Expression of p27 and c-Myc by immunohistochemistry in Breast Ductal Cancers in African American Women

Abstract

Objectives

Proteins p27 and c-myc are both key players in the cell cycle. While p27, a tumor suppressor, inhibits progression from G1 to S phase, c-Myc, a proto-oncogene, plays a key role in cell cycle regulation and apoptosis. The objective of our study was to determine the association between expression of c-Myc and the loss of p27 by immunohistochemistry (IHC) in the four major subtypes of breast cancer (BC) (Luminal A, Luminal B, HER2, and Triple Negative) and with other clinicopathological factors in a population of 202 African-American (AA) women.

Materials and Methods

Tissue microarrays (TMAs) were constructed from FFPE tumor blocks from primary ductal breast carcinomas in 202 AA women. Five micrometer sections were stained with a mouse monoclonal antibody against p27 and a rabbit monoclonal antibody against c-Myc. The sections were evaluated for intensity of nuclear reactivity (1–3) and percentage of reactive cells; an H-score was derived from the product of these measurements.

Results

Loss of p27 expression and c-Myc overexpression showed statistical significance with with ER negative (p<0.0001), PR negative (p<0.0001), triple negative (TN) (p<0.0001), grade 3 (p=0.038), and overall survival (p=0.047). There was no statistical significant association between c-myc expression/p27 loss and luminal A/B and Her2 overexpressing subtypes.

Conclusion

In our study, a statistically significant association between c-Myc expression and p27 loss and the triple negative breast cancers (TNBC) was found in AA women. A recent study found that constitutive c-Myc expression is associated with inactivation of the axin 1 tumor suppressor gene. p27 inhibits cyclin dependent kinase2/cyclin A/E complex formation. Axin 1 and CDK inhibitors may represent possible therapeutic targets for TNBC.

Introduction

Breast cancer is the most common cause of cancer morbidity and the second most common cause of cancer mortality in women worldwide. Histologically, breast neoplasia is divided into two major types, ductal and lobular. Molecular classification of ductal breast cancer by gene expression profiling has identified five major subgroups (Luminal A, Luminal B, Her-2, Normal breast like and basal phenotype) that differ in clinical behavior [1,2,3]. Luminal A and B, are estrogen and/or progesterone receptors(ER/PR) hormone receptor positive. They are generally low grade cancers with good prognosis, increased overall survival and can be treated with hormone receptor inhibitors [1,2]. Her2 overexpressing tumors are aggressive, carry poor prognosis, but have available targeted therapy. The treatment of these tumors with trastuzumab (HER2 inhibitor) has significantly improved prognosis. The triple negative breast cancers (TNBC), tumors lacking expression of ER, PR and HER2 receptors, are generally high grade ductal cancers with established aggressive clinical course, high proliferative index, decreased overall survival and increased incidence of distant metastasis[1,2]. They might be resistant to conventional chemotherapy. Currently, no targeted therapy is available for these aggressive tumors. The basaI-like TNBC subtype expresses CK5. However, all the TNBC are not basal type and vice versa.

Recent studies have shown that cell cycle dysregulation plays an important role in the pathogenesis of TNBC [17,18]. Still, the significance of c-Myc expression, p27 loss and cell cycle dysregulation in breast carcinogenesis is poorly understood. The high proliferative activity of TNBC supports the upregulation of cell cycle driver genes and the downregulation of cell cycle inhibitors as potential pathogenetic mechanisms.

In particular, c-Myc is a proto-oncogene, located on chromosome 8, that regulates the expression of many target genes involved in cell growth, cell cycle regulation, and apoptosis [14,32]. Constitutive expression of c-Myc can result in uncontrolled cell proliferation. C-Myc activation promotes formation of cyclin A/E and cyclin dependent kinase 2 complex (CDK2 and cyclinA/E), which are critical for progression from the G1 to the S phase of the cell cycle. It also downregulates p21; this inhibits progression from the G2 to the M phase [17]. A recent study has found that c-Myc stabilization by selective phosphorylation results in c-Myc with enhanced oncogenic activity due to inactivation of the axin 1 tumor suppressor gene, an important regulator of survival, growth, and stress pathways [40,41]. Protein p27 (cyclin-dependent kinase inhibitor 1B) is a tumor suppressor protein, encoded by the CDKN1B gene. It inhibits formation of CDK2/cyclin A/E complex and prevents progression of the cell cycle from the G1 phase to the S phase [9].

Notably, Breast cancers in African American (AA) women present at a younger age, have a higher grade and stage at diagnosis, and are associated with a higher mortality [38,39]. We hypothesize that the inverse expression of c-myc with p27 could be associated with more adverse and aggressive clinical phenotypes. However, no study has specifically investigated the combined expression of c-Myc and p27 in breast cancer in AA women, who manifest an increased incidence of TNBCs. For this study, we compared the immunohistochemical expression of c-Myc and p27 in the four major subtypes of breast cancer (BC) (Luminal A, Luminal B, HER2, and Triple Negative), and determined that association with other clinicopathological features including grade, stage, disease-free, and overall survival in a population of 202 AA women.

Materials and Methods

Tissue Samples

This study was reviewed and formally exempted by the Howard University Institutional Review Board (IRB-10-MED-24). We analyzed invasive breast ductal carcinomas (IDCs) from 202 AA women diagnosed and treated at the Howard University Hospital between 2000 and 2010. Demographic and clinical information was obtained through the Howard University Cancer Center Tumor Registry.

Tissue Microarrays

A series of tissue microarrays (TMAs) was constructed containing the consecutive primary IDCs (Pantomics, Inc., Richmond, CA). The TMAs consisted of 10 × 16 arrays of 1.0-mm tissue cores from well preserved, morphologically representative tumor cells in archived, formalin-fixed, paraffin-embedded (FFPE) surgical blocks. A precision tissue arrayer (Beecher Instruments, Silver Spring, MD) with two separate core needles for punching the donor and recipient blocks was used. The device also had a micrometer-precise coordinate system for tissue assembly on a multitissue block. Two separate tissue cores of IDC represented each surgical case in the TMA. Each tissue core was assigned a unique TMA location number, which was subsequently linked to an Institutional Review Board-approved database containing demographic and clinical data. Using a microtome, 5-μm sections were cut from the TMA blocks and mounted onto Superfrost Plus microscope slides.

Immunohistochemistry

Immunohistochemistry (IHC) was performed on TMA sections of FFPE tumor tissue. The polymer-HRP system was utilized for immunostaining. Following deparaffinization and rehydration of the tissue sections, heat-induced epitope retrieval at pH 9.0 was performed. Five micrometer sections were stained with mouse monoclonal antibodies against p27Kip1 (SX53G8, Cell Marque, Rocklin, CA). Additional five micrometer sections were stained with a rabbit monoclonal antibody against c-Myc (EP121, Cell Marque, Rocklin, CA). Primary antibody detection was carried out using a polymer-based detection system with staining development achieved by incubation with 3,3′-diaminobenzadine (DAB) and DAB Enhancer (Envision Plus, DAKO, Carpinteria, CA). IHC staining was performed at Quest Diagnostics (Chantilly, VA). Immunohistochemical stains were scored by two independent observers (TN and FK) blinded to the clinical outcome. The sections were evaluated for intensity of nuclear reactivity (1–3) and percentage of reactive cells. The results were entered into a secure research database. An H-score was derived from the product of these measurements. Cases were categorized as having decreased (score ≤50) or increased (score >50) nuclear expression.

Breast subtypes were defined using immunohistochemical expression of estrogen receptor (ER), progesterone receptor (PR), and HER2 based on criteria established in the literature. Luminal A was characterized by strong expression of ER and PR (H-score ≥200), Ki-67 proliferation <14%, and HER2 negativity. Luminal B was characterized by Ki-67 proliferation ≥14% and often weaker expression of ER/PR (H-score <200). Luminal B cases were HER2-negative or HER2 positive. The HER2 subtype was hormone receptor-negative with only HER2 positivity. The triple-negative subtype lacked expression of ER, PR, and HER2.

Statistical Analysis

To determine the association between c-MYC, p27 and ER, PR, HER2, subtype, grade, stage, age, overall survival, and recurrence, bivariate analysis was performed using the chi square test or Fisher’s exact test, as appropriate. To examine the correlation between variables, overall survival, and disease-free survival, Kaplan-Meier survival analyses were carried out. Estimates were considered statistically significant for two-tailed values of P< 0.05. All analyses were carried out using the SPSS 22.0 statistical program (SPSS Inc., Chicago, IL).

Results

Characteristics of the Study Population

Clinical and pathological characteristics of the study population are summarized in Table 1. There were 202 patients diagnosed with infiltrating ductal carcinomas, with adequate FFPE tumor tissue for analysis, at our institution from 2000 to 2010. The majority of the tumor blocks (67.8%) came from women over the age of 50 (mean=57.65, SD=13.03), most of whom (76.2%) had no cancer recurrence. In this population, 43%, 47.7%, and 13.9% of tumors were ER−, PR−, and HER2-positive, respectively. The most prevalent breast cancer subtype was luminal A (ER+ or PR+, HER2−; 43.5%), followed by triple-negative (ER−, PR−, HER2−; 33.7%). Greater than 70% of all tumors were stage I and II; however, they tended to be of higher grade, with Grade 3 tumors comprising 67.4% of the tumors in the study population.

Table 1.

Clinical and pathologic characteristics of study population

Characteristic	Number	%
	n=202
Age
<50	62	32.1
>50	131	67.8
Estrogen Receptor
Positive	110	57
Negative	83	43
Progesterone Receptor
Positive	92	47.7
Negative	101	52.3
HER2 Status
Positive	27	14
Negative	166	86
Subtype*
Luminal A	84	43.5
Luminal B	27	14
Her2	17	8.8
Triple Negative	65	33.7
Pathologic Stage Group
1	60	31.1
2	80	41.5
3	40	20.7
4	12	6.2
Unknown	1	0.5
Grade
I	9	4.6
II	54	28
III	130	67.4
Recurrence
None	147	76.2
Loco-regional	8	4.1
Distant	21	10.9
Never Disease free	8	4.1
Unknown	9	4.7

+N=202

ER, estrogen receptor; PR, progesterone receptor.

^*Luminal A: ER+ or PR+, HER2−; luminal B: ER+ or PR+, HER2+; triple-negative: ER−, PR−, HER2−; Her2+: ER−, PR−, HER2+

Immunohistochemistry and Analysis

Because some cores were lost in deeper TMA sections, only 197 cases were included in the analysis. The association between clinicopathological features with c-Myc and p27 expression (no expression/co-expression, c-Myc positive/p27 negative and c-Myc negative/p27 positive) can be found in table 2. Loss of p27 expression and c-Myc overexpression showed statistical significance with ER negative (p<0.0001), PR negative (p<0.0001), triple negative (TN) (p<0.0001) and grade 3 (p=0.038) breast cancers in AA women. There was no statistical significant association between c-myc expression/p27 loss and luminal A/B and Her2 overexpressing subtypes. Immunohistochemical detection of c-Myc in triple negative subtype with basal phenotype and luminal A subtype is shown in figures 1a and 1b, respectively. The triple negative subtype shows strong and diffuse expression of c-Myc and the luminal A subtype shows no expression of c-Myc. p27 expression in triple negative subtype with basal phenotype and luminal A subtype is shown in figures 2a and 2b, respectively. The triple negative subtype shows no expression of p27and the luminal A subtype shows strong expression of p27. No association was seen between c-Myc expression/p27 loss and age, tumor size, node status, or stage.

Table 2.

Association between ER, PR, molecular subtype, grade, cMyc, and p27 co-expression

	No expression or co expression	%	p27+ cMyc −	%	p27− cMyc +	%	p-Value
Estrogen Receptor Expression
Negative	28	50.91%	12	21.82%	15	27.27%
Positive	33	55.93%	25	42.37%	1	1.69%	<0.0001
Progesterone Receptor Expression
Negative	35	55.56%	13	20.63%	15	23.81%
Positive	26	50.98%	24	47.06%	1	1.96%	<0.0001
Breast Cancer Molecular Subtype
Luminal A	24	54.55%	20	45.45%	0	0.00%
Luminal B	10	62.50%	5	31.25%	1	6.25%
HER 2+	12	75.00%	2	12.50%	2	12.50%
Triple Negative Breast Cancer	15	39.47%	10	26.32%	13	34.21%	<0.0001
Grade
I	2	50.00%	2	16.67%	0	0.00%
II	21	60.00%	14	40.00%	0	0.00%
III	38	50.67%	21	28.00%	16	21.33%	0.038

Figure 1.

a) Triple negative subtype with basal-like phenotype showing diffuse and strong nuclear expression of c-Myc (X200); b) Luminal A subtype showing no expression of c-Myc (X200)

Figure 2.

a) Triple negative subtype with basal-like phenotype showing loss of expression of p27 (X200); b) Luminal A subtype showing strong expression of p27 (X200)

Discussion

Our study evaluated c-Myc and p27 expression in breast cancers in a minority population of AA women. Our results showed a significant association of c-Myc overexpression and loss of p27 in TNBCs (P<0.0001) in AA females with high sensitivity, specificity, PPV, and NPV. These findings further support the results of previous studies, which have linked either c-Myc overexpression or p27 loss to breast cancers with negative-hormone-receptor and basal-like phenotypes. No study has specifically investigated the combined expression of c-Myc and p27 in breast cancer in AA women. In our study, a statistically significant association was found between p27 loss and c-Myc overexpression in TNBCs with decreased overall survival. Earlier studies also showed worse prognosis and decreased disease-free and overall survival in c-Myc expressing breast cancers. Our study showed the association between c-Myc overexpression and p27 loss in TNBC in AA women and establishes the combined role of these key cell cycle modulators in these tumors.

Biological differences are found between breast cancers in AA and Caucasian women. In United States, the incidence of breast cancer is rising in AA women since 2008 and the overall incidence is equal in AA and Caucasian women since 2012 [37]. The breast cancer mortality is high in AA women with lower five year survival rate (77%) as compared to Caucasian women (90%) [38]. TNBC is more frequent in premenopausal AA women (39%) compared to either postmenopausal AA Women (14%) or in non-African Americans of any age (16%) [39]. More rapid progression and decreased survival in AA women may be attributed to the higher incidence of TNBCs in AA women. Approximately 70% of TNBCs are basal-like, characteristically displaying a stem cell-like phenotype, high proliferative activity, and increased incidence of lymph node and distant metastasis. In our study, 67% of TNBCs expressed CK5, a basal marker.

C-Myc, a strong proto oncogene, encodes a nuclear transcription factor that regulates expression of many genes involved in cell cycle progression, cell growth, and apoptosis through binding on Enhancer Box sequences and recruiting histone acetyltransferases [14,32]. c-Myc drives the cell cycle by promoting progression from G1 to S phase and G2 to M phase. G1 to S phase progression is facilitated by activating CDK2 and cyclin A/E complex formation, which leads to phosphorylation of the retinoblastoma protein (Rb); this causes inactivation of the Rb protein bypassing a critical checkpoint of the cell cycle [17,18,19]. G2 to M phase progression is facilitated by downregulating the p21 gene, that inhibits progression in this phase of cell cycle. A recent study found that constitutive c-Myc expression is associated with inactivation of the axin 1 tumor suppressor gene [40,41]. c-Myc is a multifunctional protein that promotes cell growth and also regulates apoptosis via downregulation of the BCL2 protein. Our study demonstrates constitutive expression of c-Myc in TNBCs when compared to luminal A and B tumors in AA women; this finding suggests that cell cycle dysregulation may play a significant role in the pathogenesis of these cancers. TNBCs are highly aggressive tumors with increased incidence of distant metastasis. They are resistant to currently available targeted therapy since they lack hormone and HER2 receptors. Axin 1 tumor suppressor gene or other target genes regulated by c-Myc may represent potential therapeutic targets for TNBCs in AA women [7].

P27 is a tumor suppressor gene that inhibits progression from G1 to S phase of cell cycle. It inhibits the formation of CDK2 and cyclin A/E complexes and keeps Rb protein in a hypophosphorylated state [9,18,21]. Loss of p27 suggests loss of this critical checkpoint of the cell cycle. Our study demonstrates loss of p27 in TNBCs when compared to luminal A and B tumors in AA women. Cyclin dependent kinase inhibitors may represent potential therapeutic targets for TNBCs in AA women.

In summary, our study shows significant association between c-Myc overexpression and p27 loss in TNBCs, a subtype, which correlates with progression and decreased survival, in AA women. TNBCs often have an aggressive clinical course, high proliferative activity, increased incidence of distant metastasis, and are resistant to targeted therapy due to lack of hormone and HER2 receptors. Breast cancers have heterogeneous carcinogenesis at the molecular level. Identification and understanding of molecular pathogenesis that underlie TNBC is critical to develop therapeutic targets. Cell cycle dysregulation with increased proliferative activity is central to the pathogenesis of TNBC. c-Myc overexpression and p27 loss cause uncontrolled activation of the cell cycle through several mechanisms. Axin 1 tumor suppressor gene stabilizers and cyclin dependent kinase inhibitors may represent potential molecular targets for therapy in triple-negative and hormone-negative breast cancers, occurring with increased incidence in minority populations.

Figure 3.

Overall survival analysis of p27 and cMyc expression

Table 3.

Mean survival for p27 and cMYC expression

	Meana
	Estimate	Std. Error	95% Confidence Interval		P-Value
			Lower Bound	Upper Bound
No expression or co expression	84.82	7.23	70.65	98.99
P27 positive cMyc negative	126.81	9.50	108.19	145.42
p27 negative cMYC positive	102.58	13.32	76.48	128.69	0.047b
Overall	103.754	6.345	91.318	116.19

^aEstimation is limited to the largest survival time if it is censored;

^bLog Rank Chi-square=6.136 (2df)