, IIA 43 (33) 18 25
GATA3-positive tumors are often ER-positive tumors, and a IIB 69 (53) 30 39
large body of studies has demonstrated that GATA3 expression is IIIA 13 (10) 5 8
signicantly associated with a favorable prognosis, suggesting that IIIB 5 (4) 3 2
GATA3 may serve as a new prognostic factor for breast cancer Tumor size 0.624a
T1 7 (5) 2 5
[12]. On the other hand, the relationship of GATA3 expression
T2 98 (75) 44 54
with response to chemotherapy has rarely been studied. Since
T3 20 (15) 7 13
GATA3 expression is associated with ER expression, which has
T4 5 (4) 3 2
been shown to be associated with a poor response to
Lymph node status 0.844b
chemotherapy [13], it is speculated that GATA3 expression is
N0 43 (33) 18 25
also associated with a poor response. Moreover, since GATA3 N1 87 (67) 38 49
induces a variety of luminal epithelial cell-related genes including Histological grade <0.001a
ER, GATA3 may be even more strongly associated with resistance 1 18 (14) 1 17
to chemotherapy. In the study presented here, we therefore tried 2 86 (66) 38 48
rst to elucidate the clinicopathological characteristics of 3 26 (20) 17 9
GATA3-positive tumors and, next, to investigate the association ER status (10%) <0.001b
of GATA3 expression with response to neoadjuvant Positive 77 (59) 13 64
chemotherapy for breast cancer. In addition, we studied whether Negative 53 (41) 43 10
or not GATA3 and FOXA1 could cooperate to induce ER in PgR status (10%) <0.001b
human breast cancer tissues since FOXA1, like GATA-3, has Positive 50 (39) 9 41
been reported to induce ER [6, 7], and also studied the Negative 80 (62) 47 33
correlation of MUC1 with response to neoadjuvant Her2 status (>2.0/FISH) 0.306b
chemotherapy since MUC1 is induced by GATA3 [8] and is Positive 38 (29) 19 19
shown to be implicated in resistance to chemotherapy [14]. Negative 92 (71) 37 55
Ki67 status (20%) 0.017b
Positive 77 (59) 40 37
patients and methods Negative 52 (40) 16 36
Unknown 1 (1) 0 1
patients MUC1 status (10%) 0.024a
Primary breast cancer patients (n = 130) who had been treated with Positive 97 (75) 36 61
neoadjuvant chemotherapy [ paclitaxel (80 mg/m2) weekly for 12 cycles Negative 24 (19) 15 9
followed by combined 5-uorouracil (500 mg/m2), epirubicin (75 mg/m2), Unknown 9 (7) 5 4
and cyclophosphamide (500 mg/m2) every 3 weeks for four cycles (P-
a
FEC)] between 2004 and 2009 at Osaka University Hospital were Fishers exact test.
b
retrospectively recruited for this study. During this period, neoadjuvant Chi-square test.
chemotherapy was indicated for the breast cancer patients with T34 and ER, estrogen receptor; GATA3, GATA binding protein 3; Her2, human
any N or any T and N13, and for those who had large tumors (3 cm) epidermal growth factor receptor 2; IDC, invasive ductal carcinoma; ILC,
relative to the breast and wished to undergo breast-conserving surgery. invasive lobular carcinoma; MUC1, mucin 1; PgR, progesterone receptor.
Figure 1 Immunohistological examination of GATA3 and FOXA1. Representative results of immunohistochemical staining of GATA3 and FOXA1 in
breast tumors and normal breast tissues (200, bar = 20 m) are shown. Nuclear staining of GATA3 and FOXA1 is seen in breast cancer cells ( panels A and
C, respectively) and normal luminal epithelial cells ( panels B and D, respectively). FOXA1, forkhead box A1; GATA3, GATA binding protein 3.
GATA3 for prediction of pathological response clinicopathological parameters in order to elucidate the
pCR was observed in 8 (11%) of 74 GATA3-positive tumors characteristics of GATA3-positive tumors.
and in 22 (39%) of 56 GATA3-negative tumors (P < 0.001) Immunohistochemical examination showed that such tumors
(Supplemental Figure S1, available at Annals of Oncology were more likely to be ER-positive and PgR-positive tumors
online). Univariate analysis of clinicopathological parameters and were more likely to be luminal A tumors as determined by
showed that tumor size (P = 0.01), ER (P < 0.001), PgR gene expression proling. These ndings are consistent with
(P = 0.004), HER2 (P = 0.004), Ki67 (P = 0.027), and GATA3 the function of GATA3 in which it plays a pivotal role in
(P < 0.001), but not FOXA1 and MUC1, were signicantly differentiation into luminal epithelial cells. Similar results for
associated with pCR rate (Table 3). Multivariate analysis the preferential expression of GATA3 in luminal A tumors
identied tumor size (P = 0.037), HER2 (P = 0.027), and have also been reported by Chou et al. [22]. Interestingly, we
GATA3 (P = 0.036) as independent predictors of pCR rate found that all inltrating lobular cancers were GATA3-positive
(Table 3). as was also found by Albergaria et al. [23]. It has been reported
that GATA3 expression is relatively high in the terminal end
buds from which lobular carcinomas are thought to stem, so
differentially expressed genes in GATA3-positive that inltrating lobular cancers are considered to be breast
and GATA3-negative tumors tumors, which have highly differentiated into luminal cells. In
The 37 genes, corresponding to the 57 probes and differentially fact, all inltrating lobular cancers in our study were classied
expressed in GATA3-positive and GATA-negative tumors, were as luminal A tumors.
selected by using the P value (9.15 107) determined after Since GATA3 and FOXA1 have been reported to induce ER
Bonferroni correction. The list of these differentially expressed synergistically in in vitro studies [22, 24, 25], we investigated
genes with annotations is shown in Supplemental Table S3 whether such a cooperation can also be observed in human
(available at Annals of Oncology online). Of the 57 probes, 21 breast cancers. We found that both GATA3- and FOXA1-
(37%) were related to metabolism, 9 (16%) to gene positive tumors showed the highest ER positivity and that both
transcription, 6 (11%) to signal transduction, 5 (9%) to mucin GATA3- and FOXA1-negative tumors showed the lowest
secretion, 2 (4%) to proliferation, 2 (4%) to DNA repair, and positivity, while either GATA3- or FOXA1-positive tumors
12 (21%) were unknown. showed intermediate positivity. These results seem to be
consistent with the in vitro ndings that GATA3 and FOXA1
cooperate to induce ER in breast cancer.
discussion It has been reported that 4%5% of breast cancers harbor
For current study, we rst studied the associations between somatic mutations of GATA3, which are predominantly
GATA3 expression and the various conventional located in exons 46 [10, 11]. Consistent with this nding, we
Figure 2 Intrinsic subtyping of breast tumors. Classication of 123 breast tumors into intrinsic subtypes (luminal A, luminal B, HER2 enriched, basal-like,
and normal breast like) was done by DNA microarray analysis of gene expression with a previously described method [20]. Hierarchal cluster analysis was
done in each subtype and the results are shown as a heatmap. The genes used for classication is also shown in the Supplementary Table 2 (available at
Annals of Oncology online). Bars at the bottom show the immunohistochemical results for ER, PR, HER2, Ki67, GATA3, and FOXA1 as well as the
pathological response. ER, estrogen receptor; FOXA1, forkhead box A1; GATA3, GATA binding protein 3; HER2, human epidermal growth factor receptor
2; OR, odds ratio; pCR, pathological complete response; PR, progesterone receptor.
Table 3. Univariate and multivariate analysis of various predictive factors for response to chemotherapy
tumors (5%) were found to harbor somatic mutations and all 37 genes implicated in apoptosis, DNA repair, cell cycle
these tumors were ER positive [10]. Since GATA3 causes regulation, and metastasis (Supplemental Table S3, available at
invasive breast cancer cells to undergo reversal of epithelial Annals of Oncology online). The inclusion of trefoil factor 1
mesenchymal transition, leading to the suppression of cancer (TFF1) in these genes was noteworthy since it was signicantly
metastasis [26], and it regulates tumor differentiation and highly expressed in GATA3-positive tumors and is known to
suppresses tumor dissemination in breast cancer [5], GATA3 is inhibit the cytochrome C- or Fas-induced activation of caspase
thought to have a tumor suppressive effect. Putting these 8 [27]. This suggests that TFF1 may play a signicant role in
results together leads to the speculation that loss of GATA3 the resistance to chemotherapy through the inhibition of
function is implicated in the pathogenesis of luminal-type apoptosis. It has also been suggested that phosphoserine
breast cancer even though its incidence is very low (< 5%). aminotransferase 1 (PSAT1) is implicated in chemoresistance
It is well known that ER positivity is associated with a poor by decreasing the apoptotic response in colon cancer cell [28].
response to chemotherapy, and, indeed, we found in this study Moreover, anterior gradient 2 homolog (AGR2) is reportedly
that the pCR rate of ER-positive tumors (12%) was implicated in resistance to gemcitabine in pancreatic cancer
signicantly lower than that of ER-negative tumors (40%). The cell lines [29] and cyclin D1 (CCND1) is also reported to play
precise reason for this association still remains to be a signicant role in resistance to docetaxel in in vitro [30]. In
investigated but one hypothesis is that ER serves as a marker of addition, high expression of CCND1 in GATA3-positive
luminal epithelial differentiation and that well-differentiated tumors is compatible with the recent nding that CCND1 is a
tumors are generally thought to be less likely to respond to direct transcriptional target of GATA3 in neuroblastoma tumor
chemotherapy than less differentiated tumors. We next cells [31]. However, the precise mechanism of the resistance of
examined the relationship between GATA3 expression and GATA3-positive tumors to chemotherapy still needs to be
pCR rate because GATA3 is a master gene for luminal investigated.
differentiation and can thus be expected to reect the luminal A limitation of this study is that the GATA3 cut-off value
differentiation status more accurately than ER expression and was decided so that the P value for the correlation between
may serve as a better marker of resistance to chemotherapy GATA3 and pathological response would become minimal,
than ER. We were able to demonstrate that GATA3-positive implying that this could be a hypothesis-raising study and thus
tumors were signicantly less likely to achieve pCR, and the predictive value of GATA3 is overrated. Our present
multivariate analysis results showed that GATA3 positivity was observation needs to be validated in future. However, the fact
signicantly associated with a lower pCR rate as well as that that the median value of GATA3 was also 10% seems to
this association was independent of other clinicopathological indicate that the association of GATA3 expression with
parameters including ER. To the best of our knowledge, ours is resistance to chemotherapy may be plausible since
the rst report to demonstrate that GATA3 is a better predictor dichotomization using a median value is a less biased method.
than ER of response to chemotherapy. In conclusion, our results showed that GATA3-positive
Since GATA3 induces a variety of genes during luminal tumors possessed a phenotype of luminal A tumors and
differentiation, we speculated that some of these might play a GATA3 mutations were found in only 3% of breast tumors.
signicant role in the resistance to chemotherapy. We therefore Furthermore, GATA3 was found to be an independent
investigated the genes differentially expressed in GATA3- predictor of response to chemotherapy for human breast
positive and GATA3-negative tumors and were able to identify cancer, suggesting that GATA3 may be clinically useful as a