Expression of CD74 in Invasive Breast Carcinoma: Its Relation to Nottingham Prognostic Index,Hormone Receptors,and HER2 Immunoprofile

Abstract

Purpose

To study the immunohistochemical expression of CD74 in series of invasive breast carcinomas classified according to their estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) immunoprofile and explore its correlation to Nottingham Prognostic Index (NPI) and tumor pathologic stage to determine if it has a prognostic value.

Methods

A total of 160 cases of mammary carcinoma were classified broadly according to their ER, PR, and HER2 expression into luminal, HER2-positive, and triple-negative groups. The NPI was calculated and pathologic stage was recorded for each individual case and cases were classified into different prognostic groups. The CD74 expression was evaluated immunohistochemically and correlated to different prognostic variables.

Results

The CD74 immunohistochemical expression in invasive breast carcinoma was significantly higher in triple-negative tumors, higher tumor grades, presence of lymph nodal metastasis, higher tumor stages, and higher NPI scores.

Conclusions

The CD74 might be a useful prognostic indicator predicting poor outcome of patients with breast carcinoma. Its consistent expression in triple-negative breast carcinomas points to the need of further studies to test the possibility if it can be targeted in treatment of breast carcinoma, especially in such groups.

Introduction

Despite early detection, treatment options, and survival improvement, the morbidity and mortality of invasive breast carcinoma continue to increase and many patients develop metastatic diseases that lead to death. Thus, there is an urgent need to identify novel prognostic biomarkers (1).

Clinically, this heterogeneous disease is broadly categorized into 3 basic molecular groups. The estrogen receptor (ER)-positive group is the most numerous and diverse. The human epidermal growth factor receptor 2 (HER2)-amplified group has great clinical success because of effective therapeutic targeting of HER2. Triple-negative breast cancers represent approximately 15% of the newly diagnosed cancers worldwide. The last group represents an important clinical challenge because these cancers do not respond to endocrine therapy or other available targeted agents, thus it is urgent to find active agents in this field of non-hormone-responsive lesions (2, 3).

Major histocompatibility complex (MHC) class II-associated invariant chain (Ii; CD74) is a type II transmembrane glycoprotein. It connects with the MHC II a and b chains and guides the convey of the abII complexes to intracellular endosomes and lysosomes, thus launching antigen presentation for immune response (4). With respect to these biological functions, it is possibly pertinent that CD74 is also expressed on a diversity of malignant cells.

CD74 expression has been described in hematologic (5, 6) and nonhematologic cancers, including gastrointestinal (7-8-9), renal (10), non-small-cell lung (11), and recently, glioblastoma cell lines (12). In many of these cancers, expression of CD74 has been proposed to be a prognostic factor, with its higher relative expression of CD74 correlating with tumor progression or poor clinical outcome (13, 14). The biological functions of CD74 together with its expression on malignant cells and limited expression on normal tissues suggest CD74 as a probable therapeutic target worthy to be studied further in other types of cancers (15).

Few studies explored CD74 expression in breast carcinoma (1, 16), so this work aims to study the immunohistochemical expression of CD74 in series of invasive breast carcinomas classified according to their ER, progesterone receptor (PR), or HER2 immunoprofile and explore its correlation to Nottingham Prognostic Index (NPI) and tumor pathologic stage to determine if it has prognostic value.

This retrospective study followed the ethical standards of and was approved by the Ethical Committee at Tanta Faculty of Medicine, Egypt.

Materials

The present study was carried out on 160 specimens of invasive breast carcinoma. Cases with hormonal profile and HER2 status were those to be included in this study provided that the blocks were of primary tumor from patients who underwent radical mastectomy, modified radical mastectomy, or conserving surgery including sentinel and/or axillary lymph node dissection, while patients who received neoadjuvant chemotherapy or who underwent preceding treatment at another institution or patients with second primary tumor were excluded. Data regarding primary tumor size, lymph node status, and pathologic stage (pTNM) according to Lakhani et al (17) were retrieved from pathology archives at Tanta University together with hormonal profile and HER2 status for each included case. Data about the real outcome of patients included in this study were missing as our cases were from archives as the regimen of treatment now differs in our institution and most patients receive neoadjuvant chemotherapy before surgery, which conflicts with our inclusion criteria.

Methods

Paraffin blocks from the primary tumor were serially sectioned into 5 µm thickness and stained with hematoxylin & eosin. Stained sections were reviewed to assess the histologic grade according to the Elston and Ellis grading system (18).

Nottingham Prognostic Index was calculated for each individual case taking into consideration the tumor size, lymph node status, and histologic grade. Nottingham Prognostic Index was calculated as follows: NPI = tumor size in cm × 0.2 + histologic grade (1-3) + lymph node status (1-3) (1 = 0 lymph nodes, 2 = 1-3 lymph nodes, 3 = ≥4 lymph nodes).

According to the score value, the cases were stratified into one of 4 prognostic groups where the higher the value, the worse the prognosis: excellent, NPI score <2.41; good, NPI score <3.41; moderate, NPI score 3.41-5.4; poor, NPI score >5.4 (19).

Immunohistochemistry

Estrogen receptor, PR, and HER2 immunohistochemical stained slides (immunohistochemical assay for all was from Dako Denmark A/S, Glostrup) of the studied cases were retrieved from Pathology Department archives for reevaluation and confirmation of scoring recorded. Then, cases were broadly classified according to these markers’ scores into 3 groups: luminal group (ER-positive, PR-positive or negative, HER2-positive or negative), HER2-positive group (ER-negative, PR-negative, HER2-positive), and triple-negative group (ER-negative, PR-negative, HER2-negative) according to Prat et al (20).

For CD74 immunohistochemical staining, 3-mm sections from primary tumors were deparaffinized in xylene for 30 minutes and rehydrated with a graded alcohol series. Sections were then subjected to antigen retrieval in 10 mM citrate buffer, pH 6.0 (Lab Vision Catalogue #AP-9003, Fremont, CA, USA) in a pressure cooker for 10 minutes followed by cooling at room temperature for 20 minutes and rinsing with phosphate-buffered saline (PBS) for 1 minute. Endogenous peroxidase was blocked by immersion of the sections in 3% hydrogen peroxide solution for 10 minutes, then washing them in PBS. Immunohistochemical staining was performed using the Lab Vision's UltraVision Detection Kit (TP-015-HD) according to the manufacturer's protocol. Sections were incubated for 10 minutes with Ultra V block to prevent nonspecific background staining. Then, sections were rinsed is PBS followed by an overnight incubation in a humidity chamber with mouse monoclonal antibody against CD74 (Lab Vision Catalogue #MS-131-R7 ready to use), then washing in PBS. Sections were then covered with 4-5 drops of UltraVision biotinylated goat anti-polyvalent secondary antibody, incubated at room temperature for 10 minutes, then washed in PBS, followed by incubation with streptavidin peroxidase solution for 10 minutes at room temperature, then rinsing with PBS. Sections were then covered for 15 minutes by adding 1 drop of 3-30-diamino-benzidine-tetrahydrochloride (DAB) chromogen mixed with 2 mL of DAB substrate. Finally, sections were counterstained with Mayer's hematoxylin, dehydrated in alcohol, and mounted in distyrene, plasticizer, and xylene. Sections of lymph node were used as positive control for CD74. Negative controls had primary antibody replaced by buffer.

Immunohistochemical evaluation

CD74 expression was scored for pattern of distribution: negative, <1% of tumor cells were labeled; focal, 1%-25% of tumor cells were labeled; diffuse, >25% of tumor cells were labeled and intensity (scale of +1 to +4). Only the carcinoma cells were considered for assessment of CD74 expression (4). Conventional H scoring was obtained by multiplying percentage of positive cells by the factor representing the intensity of staining (21).

Statistical analysis

The difference in studied marker expression among different studied tumor groups was statistically analyzed using SPSS (Chicago, IL) software statistical computer package version 15. Scores are expressed as mean ± SD. The difference in frequency was statistically analyzed using chi-square testing; comparing means of CD74 H-score and NPI among more than 2 different groups was carried out using analysis of variance (ANOVA); post hoc Tukey test was used for comparing 2 groups; and Spearman rank correlation coefficient was used to assess if there was a correlation between CD74 expression scores and NPI score in studied cases. A probability value of less than 0.05 was considered statistically significant.

Results

The present study included 160 specimens of invasive breast carcinoma. Forty cases (25%) were classified as luminal group (Fig. 1), 72 cases (45%) were classified as HER2-positive group (Fig. 2), and the remaining 48 cases (30%) were classified as triple-negative group (Fig. 3).

Fig. 1

A case from the luminal group shows positivity to estrogen receptor (ER) and progesterone receptor (PR) receptors and negativity to human epidermal growth factor receptor 2 (HER2) (immunoperoxidase ×400).

Fig. 2

A case from the human epidermal growth factor receptor 2 (HER2)-positive group shows negativity to estrogen receptor (ER) and progesterone receptor (PR) receptors and positivity to HER2 (immunoperoxidase (400 [ER, HER2] and 4100 [PR]).

Fig. 3

A case from the triple-negative group shows negativity to estrogen receptor (ER) and progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) (immunoperoxidase ×400).

Table I demonstrates distribution of cases in each studied tumor group as regards the different studied clinicopathologic parameters. Taking into consideration the clinicopathologic data, NPI for each individual case was calculated. Accordingly, studied cases were stratified into 3 prognostic groups: 5 cases (3.1%) had NPI ranging from 2.41-3.41 and these were categorized as good prognostic group, 72 cases (45%) had NPI ranging from 3.41-5.4 and these were categorized as moderate prognostic group, and 83 (51.9%) cases had NPI more than 5.4 and these were categorized as poor prognostic group.

Table I

Clinicopathologic variables in different studied groups

Clinicopathologic variant	Groups						Chi-square	p Value
	Luminal		HER2-positive		Triple-negative
	No.	%	No.	%	No.	%
Grade							26.154	<0.001^a
2	24	60	44	61.1	8	16.7
3	16	40	28	38.9	40	83.3
Tumor size, mm							9.586	0.008^a
2-5	32	80	68	94.4	36	75
>5	8	20	4	5.6	12	25
Lymph node metastasis							29.084	<0.001^a
0	8	5	12	7.5	0	0
1-3	24	15	52	32.5	24	15
>3	8	5	8	5	24	15
TNM stage							38.003	<0.001^a
2A	8	20	16	22.2	0	0
2B	24	60	48	66.7	20	41.7
3A	8	20	8	11.1	28	58.3
NPI prognostic group							50.098	<0.001^a
Good	3	7.5	2	2.8	0	0
Moderate	24	60	45	62.5	3	6.25
Poor	13	32.5	25	34.7	45	93.75

NPI = Nottingham Prognostic Index.

Significant.

CD74 immunohistochemical expression

CD74 was expressed mostly as brown granular cytoplasmic staining that was diffuse and/or perinuclear in tumor epithelial cells. Cell surface expression was observed in some cells as well (Fig. 4).

Fig. 4

A case of invasive breast carcinoma from the triple-negative group shows moderate membranous (black arrows), diffuse cytoplasmic (arrowhead), and perinuclear (white arrows) CD74 staining in tumor cells (immunoperoxidase g1,000).

As regards CD74 pattern of staining, 8 cases (5%) were negative, 56 cases (35%) showed focal expression, and the remaining 96 (60%) cases showed diffuse expression (Fig. 5). CD74 pattern frequencies varied significantly among different studied groups (p<0.001). Of the 152 cases that expressed CD74, 28 (17.5%) showed mild (+1) intensity, 64 (40%) showed moderate (+2) intensity, and 60 (37.5%) cases showed intense (+3) staining (Fig. 6). The frequency of CD74 different intensities also varied significantly among different studied groups (p<0.001). On combining both intensity and percentage of stained cells into H-score, 70 cases (43.75%) had H-score less than 75 and considered as negative and 80 cases (56.25%) had H-scores equal to or more than 75 and were considered as positive. The frequency of CD74 H-score negativity and positivity was significantly different among different studied groups (p<0.001). Table II demonstrates the distribution of cases in different studied groups as regards CD74 pattern, intensity, and H-score.

Table II

CD74 staining pattern, intensity, and H-score among different studied groups

CD74 expression	Groups						Chi-square	p value
	Luminal		HER2-positive		Triple-negative
	No.	%	No.	%	No.	%
CD74%							50.222	<0.001^a
Negative	4	10	4	5.6	0	0
1%-25% (focal staining)	24	60	32	44.4	0	0
>25% (diffuse staining)	12	30	36	50	48	100
CD74 intensity score							47.408	<0.001^a
Negative	4	10	4	5.6	0	0
+1	0	0	24	33.3	4	8.3
+2	20	50	32	44.4	12	25
+3	16	40	12	16.7	32	66.7
CD74 H-score							37.185	<0.001^a
Negative (<75)	27	67.5	38	52.8	5	10.4
Positive (≥75)	13	32.5	34	47.2	43	89.6

Significant.

Fig. 5

Different cases of invasive breast carcinoma show negative (A), focal (B), and diffuse (C) expression of CD74 (immunoperoxidase ×400 [a, b], ×4200 [c]).

Fig. 6

Different cases of invasive breast carcinoma show different intensities of CD74 expression: mild (A), moderate (B), and strong (C) (immunoperoxidase ×200 [a], ×2400 [b, c]).

On performing ANOVA (Tab. III) to compare between means of CD74 H-scores in different studied groups, the difference was of high statistical significance (p<0.001) with significantly higher scores in the triple-negative group (201.98 ± 71.83) when compared to luminal (77.08 ± 71.39) and HER2-positive (85.06 ± 84.89) groups (p<0.001 for both) and the latter 2 groups did not differ significantly in their H-scores (p = 0.862) as evidenced by the post hoc test.

Table III

Comparison between means of CD74 staining H-scores in different studied groups

	No.	Mean	SD	Minimum	Maximum	Analysis of variance
	No.	Mean	SD	Minimum	Maximum	F	p value
Luminal	40	77.0750	71.39826	0	270	39.838	<0.001^a
HER2-positive	72	85.0556	84.89710	0	270
Triple-negative	48	201.9792	71.82825	20	270
Total	160	118.1375	95.05665	0	270
Luminal vs HER2-positive, Tukey test			Luminal vs triple-negative, Tukey test			HER2-positive vs triple-negative, Tukey test
0.862			<0.001^a			<0.001^a

Significant.

On studying frequency of CD74 expression patterns in different clinicopathologic parameters, a significant difference was present with tumor grade, lymph node metastasis status, TNM stage, and NPI grouping, but not with tumor size (Tab. IV). On the other hand, frequency of CD74 different intensities varied significantly with tumor grade and NPI grouping only but not with other clinicopathologic parameters, as demonstrated in Table V. CD74 H-score categories showed significantly different frequencies with all studied clinicopathologic parameters except for tumor size (Tab. VI).

Table IV

Frequency of CD74 staining patterns within studied clinicopathologic variables

Clinicopathologic variable	CD74 pattern						Chi-square	p value
	Negative		Focal		Diffuse
	No.	%	No.	%	No.	%
Grade							24.142	<0.001^a
2	8	10.5	36	47.4	32	42.1
3	0	0	19	22.6	65	77.4
Tumor size, mm							0.432	0.806
2-5	7	5.1	48	35.3	81	59.6
>5	1	4.2	7	29.2	16	66.7
Lymph node metastasis							35.390	<0.001^a
0	2	10	14	70	4	20
1-3	5	5	40	40	55	55
>3	1	2.5	1	2.5	38	95
TNM stage							0.379	<0.001^a
2A	2	8.3	15	62.5	7	29.2
2B	5	5.4	37	40.2	50	54.3
3A	1	2.3	3	6.8	40	90.9
NPI prognostic group							51.382	<0.001^a
Good	0	0	5	100	0	0
Moderate	8	11.1	38	52.8	26	36.1
Poor	0	0	12	14.5	71	85.5

Significant.

Table V

Frequency of CD74 staining intensities within studied clinicopathologic variables

Clinicopathologic variable	CD74 intensity								Chi-square	p value
	Negative		+1		+2		+3
	No.	%	No.	%	No.	%	No.	%
Grade									19.191	<0.001^a
2	8	10.5	20	26.3	24	31.6	24	31.6
3	0	0	8	9.5	40	47.6	36	42.9
Tumor size, mm									3.800	0.284
2-5	7	5.1	26	19.1	56	41.2	47	34.6
>5	1	4.2	2	8.3	8	33.3	13	54.2
Lymph node metastasis									8.047	0.235
0	2	10	4	20	8	40	6	30
1-3	5	5	22	22	37	37	36	36
>3	1	2.5	2	5	19	47.5	18	45
TNM stage									5.092	0.532
2A	2	8.3	4	16.7	11	45.8	7	29.2
2B	5	5.4	18	19.6	38	41.3	31	33.7
3A	1	2.3	6	13.6	15	34.1	22	50
NPI prognostic group									20.256	0.002^a
Good	0	0	1	20	2	40	2	40
Moderate	8	11.1	19	26.4	25	34.7	20	27.8
Poor	0	0	8	9.6	37	44.6	38	45.8

Significant.

Table VI

Correlation of CD74 H-score to studied clinicopathologic variables

Clinicopathologic variable	CD74 H-score				Chi-square	p value
	Negative (<75)		Positive (≥75)
	No.	%	No.	%
Grade					19.628	<0.001^a
2	47	61.8	29	38.2
3	23	27.4	61	72.6
Tumor size, mm					1.272	0.187
2-5	62	45.6	74	54.4
>5	8	33.3	16	66.7
Lymph node metastasis					32.211	<0.001^a
0	15	75	5	25
1-3	51	51	49	49
>3	4	10	36	90
TNM stage					22.694	<0.001^a
2A	16	66.7	8	33.3
2B	47	51.1	45	48.9
3A	7	15.9	37	84.1
NPI prognostic group					44.289	<0.001^a
Good	4	80	1	20
Moderate	50	69.4	22	30.6
Poor	16	19.3	67	80.7

Significant.

On performing Spearman rank correlation coefficient to compare between CD74 expression and NPI scores, it was found that there is a significant positive correlation between CD74 intensity of staining, percentage of stained cells, H-scores, and NPI score (r = 0.253, 0.547, 0.501 and p = 0.01, <0.001, <0.001, respectively); thus the higher CD74 expression the higher the NPI score and consequently the worse the expected prognosis. The correlation between CD74 expression and NPI score is demonstrated in the graphs of Figure 7.

Fig. 7

Scattergraph demonstrates the positive correlation among CD74 expression intensity, percentage of stained cells, H-scores, and Nottingham Prognostic Index (NPI) scores in studied cases.

Discussion

Prognostic factors can be used to distinguish patients likely to have recurrences after treatment of their primary tumor from those with low risk of recurrence, and those likely to benefit from adjuvant therapy from those whose disease is likely to be resistant to treatment. So, these factors influence the layout, direction, and analysis of clinical trials of breast cancer (22). The statuses of ER, PR, and HER2 are critical in the management of patients with invasive breast carcinoma (23). In patients whose tumors show no expression of ER and PR, any form of endocrine therapy is considered ineffective, and trastuzumab is not considered in those whose tumors exhibit less than 2+ staining of HER2 (24).

This study included 160 cases of invasive breast carcinoma that were classified according to their ER, PR, and HER2 immunohistochemical expression. Twenty-five percent of the studied cases were designated as luminal group, 45% designated as HER2-positive group, and the remaining 30% of cases designated as triple-negative group.

Tumor grade and size, which have a significant prognostic value in the luminal/ER-positive classes, show a limited prognostic power in HER2-positive and basal-like tumors. Thus, clinicopathologic prognostic factors behave differently in the different molecular subclasses (25, 26).

Accordingly, Rakha et al (27) reported that enhanced tailoring of treatment for breast carcinoma needs integration of clinical, pathologic, and cancer biological information to make sure that all known variables that could potentially control patient outcome and response to therapeutic treatments are considered. Subsequently, there has been increasing interest in the clinical utility of NPI, which is based on a combination of histopathologic examination of tumor size, lymph node stage, and tumor grading assembled in a prognostic index formula.

In this study, data about the real outcome of patients included in this study were missing as the blocks were from archives, in which the cases did not receive neoadjuvant chemotherapy before surgery. Thus we depended on the expected outcome of cases using NPI that classified cases into different prognostic groups. Most triple-negative group cases (93.75%) had high NPI and were considered of poor prognosis and the remaining cases of this group (6.25%) were of moderate prognosis. In the HER2-positive group, 34.7% of cases had high NPI and were considered of poor prognosis, 62.5% of cases had lower NPI and were considered of moderate prognosis, and the remaining 2.8% only had the lowest NPI and were considered of good prognosis. As regards the luminal group, 32.5% of cases had high NPI and considered of poor prognosis, 60% of cases had lower NPI and were of moderate prognosis, and 7.5% of cases had much lower NPI and were considered of good prognosis.

Dunkler et al (28) reported that the use of a formula like NPI not only overcomes problems associated with the variable prognostic power of each individual clinicopathologic factor in the different molecular classes but also provides a way of incorporating biological and clinical variables in a scientifically and clinically relevant way. Soria et al (29) added that the biological characteristics of breast carcinoma and incorporation into the NPI could significantly improve the delivery of personalized medicine in breast carcinoma patients. Baker et al (30) found a significant correlation between the expected outcome as determined by NPI and the real outcome. Meteoglu et al (31) also reported that high PR positivity had a meaningful correlation with low NPI values. These findings explain why we stratified cases of this study according to their NPI.

The tendency of having poor prognosis in this study was significantly more frequent in triple-negative (93.75% of cases) than HER2-positive (34.7% of cases) and luminal groups (32.5% of cases). This finding signifies the urgent requirement to search for and identify novel prognostic biomarkers for such group and this is consistent with what was reported by others (32).

CD74, the γ subunit of MHC class II, is frequently overexpressed in malignant tumors of epithelial and mesenchymal origin (33). The CD74 expression in this study was detected mostly as granular cytoplasmic staining that was diffuse and/or perinuclear in tumor epithelial cells, with cell-surface expression observed in some cases as well. Similar staining pattern of expression was reported in gastrointestinal cancers by Gold et al (4). This finding is in contrast to hematologic tumors that show CD74 cell-surface expression as reported by Burton et al (5) and Starlets et al (34).

Gold et al (4) explained the variable expression of CD74 by the fact that their study, like ours, “evaluated a static presentation of antigen rather than a dynamic, time-dependent, profile.” They added that CD74 is possibly present within the membrane of tumors with cytoplasmic expression at densities much less than the detection limits of the technologies employed or it is possible that this large intracellular aggregation of CD74 allows greater chance for maintenance of surface-expressed CD74 receptor at low amount.

In this study, CD74 immunohistochemical expression in tumor epithelial cells was evaluated as regards pattern, intensity of staining, and H-score. For pattern of staining, 34.4% and 60% of cases had focal and diffuse CD74 staining, respectively, while only 5% of cases were negative. All cases in the triple-negative group of this study showed diffuse staining. In the HER2-positive group, 5.6%, 44.4%, and 50% of cases showed negative, focal, and diffuse staining, respectively. The luminal group showed negative, focal, and diffuse staining in 10%, 60%, and 30%, respectively. The pattern of CD74 staining varied significantly among different studied groups with tendency of diffuse expression to be higher in the triple-negative group than the HER2-positive and luminal groups.

These findings are similar to that of Tian et al (1), who also reported that CD74 was located in the cytoplasm and membrane of breast cancer cases of their study. They studied the pattern of expression and found that it related significantly to triple negativity of breast cancer.

As regards CD74 intensity of staining in this study, 17.5% of cases showed mild staining, 40% of cases showed moderate staining, and 37.5% of cases showed intense staining. The triple-negative cases showed intense, moderate, and mild staining in 66.7%, 25%, and 8.3% of cases, respectively. The HER2-positive group cases showed intense, moderate, and mild staining in 16.7%, 44%, and 33.3% of cases, respectively. The luminal group cases showed intense and moderate staining in 40% and 50% of cases, respectively. The frequency of different CD74 expression intensities varied significantly between groups (p<0.001), with highest frequency of intense expression observed in the triple-negative group.

On calculating H-score of CD74 expression in studied cases by combining intensity of staining and percentage of stained cells, cases were finally categorized into negative cases (43.75%) with CD74 H-score less than 75 and into positive cases (56.25%) with CD74 H-score more than or equal to 75. The frequency of cases in these 2 categories varied significantly among different studied groups with highest frequency of positivity observed in the triple-negative group (89.6% of cases).

Greenwood et al (35) reported that CD74 was consistently expressed in the triple-negative subgroup of breast cancer in their study and correlated with clinicopathologic and immunohistochemical characteristics.

In this study, CD74 pattern of expression and H-score correlated significantly with the presence of lymph node metastasis (p<0.001 for both). This finding is similar to others who reported that CD74 overexpression is linked to increased invasion and metastasis of breast tumors, particularly tumors of the triple-negative phenotype (1, 33, 36).

In this study, neither CD74 pattern nor intensity of expression nor H-score showed significant correlation with tumor size (p = 0.806, 0.284, 0.187, respectively), similar to Tian et al (1).

Our study shows that CD74 pattern, intensity of staining, and H-score correlated significantly to NPI score as demonstrated by differences in frequency of expression in different prognostic groups and by Spearmen rank correlation coefficient, where the higher the CD74 expression, the higher the NPI and thus the poorer the expected prognosis. This finding is consistent with that reported by Tian et al (1); survival analysis in their study revealed that patients with CD74 expression experienced worse postoperative survival compared to those without CD74 expression.

Others have also suggested an association between CD74 expression and tumor behavior and added that this may correlate with prognosis (37-38-39). Gold et al (4) reported that the presentation of tumor antigens by MHC II may be impossible by increased CD74 expression with an eventual decrease of tumor immunogenicity. They added that more effective MHC II peptide-binding cleft blocking can be provided by high intracellular concentrations of CD74 that will prevent uptake of endogenous tumor-related peptides and presentation of immunogen to CD4+ T cells. Other studies have also suggested another important role for the CD74 molecule in catalyzing cell survival pathways including the ERK-1/2 MAP kinase signaling cascade and inducing cell proliferation by increasing cyclins and expression of other regulatory factors (40, 41).

In accordance with these findings, this study shows that CD74 expression pattern and H-score varied significantly (p<0.001 for both) among different studied tumor stages where tumors of higher stages show higher frequencies of increased CD47 expression. These findings are in contrast to Tian et al (1), who found that the correlation between CD74 and tumor stage was of no significance.

CD74 expression is also linked with several forms of cancer (15, 42-43-44-45-46-47-48). Zhang et al (42) found that CD74 overexpression in pancreatic ductal adenocarcinoma patients was associated with poor prognosis even if curative resection was done. They concluded that CD74 can be used as a prognostic indicator for resectable pancreatic ductal adenocarcinoma. Moreover, a study carried out by Otterstrom et al (49) revealed that high expression levels of CD74 have positive correlation to poor prognosis in mesothelioma.

Borghese and Clanchy (50) outlined the role of CD74 signaling in carcinogenic processes and its use as a therapeutic target in cancer. The upregulation of CD74 in gastric carcinoma was found to be associated with increasing clinical stage and provided an opportunity as novel gastric cancer chemoprevention and/or treatment strategy (51). Cheng et al (52) reported that CD74 overexpression in thyroid cancer is associated with advanced tumor stage and may serve as a therapeutic target. Some studies have demonstrated that CD74 suppression can transform tumor cells into potent vaccines. The MHC II molecules can bind and present endogenous peptides as immunogenic tumor antigens in the absence of the CD74 chaperone, and this can be used in cancer immunotherapy (53, 54).

In summary, this study shows that CD74 immunohistochemical expression in invasive breast carcinoma was significantly higher in triple-negative tumors, higher tumor grade, presence of lymph node metastasis, higher tumor stage, and higher NPI score. These findings indicate that CD74 might be considered as a useful prognostic indicator predicting poor outcome of patients with invasive breast carcinoma and its expression on malignant cells especially in the triple-negative group suggest CD74 as a potential therapeutic target worthy to be studied further in such cancers. Few studies have worked on evaluation of CD74 expression in breast carcinoma with its different molecular and prognostic subclasses. Consequently, effort should be focused on understanding the clinical significance of this marker aiming at optimal utilization of available therapies and development of novel therapies based on improved cancer models.

Footnotes

Acknowledgment

Authors greatly appreciate the support of Prof. Dr. Hussein Zaki Ghoraba and Dr. Mohamed Elrashidy during delivering this work.

Financial support: No financial support was received for this submission.

Conflict of interest: None of the authors has conflict of interest with this submission.

Meeting presentation: This work has been accepted as poster presentation in the XXXI International Congress of the International Academy of Pathology and the 28^th Congress of the European Society of Pathology that will be held, from 25 September to 29 September 2016, in the KölnMesse in Cologne, Germany.

References

Tian

Zhang

Liu

Dong

CD74: a potential novel target for triple-negative breast cancer. Tumour Biol. 2012;33 (6): 2273–2277.

Perou

Molecular stratification of triple-negative breast cancers. Oncologist. 2011;16 (Suppl 1):61–70.

Zagon

Porterfield

McLaughlin

Opioid growth factor - opioid growth factor receptor axis inhibits proliferation of triple negative breast cancer. Exp Biol Med (Maywood). 2013;238 (6):589–599.

Gold

Stein

Burton

Goldenberg

Enhanced expression of CD74 in gastrointestinal cancers and benign tissues. Int J Clin Exp Pathol. 2010;4 (1):1–12.

Burton

Ely

Reddy

CD74 is expressed by multiple myeloma and is a promising target for therapy. Clin Cancer Res. 2004;10 (19):6606–6611.

Stein

Cardillo

Antiproliferative activity of a humanized anti-CD74 monoclonal antibody, hLL1, on B-cell malignancies. Blood. 2004;104 (12):3705–3711.

Ishigami

Natsugoe

Tokuda

Invariant chain expression in gastric cancer. Cancer Lett. 2001;168 (1):87–91.

Hustinx

Cao

Maitra

Differentially expressed genes in pancreatic ductal adenocarcinomas identified through serial analysis of gene expression. Cancer Biol Ther. 2004;3 (12): 1254–1261.

Cuthbert

Wilson

Scott

Coletta

Hull

Differential CD74 (major histocompatibility complex Class II invariant chain) expression in mouse and human intestinal adenomas. Eur J Cancer. 2009;45 (9):1654–1663.

10.

Young

Amin

Moreno

Expression profiling of renal epithelial neoplasms: a method for tumor classification and discovery of diagnostic molecular markers. Am J Pathol. 2001;158 (5):1639–1651.

11.

McClelland

Zhao

Carskadon

Arenberg

Expression of CD74, the receptor for macrophage migration inhibitory factor, in non-small cell lung cancer. Am J Pathol. 2009;174 (2):638–646.

12.

Kitange

Carlson

Schroeder

Expression of CD74 in high grade gliomas: a potential role in temozolomide resistance. J Neurooncol. 2010;100 (2):177–186.

13.

Chamuleau

Souwer

Van Ham

Class II-associated invariant chain peptide expression on myeloid leukemic blasts predicts poor clinical outcome. Cancer Res. 2004;64 (16):5546–5550.

14.

Koide

Yamada

Shibata

Establishment of perineural invasion models and analysis of gene expression revealed an invariant chain (CD74) as a possible molecule involved in perineural invasion in pancreatic cancer. Clin Cancer Res. 2006; 12 (8):2419–2426.

15.

Stein

Mattes

Cardillo

CD74: a new candidate target for the immunotherapy of B-cell neoplasms. Clin Cancer Res. 2007;13 (18 Pt 2):5556s–5563s.

16.

Verjans

Noetzel

Bektas

Dual role of macrophage migration inhibitory factor (MIF) in human breast cancer. BMC Cancer. 2009;9 (1):230.

17.

Lakhani

Ellis

Schnitt

Tan

van de Vijver

WHO classification of tumours of the breast; International Agency for Research on Cancer; World Health Organization. Lyon

International Agency for Research on Cancer

2012; 1-240.

18.

Elston

Ellis

Pathological prognostic factors in breast cancer. I. The value of histological grade in breast cancer: experience from a large study with long-term follow-up. Histopathology. 1991;19 (5):403–410.

19.

Rakha

El-Sayed

Lee

Prognostic significance of Nottingham histologic grade in invasive breast carcinoma. J Clin Oncol. 2008;26 (19):3153–3158.

20.

Prat

Parker

Karginova

Phenotypic and molecular characterization of the claudin-low intrinsic subtype of breast cancer. Breast Cancer Res. 2010;12 (5):R68.

21.

McCarty

Jr Szabo

Flowers

Use of a monoclonal anti-estrogen receptor antibody in the immunohistochemical evaluation of human tumors. Cancer Res. 1986;46 (8) (Suppl): 4244s–4248s.

22.

Okugawa

Yamamoto

Uemura

Prognostic factors in breast cancer: the value of the Nottingham Prognostic Index for patients treated in a single institution. Surg Today. 2005;35 (11):907–911.

23.

Goldhirsch

Wood

Coates

Gelber

Thürlimann

Senn

Panel members. Strategies for subtypes—dealing with the diversity of breast cancer: highlights of the St. Gallen International Expert Consensus on the Primary Therapy of Early Breast Cancer 2011. Ann Oncol. 2011;22 (8):1736–1747.

24.

Mann

Fahey

Feleppa

Buchanan

Reliance on hormone receptor assays of surgical specimens may compromise outcome in patients with breast cancer. J Clin Oncol. 2005;23 (22):5148–5154.

25.

Foulkes

Smith

Reis-Filho

Triple-negative breast cancer. N Engl J Med. 2010;363 (20):1938–1948.

26.

Rakha

Reis-Filho

Baehner

Breast cancer prognostic classification in the molecular era: the role of histological grade. Breast Cancer Res. 2010;12 (4):207–218.

27.

Rakha

Soria

Green

Nottingham Prognostic Index Plus (NPI+): a modern clinical decision making tool in breast cancer. Br J Cancer. 2014;110 (7):1688–1697.

28.

Dunkler

Michiels

Schemper

Gene expression profiling: does it add predictive accuracy to clinical characteristics in cancer prognosis?

Eur J Cancer. 2007;43 (4):745–751.

29.

Soria

Garibaldi

Ambrogi

A methodology to identify consensus classes from clustering algorithms applied to immunohistochemical data from breast cancer patients. Comput Biol Med. 2010;40 (3):318–330.

30.

Baker

Stephenson

Reed

Brown

Expression of proteinases and inhibitors in human breast cancer progression and survival. Mol Pathol. 2002;55 (5):300–304.

31.

Meteoglu

Dikicioglu

Erkus

Breast carcinogensis Transition between hypersensitivity and invasive lesions. Saudi Med J. 2005;26 (12):1889–1896.

32.

Rakha

El-Sayed

Green

Lee

Robertson

Ellis

Prognostic markers in triple-negative breast cancer. Cancer. 2007;109 (1):25–32.

33.

Sapra

Stein

Pickett

Anti-CD74 antibody-doxorubicin conjugate, IMMU-110, in a human multiple myeloma xenograft and in monkeys. Clin Cancer Res. 2005;11 (14):5257–5264.

34.

Starlets

Gore

Binsky

Cell-surface CD74 initiates a signaling cascade leading to cell proliferation and survival. Blood. 2006;107 (12):4807–4816.

35.

Greenwood

Metodieva

Al-Janabi

Stat1 and CD74 overexpression is co-dependent and linked to increased invasion and lymph node metastasis in triple-negative breast cancer. J Proteomics. 2012;75 (10):3031–3040.

36.

Metodieva

Nogueira-de-Souza

Greenwood

CD74-dependent deregulation of the tumor suppressor scribble in human epithelial and breast cancer cells. Neoplasia. 2013;15 6: 660–668.

37.

Ishigami

Natsugoe

Tokuda

Invariant chain expression in gastric cancer. Cancer Lett. 2001;168 (1):87–91.

38.

Cuthbert

Wilson

Scott

Coletta

Hull

Differential CD74 (major histocompatibility complex Class II invariant chain) expression in mouse and human intestinal adenomas. Eur J Cancer. 2009;45 (9):1654–1663.

39.

Koide

Yamada

Shibata

40.

Leng

Metz

Fang

MIF signal transduction initiated by binding to CD74. J Exp Med. 2003;197 (11):1467–1476.

41.

Shi

Leng

Wang

CD44 is the signaling component of the macrophage migration inhibitory factor-CD74 receptor complex. Immunity. 2006;25 (4):595–606.

42.

Zhang

Hua

Liu

Huo

Sun

Effect of CD74 on the prognosis of patients with resectable pancreatic cancer. Hepatobiliary Pancreat Dis Int. 2014;13 (1):81–86.

43.

Datta

Shahsafaei

Nadler

Freeman

Dorfman

Expression of MHC class II-associated invariant chain (Ii;CD74) in thymic epithelial neoplasms. Appl Immunohistochem Mol Morphol. 2000;8 (3):210–215.

44.

Meyer-Siegler

Iczkowski

Vera

Further evidence for increased macrophage migration inhibitory factor expression in prostate cancer. BMC Cancer. 2005;5 (1):73.

45.

Meyer-Siegler

Iczkowski

Leng

Bucala

Vera

Inhibition of macrophage migration inhibitory factor or its receptor (CD74) attenuates growth and invasion of DU-145 prostate cancer cells. J Immunol. 2006;177 (12):8730–8739.

46.

Binsky

Haran

Starlets

IL-8 secreted in a macrophage migration-inhibitory factor- and CD74-dependent manner regulates B cell chronic lymphocytic leukemia survival. Proc Natl Acad Sci USA. 2007;104 (33):13408–13413.

47.

McClelland

Zhao

Carskadon

Arenberg

Expression of CD74, the receptor for macrophage migration inhibitory factor, in non-small cell lung cancer. Am J Pathol. 2009;174 (2):638–646.

48.

Nagata

Jin

Yoshizato

CD74 is a novel prognostic factor for patients with pancreatic cancer receiving multimodal therapy. Ann Surg Oncol. 2009;16 (9):2531–2538.

49.

Otterstrom

Soltermann

Opitz

CD74: a new prognostic factor for patients with malignant pleural mesothelioma. Br J Cancer. 2014;110 (8):2040–2046.

50.

Borghese

Clanchy

CD74: an emerging opportunity as a therapeutic target in cancer and autoimmune disease. Expert Opin Ther Targets. 2011;15 (3):237–251.

51.

Zheng

Yang

Rong

CD74 and macrophage migration inhibitory factor as therapeutic targets in gastric cancer. World J Gastroenterol. 2012;18 (18):2253–2261.

52.

Cheng

Liu

Chen

CD74 expression and its therapeutic potential in thyroid carcinoma. Endocr Relat Cancer. 2015;22 (2):179–190.

53.

Hillman

Kallinteris

Generating MHC Class II+/Ii- phenotype after adenoviral delivery of both an expressible gene for MHC Class II inducer and an antisense Ii-RNA construct in tumor cells. Gene Ther. 2003;10 (17):1512–1518.

54.

Kallinteris

Tumor immunotherapy by converting tumor cells to MHC class II-positive, Ii protein-negative phenotype. Cancer Immunol Immunother. 2003;52 (10):592–598.