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AMACR Expression is a Potential Diagnostic Marker in Apocrine Lesions of Breast, and is Associated with High Histologic Grade and Lymph Node Metastases in Some Invasive Apocrine Breast Cancers
Address for correspondence: Gabriel Lerner, MD, MS, [email protected], Department of Surgical Pathology, Yale University School of Medicine, New Haven, CT.
Affiliations
Department of Surgical Pathology, Yale University School of Medicine, New Haven, CT
Carcinoma with apocrine differentiation (AC) is a subtype of breast carcinoma with apocrine features in >90% of the tumor. Molecular studies demonstrate AC has high expression of androgen receptor (AR) mRNA. Pure AC lack estrogen receptor (ER), progesterone receptor (PR), and express AR, with variable human epidermal growth factor 2 (HER2) status. Currently, in triple negative AC, no targetable therapies or specific diagnostic markers exist.
Materials and Methods
α-Methylacyl CoA racemase (AMACR) expression was investigated as a marker of apocrine differentiation using a single-plex immunoperoxidase stain, and a novel AMACR/p63 dual stain in a subset of cases, across 1) benign apocrine lesions (apocrine metaplasia, adenosis) 2) apocrine DCIS (ADCIS), 3) AC/ invasive ductal carcinoma (IDC) with apocrine features, 4) non-apocrine triple negative breast cancer (TNBC) and 5) IDC, no special type. A sub-set of cases were evaluated by tissue microarray.
Results
AMACR expression was increased in both AC and ADCIS, with minimal expression in benign breast tissue, TNBC and IDC, NST cases. In invasive cases, those with positive AMACR (>5% positivity) were significantly associated with higher histologic grade (P = .006), initial N stage (chi squared 0.044), and lack of ER or PR expression (both P < .001), with no correlation with overall survival. Analysis of TCGA breast cancer datasets revealed AMACR expression was significantly higher in molecularly defined apocrine carcinomas relative to basal and luminal subtypes. Moreover, high AMACR expression predicted worse relapse-free and distant-metastasis free survival, among both ER-/PR-/Her2- and ER-/PR-/Her2+ breast cancer cohorts (log-rank P = .081 and .00011, respectively).
Conclusion
AMACR represents a promising diagnostic and prognostic marker in apocrine breast lesions. Further study is needed to determine the biologic and clinical significance of this protein in AC.
Apocrine differentiation in breast lesions, defined by cuboidal or columnar cells with low nuclear-cytoplasmic ratio, abundant eosinophilic cytoplasm, prominent apical granules, and round nuclei with pale chromatin and often prominent nucleoli, are commonly encountered in daily breast pathology practice.
The histologic spectrum of these lesions includes apocrine metaplasia, apocrine adenosis, ductal hyperplasia, carcinoma in situ, and invasive carcinoma. Invasive apocrine breast carcinomas (AC) are rare – by some estimates 0.3% to 4% of all invasive ductal carcinomas (IDC), and have been defined as lesions showing >90% apocrine morphology in tumor cells.
Apocrine lesions of the breast: part 2 of a two-part review. Invasive apocrine carcinoma, the molecular apocrine signature and utility of immunohistochemistry in the diagnosis of apocrine lesions of the breast.
Recently, however, the field has now moved to classify these lesions via molecular rather than morphologic criteria, as a subset of the “triple negative” breast cancers lacking appreciable steroid receptor expression (estrogen receptor [ER], progesterone receptor [PR], and human epidermal growth factor 2 [HER2]), with positivity for the androgen receptor (AR).
A recent study of invasive apocrine carcinomas in a large national database showed that while these cancers more likely to present with aggressive clinicopathologic features than non-apocrine carcinomas, breast cancer-specific survival was the same, and among triple-negative apocrine carcinomas, survival was better than non-apocrine triple negative carcinomas.
Therefore, accurate diagnosis of these lesions is of paramount importance for practicing surgical pathologists.
Apocrine morphology may be encountered in tumors from organ systems outside of the breast, including primary cutaneous apocrine (sweat gland) carcinoma of the skin, apocrine salivary duct carcinoma, papillary renal cell carcinoma, urothelial cell carcinoma in situ, and rare reports of apocrine carcinoma arising within ovarian teratoma.
In patients presenting with axillary lymphadenopathy or an axillary mass of unknown primary, without adequate clinical history, differentiating primary cutaneous apocrine carcinoma and metastatic apocrine carcinoma of the breast may be virtually impossible on morphologic or immunohistochemical grounds alone – representing an important challenge for practicing breast surgical pathologists.
We recently encountered such a case at our institution, which stained strongly and diffusely for the traditional apocrine markers AR and gross cystic disease fluid protein 15 (GCDFP15), as well as α-methylacyl CoA racemase (AMACR; Figure 1). Additional immunohistochemical markers supported an invasive breast primary, including diffuse GATA3 and cytokeratin 7 positivity and negativity for cytokeratin 20, TTF-1, PSA, CA IX, mammaglobin, S100, SOX10, and PAX8, and p63, with absence of myoepithelial markers p63, CK5/6 and calponin as well (not shown). Ultimately, primary apocrine breast carcinoma was favored.
Figure 1Metastatic poorly differentiated apocrine carcinoma involving subcutaneous tissue. A. H&E stain shows tumor nodules with solid, papillary and nested growth pattern with large tumor cells with eosinophilic cytoplasm and enlarged nuclei in the axillae, directly under overlying epidermis; B. diffuse granular AMACR expression in the cytoplasm, as well as diffuse positivity for C. GCDFP15 and D. AR (all 40x).
Many prior researchers have sought to identify markers that might aid in routine identification of breast carcinoma (p63, CK5/6, GATA3, mammaglobin), as well as specific identification of primary apocrine breast carcinoma (GCDFP, AR, 15-prostaglandin dehydrogenase [15-PGDH], TRPS1, acyl-CoA synthetase medium-chain family member 1 [ACSFM1]), forkhead box protein A1 (FOXA1), and the steroid hormone receptors ER, PR, and HER2 – however, no single reliable marker with adequate sensitivity and specificity has emerged.
In our study, we found strong, granular cytoplasmic expression of AMACR (P504S) in the majority (58%) of apocrine breast carcinoma cases, a tumor marker previously known for overexpression in prostatic adenocarcinoma.
AMACR is a 382-amino acid protein involved in β-oxidation of branched-chain fatty acids, which was first identified by Xu et al in 2000 using RNA subtraction and DNA microarray techniques.
Jiang and colleagues proposed its use as a diagnostic marker in prostatic adenocarcinoma in 2004, after finding AMACR to display both high specificity and sensitivity for prostatic carcinoma, in both resection and core-needle biopsy specimens, regardless of Gleason grade.
AMACR has now been in clinical use for decades as part of the so-called “PIN4” antibody cocktail (along with the basal cell markers p63 and high-molecular weight keratin).
More recently, Nakamura and colleagues have studied AMACR in AC, finding significant expression in both invasive and in situ apocrine proliferations, and lower expression non-apocrine breast carcinomas.
Our study expands upon these data in a large single-institution retrospective cohort study, using immunohistochemistry for AMACR as well as relevant molecular data available in the Cancer Genome Atlas (https://portal.gdc.cancer.gov). We also developed a novel dual AMACR-p63 immunohistochemical stain, which was well-suited for evaluation of AMACR (pink) and p63 (brown, a myoepithelial marker) protein expression for differentiation of invasive or in situ lesions. Our aim was to assess the potential utility of AMACR as a diagnostic tumor marker in apocrine breast lesions, and secondly to assess whether AMACR expression was associated with clinical outcome.
Materials and Methods
Study Cohort
Institutional Review Board approval for this study was obtained from Yale New Haven Hospital. A review of the pathology archives at our hospital was performed to identify patients with any diagnosis of apocrine lesions of the breast. All samples were obtained from breast core biopsy, lumpectomy or mastectomy specimens from the case files of the Yale Department of Surgical Pathology, with cases signed out between 2001 and 2022.
In this study, AMACR expression was evaluated and compared to that of AR and GCDFP15 across a spectrum of apocrine breast lesions, as defined by WHO Classification of Breast Tumors, fifth edition, 2019. These included 13 cases of apocrine metaplasia, 4 cases of apocrine adenosis (2 atypical), 30 cases of apocrine ductal carcinoma in situ (ADCIS), 70 cases of invasive ductal carcinoma with apocrine features/AC, 73 cases of non-apocrine triple negative breast cancer (TNBC), and 4 cases of pleomorphic apocrine LCIS. Clinical information was incomplete for a small subset of AC cases. Additionally, 79 cases of benign breast tissue, and 199 cases of invasive ductal carcinoma, no special type (IDC, NOS), were evaluated for AMACR expression using a previously constructed tissue micro-array (TMA).
Hematoxylin and eosin-stained slides were re-reviewed for all cases by 2 pathologists (GL and MH) which included AC and ADCIS, to confirm that the cases met the diagnostic criteria for pure apocrine histology in >90% of the carcinoma as defined by WHO morphologic criteria.
Immunohistochemical staining was performed on 5-micron thick 10% buffered formalin-fixed paraffin embedded tissue blocks in the clinical Yale immunohistochemistry laboratory. Standard automated immunohistochemical techniques were performed, which included previously validated antibodies for clinical use, monoclonal antibodies against AR (Dako/Agilent, Santa Clara, CA, 1:150 dilution, clone AR441, ER 2 20′ antigen retrieval, on Leica Bond), GCDFP (Biocare Medical, Pacheco, CA, clone D6, neat dilution, CC1 antigen retrieval on the Ventana Ultra platform), and AMACR (Dako/Agilent, Santa Clara, CA, rabbit monoclonal M3616, dilution 1:200, CC1 high pH antigen retrieval on Ventana Ultra).
The Refine staining detection kit was utilized with the Leica Bond and Ultraview DAB and the Ultraview Ultrafast Red detection kit was used with the Ventana Ultra. Additionally, an AMACR/p63 dual stain was modified from an existing clinically-validated PIN4 protocol excluding the CK903 in the cocktail, as CK903 may be expressed by some breast cancers. There is limited data and no standardized assessment method of AMACR in breast lesions. AMACR expression was quantitatively evaluated in apocrine cells only for all cases, using the H scoring method (as the product of percentage of cell staining positivity and stain intensity category
AMACR staining intensity was graded as weak, moderate or strong. As previously published in AMACR validation studies in prostate, AMACR expression was evaluated semi-quantitatively using a proportion score, with cell positivity at any intensity taken as positive as follows: >75% of evaluated cells positive as strong; 50% to 74% of cells as moderate; 5% to 49% of cells as weak, and negative staining if there were 0% to <5% of cells staining.
Statistical analysis was performed using StataCorp. 2019. Stata Statistical Software: Release 16. College Station, TX: StataCorp LLC. Significance level was set at 0.05.
To assess the degree of association between AMACR expression, GCDFP15 expression, and AR expression, Pearson's correlation coefficient (r) was estimated. A non-paired 2-sample Student's t test assuming equal variance was used to compare AMACR, GCDFP15 and AR expression in cases of AC/IDC with apocrine features, apocrine adenosis (atypical apocrine hyperplasia and apocrine adenosis) and apocrine metaplasia. Chi-squared test and Fisher's exact test were used for analysis of all contingency tables. For tables with small sample sizes (Tables 2 and 3), results of both tests were provided, otherwise the Chi-squared test result was provided. Kaplan Meier survival curve and cox proportional hazards model were used for survival analysis of cases of AC/IDC with apocrine features.
Kaplan-Meier Database Analysis
Kaplan-Meier plotter (KM plotter; http://kmplot.com/analysis/) was used to determine the prognostic value of AMACR in a subset of breast cancers.
KM plotter database contains gene expression data and survival information derived from Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/), European Genome-Phenome Archive (https://ega.crg.eu/) and TCGA containing a total of 4929 patients with breast cancer with survival data. The ideal AMACR probe ID was identified using Jetset and the association of AMACR expression and relapse-free (RFS) as well as distant metastasis-free survival (DMFS) was calculated for ER negative/PR negative breast cancer, further split by HER2 status (ER−, PR− HER2+ vs. ER− PR− HER2−).
The patients were split into AMACR mRNA-high and low expression cohort using an automatically calculated cutoff which was determined as the best performing cutoff value between the lower and upper quartile. The number of cases, hazard ratios (HRs), 95% confidence intervals (CIs) and log rank P-values were obtained from the webpage of the KM plotter.
Breast Cancer Expression Datasets Analysis
Previously published gene expression dataset from apocrine, basal and luminal breast cancer subtype was analyzed for AMACR, AR, GCDFP, and FOXA1 expression and plotted as box plot.
Box plots were created using a webtool BoxPlotR (http://shiny.chemgrid.org/boxplotr/). Co-expression of AMACR, AR, GCDFP, and FOXA1 was analyzed using the TCGA data and visualized using cbioportal webtool (https://www.cbioportal.org/).
Results
Clinical pathologic features and diagnostic history of cases of ADCIS, AC, and TNBC, and treatment information for AC, are summarized in Table 1. Immunohistochemical staining for AMACR, AR, and GCDFP15 expression was evaluated on all cases; 20 cases of ADCIS and AC cases were evaluated using the AMACR-p63 dual stain, to assist in differentiation of in situ and invasive apocrine lesions in cases with nested and solid growth (Figure 2). Assessment of AMACR expression and clinicopathologic features was performed, summarized in Table 2. Information on initial M stage of invasive cases was not collected. In multivariate analysis, there was statistically significant interaction between node stage and histologic grade, after stratification by AMACR positivity (P = .012). The odds of cases with N0 and histologic grade 3 being AMACR positive was 3.7 times higher than the odds of cases with N0 and histologic grades 1 or 2. The odds of cases with N1 or higher and histologic grade 3 being AMACR positive was 22.3 times higher than the odds of cases with N0 and histologic grades 1 or 2. AMACR was not independently significant when compared to other known prognostic variables such as tumor size or stage.
Table 1Summary of Selected Clinical Features for Study Cases, Including Mean Subject Age at Diagnosis, and Mortality and Average Survival
Figure 2AMACR/p63 multiplex staining in apocrine breast lesions. AMACR expression appears pink, and p63 highlights intact myoepithelial cells. This dual stain highlights invasive apocrine foci and allows for differentiation of A. in situ lesions and benign breast tissue from B. invasive apocrine lesions, with absence of p63 expression in the latter (red arrow). Note lack of AMACR expression in background benign breast tissue (black arrow).
Table 2Comparison of AMACR Expression and Clinicopathologic Features in Cases of invasive Apocrine Carcinoma/Invasive Ductal Carcinoma with Apocrine Features (AC; n = 69)
AC, AMACR Negative (<5% Staining; n = 30)
AC, AMACR Positive (>5% Staining; n = 39)
P value
Age at first diagnosis (median, range)
60.5, 31-86
67, 31-98
.26
Laterality
Right
15
21
.75
Left
15
18
Race
White/Caucasian
23
25
.54
Black/African American
5
11
Asian
1
0
Hispanic
1
2
Other
0
1
BMI (median, range)
26.3, 18.7-47.7
27, 17.3-54.8
.88
Tumor size (median, range)
1.05, 0.1-4.5
1.6, 0.25-11.9
.037 (one sided; invasive apocrine carcinoma with larger size)
Histologic grade
1
6
1
.006
2
15
13
3
9
25
Initial T stage
1 or is or 0
21
24
.433
2
6
12
3
0
2
Initial N stage
0
19
16
.044 (chi2)
1
3
13
2+
2
5
Vital status
Alive
23
31
.778
Deceased
7
8
Follow up time (median, range)
Alive
1440, 318-4989, n = 23
1936, 222-6492, n = 31
.29
Deceased
1395, 79-1558, n = 7
1544.5, 167-7730, n = 8
ER
Negative
15
35
<.001
Positive
15
4
PR
Negative
19
37
.001
Positive
11
2
Her2
Negative
23
30
.98
Positive
7
9
AR
Negative
4
9
.305
Positive
26
30
AMACR expression was significantly correlated with tumor histologic grade as assessed by Nottingham System criteria (P = .006) and initial lymph node status (Chi-squared test 0.044).
Bold-face P values were statistically significant.
AMACR expression was seen in the majority of malignant apocrine lesions and was also expressed across a spectrum of benign apocrine lesions (58% of AC, and 48% of ADCIS; Figure 3A, B). There was minimal AMACR positivity in non-apocrine TNBC cases, (∼4% of 73 cases evaluated), as well as cases of ICD, NST (Figure 3A, B). Of 199 ICD, NST cases evaluated for AMACR by TMA, only 10 (∼5%) displayed weak positivity. Of those, 3 showed apocrine morphology. AMACR expression was not observed in benign breast tissue (Figure 3A). The AMACR staining intensity was found to be strong (>75% cells with positivity granular cytoplasmic staining) in ∼30% of cases of AC or IDC with apocrine features, ∼17% of apocrine DCIS cases, and ∼8% of cases of apocrine metaplasia (Figure 3B). The sensitivity and specificity of AMACR positivity (>5% staining) for apocrine IDC cases, was 0.58 and 0.82, respectively.
Figure 3A. AMACR expression by tissue type in apocrine and non-apocrine breast lesions. B. Percentage AMACR staining intensity (none, <5%; weak, 5%-49%; moderate, 50%-74%, strong >75%) in apocrine and non-apocrine breast lesions.
AMACR expression was also compared with staining with other known markers of apocrine differentiation, including AR and GCDFP15, on whole slides of study cases (Table 3). AR and GCDFP15 were highly expressed across all apocrine lesions, including both benign and malignant lesions. Among cases of ADCIS, AMACR and AR, and AMACR and GCDFP15 were not statistically differentially expressed (Table 3). For invasive apocrine carcinomas, AMACR expression was not statistically correlated with AR or GCDFP15 expression (chi squared 0.305 and 0.401, Table 3). Expression of AMACR did not differ between cases of ADCIS and AC (Pearson correlation >0.1). A subset of invasive apocrine cases with available lymph node metastasis tissue were stained for AMACR (n = 14; Figure 4A, B). AMACR expression was statistically decreased in metastatic tissue deposits, when compared with paired primary tumor samples (paired t test = 0.047).
Table 3Expression of AMACR, AR, and GCDFP-15 in Apocrine Breast Lesions
Category (Total Cases)
Cases
AR Positive
AR Negative
P value
GCDFP Positive
GCDFP Negative
P value
Apo metaplasia and adenosis (n = 17)
AMACR-
13
13
0
NA
13
0
NA
AMACR+
4
4
0
4
0
Apocrine DCIS (n = 30)
AMACR-
17
17
0
NA
16
1
1
AMACR+
13
13
0
13
0
AC or IDC with apocrine features (n = 70)
AMACR-
30
26
4
.305
37
10
.401
AMACR+
39
30
9
16
7
AR and GCDFP expression stratified by AMACR positivity (as assessed by >5% cell staining), across a spectrum of benign apocrine lesions (apocrine metaplasia and adenosis; n = 17), apocrine DCIS (n = 30), and invasive apocrine lesions (AC/ IDC with apocrine features; n = 70). AR and GCDFP was highly expressed in all apocrine lesions. AMACR expression in invasive apocrine lesions was not correlated with AR or GCDFP expression (chi squared 0.305 and 0.401, respectively).
Figure 4AMACR expression in invasive apocrine carcinoma metastatic to lymph node. A. H&E stain shows tumor nodules with solid and nested growth pattern with large tumor cells with eosinophilic cytoplasm and enlarged nuclei within lymph node tissue; as well as B. diffuse granular cytoplasmic AMACR expression.
There was no statistically significant difference in survival in patients from the institutional cohort with invasive carcinomas stratified by AMACR staining, however, there was a trend of association of AMACR expression and worse outcome (>5% positivity positive cutoff; Figure 5A). To complement the institutional cohort, we analyzed a larger cohort of ER-/PR- breast cancer patients contained in TCGR (n = 677), stratified by AMACR expression. Our analysis revealed clinically as well as statistically significant negative correlation between AMACR expression and RFS (median of 32 months for low-expression cohort vs. 22.5 months for high expression cohort, P-value .0037) as well as DMFS (median of 32.8 months for low-expression cohort vs. 24 months for high expression cohort, P value .013; Figure 5B). Furthermore, we analyzed survival among institutional cohorts of AC patients with HER2 positivity by immunohistochemistry, stratified by AMACR status. We found that the negative association of AMACR expression and survival in the HER2+ cohort approached but did not reach statistical significance (log rank P > .05; n = 16), and was not statistically different among AR positive cases stratified by AMACR status (log rank P ≥ .05; n = 55; Supplementary Figure 1A and B). Among the ER negative invasive apocrine carcinoma cases (n = 50), those with HER2 amplification by FISH testing were more likely to be AMACR positive (8 of 13 positive for AMACR; 1-sided Fisher's exact test 0.092).
Figure 5Kaplan Meier survival curves for patients with AC, stratified by AMACR expression. A. Overall survival (log rank P = .2907; n = 69), and for B. relapse free survival and distant metastasis free survival of molecular subsets of breast cancer (TCGA data).
To further analyze the association of AMACR expression with patient survival, we analyzed the TCGA cohort of ER-/PR- patients split into HER2+ and HER2- and stratified by AMACR expression. We found that the negative association of AMACR expression and RFS and DMFS was even more pronounced in HER2-amplified subset of ER/PR negative breast cancer patients, compared to the complete ER-/PR- cohort (RFS: median of 171 months for low expression cohort vs. 42 months for high expression cohort, P-value .00011; Figure 5B).
Finally, we analyzed the association of AMACR expression and the systemic therapy outcome. Given that our institutional cohort had only a limited number of cases that prevented any meaningful statistical analysis, we therefore analyzed the TCGA data. Analysis of the chemotherapy-treated TCGA cohort of breast cancer patients compared to non-treated showed negative association of AMACR expression and therapy outcome (RFS: median of 41.6 months for low expression cohort vs. 28 months for high expression cohort, P-value .045) (Supplementary Figure 2A, left). Interestingly, when comparing hormone therapy-treated patients with non-treated, the relationship between AMACR expression and therapy outcome was inverted (RFS: median of 72.5 months for low expression cohort vs. 107.3 months for high expression cohort, P-value .031; Supplementary Figure 2A, right). However, further analysis of ER-/PR- cohort of breast cancer patients revealed a strong negative association of AMACR expression and therapy outcome (RFS: median of 171.43 months for low expression cohort vs. 58.15 months for high expression cohort, P-value .0067), most pronounced in the HER2+ subset of patients (RFS: median of 171.43 months for low expression cohort vs. 24 months for high expression cohort, P-value .0034; Supplementary Figure 2B).
To associate the expression of AMACR with previously described markers of apocrine breast cancer, we analyzed the mRNA expression data from publicly available datasets, for expression of AMACR, AR, GCDFP15, and FOXA1 (TCGA; Figure 6A).
Expression of AMACR, AR, and GCDFP15 was significantly increased in apocrine carcinomas in comparison to basal and luminal carcinoma subtypes although the expression of FOXA1 was similar increased in apocrine carcinoma and luminal subtype in comparison to basal subtype (Figure 6A). Similarly, the TCGA expression data of ER/PR negative, HER2-amplified breast cancer samples showed highly significant co-expression of AMACR with all 3 markers of apocrine carcinoma, namely AR, GCDFP, and FOXA1 (Figure 6B).
Figure 6Biomarker expression in breast carcinoma. A. Expression of AMACR, androgen receptor (AR), gross cystic disease fluid protein 15 (GCDFP15) and forkhead box protein 1 (FOXA1) mRNA was analyzed using previously published expression data in apocrine breast cancer (Farmer et al, 2005; basal-type breast cancer and luminal-type breast cancer, ***P < .001, **** P < .0001). B. Biomarker co-expression in HER2+ ER/PR- breast carcinoma. Co-expression of AMACR and AR, GCDFP15 and FOXA1 mRNA was analyzed using TCGA expression data (RNA Seq V2 RSEM).
The current study identified positive AMACR protein expression, defined as >5% of cell staining, in ∼58% of AC cases, 48% of ADCIS cases, ∼4% of non-apocrine TNBC cases, 5% of IDC, NST, and no expression in normal breast tissue. In our study, although AMACR expression did not increase in a statistically significant manner across ADCIS and AC cases, it was differentially expressed in malignant apocrine lesions when compared to normal breast tissue, and a large cohort of non-apocrine invasive breast cancers. Similar to normal prostate tissue, in which minimal expression was observed; AMACR expression was not seen in normal breast tissue.
Statistical analysis of invasive cases revealed significant correlation between AMACR expression and certain clinicopathologic features, including AR positivity, lack of ER and PR expression, and higher initial N stage and histologic grade - suggesting at least in this subset of apocrine lesions, an association with increased aggressive behavior exists. Multivariate analysis revealed statistically significant interaction between node stage and histologic grade, after stratification by AMACR positivity, with AMACR positivity significantly more likely in cases with lymph node involvement (N1), as well as histologic grade 3 (P = .012). However, there was no difference in overall survival when stratifying by AMACR expression, nor was there a difference in tumor size, patient BMI, initial T stage, or demographic features.
Nonetheless, AMACR may represent a promising diagnostic immunohistochemical marker for malignant apocrine lesions. Use of a novel AMACR-p63 dual stain in a subset of study cohort cases aided in distinguishing challenging in situ from invasive apocrine lesions, as some apocrine invasive carcinomas had a deceptive nodular and nested growth pattern, mimicking an in situ process. Further systematic blinded study of a larger number of cases is required to assess the impact on diagnostic accuracy in routine surgical pathology practice. These findings substantiate those of previous groups evaluating AMACR expression in apocrine breast lesions. The research efforts of Nakamura and colleagues demonstrated virtually universal AMACR expression in invasive carcinomas with apocrine differentiation, as well as cases of ADCIS (38 of 39 carcinomas, 97.4%; and 27 of 28, 96.4%, respectively), with much lower expression (32 of 145; 22%) in non-apocrine carcinomas.
Additionally, Vranic and Gatalica also found almost universal AMACR expression in invasive apocrine cases, as well expression in non-apocrine breast lesions.
Potential reasons for discrepancy with these prior findings regarding AMACR expression in invasive cases include heterogeneity of AMACR expression across multiple areas of the tumor; only a single slide was examined per case and therefore stronger areas may have been missed. Additionally, carcinomas originally signed out as “invasive ductal carcinoma with apocrine features” included in this study, may have had focal apocrine differentiation but in fact represent molecularly defined non-apocrine invasive ductal carcinoma. It is a well-recognized fact that molecularly defined apocrine carcinoma does not necessarily correlate with morphologically and immunohistochemically defined (ER-/AR+) apocrine carcinomas, with substantial overlap in 70% to 80% of cases. Furthermore, the vast majority of the luminal androgen receptor carcinomas have a triple negative phenotype, while 30% to 60% of morphologically and immunohistochemically defined apocrine carcinomas may overexpress HER2/neu.
We did also note a subset of AC cases in our cohort were ER positive (18 of 69; 26%). However, we retained these cases; their presence is likely due to the retrospective nature of this study. By the strict definition of molecular apocrine carcinoma (ER-, PR-, Her2-, AR+), these cases should be excluded. However, several prior studies, including 2 SEER based studies, have documented ER+ apocrine carcinomas.
Characteristics and prognosis of 17 special histologic subtypes of invasive breast cancers according to World Health Organization classification: comparative analysis to invasive carcinoma of no special type.
Clinicopathological characteristics and prognosis of breast cancer with special histological types: a surveillance, epidemiology, and end results database analysis.
We confirmed apocrine histology on re-review of tumor slides, and also confirmed the majority of these cases expressed the apocrine marker GCDFP, as well as AR (100% of cases positive; Supplementary Table 1).
We note that although there are clear anatomic and functional differences, important similarities exist between cancers of the prostate and breast.
Both organs have exocrine function, with normal physiologic functions largely steroid (estrogen and androgen) dependent – leading to the important therapeutic hormone inhibition strategies currently part of clinical practice today for these cancer types.
Jiang and colleagues studied AMACR expression in multiple organ systems, finding minimal or negative AMACR expression in a limited sampling of both benign and neoplastic breast tissue (0/12 and 9 of 61 cases) – although importantly no specification as to invasive tumor morphology was indicated.
Others have evaluated AMACR expression via tissue microarray, finding increased AMACR expression in colorectal (20 of 24 cases; 83%), prostate (16 of 16 cases; 100%), ovarian, as well as limited expression in breast carcinoma (52 total cases; ∼50% positivity) - although similarly, no histotype-specific information was provided.
Diets high in fat have been shown in both human studies and mouse models, to activate peroxisomal (peroxisome proliferator-activated receptor-mediated) pathways, affect intracellular oxidant balance, and potentially mediate early uncontrolled cell proliferation in these cancers.
Fatty acid metabolism has been implicated in prior studies of breast carcinoma; AMACR's known role in β-oxidation of branched-chain fatty acids therefore makes it an attractive potential diagnostic biomarker and potential therapeutic target in this cancer.
The relevance of AMACR over-expression as a tumor marker in in other cancer types has been explored. In prostate cancer, over-expression was shown to be highest in localized disease, with decreased but still elevated expression in metastatic prostate cancer - which was shown to be associated with increased risk of biochemical recurrence and cancer-specific death, although this is not used in routine practice.
Decreased alpha-methylacyl CoA racemase expression in localized prostate cancer is associated with an increased rate of biochemical recurrence and cancer-specific death.
Cancer Epidemiol Biomarkers Prev.2005; 14: 1424-1432
One study showed AMACR expression in 75% (123 of 163) colorectal carcinomas, with lack of staining or low intensity staining correlating with poor tumor differentiation, and LVI, as well as worse 5-year disease-specific survival rates.
In hepatocellular carcinoma, a tissue microarray study of 158 cases found that lower AMACR expression independently also predicted worse survival of these patients, as well as increased tumor capsule involvement and portal vein thrombosis.
AMACR expression has been used as part of a diagnostic panel for ovarian clear cell carcinoma, although its expression has not been equivocally shown to be associated with survival.
Comparative analysis of Napsin A, alpha-methylacyl-coenzyme A racemase (AMACR, P504S), and hepatocyte nuclear factor 1 beta as diagnostic markers of ovarian clear cell carcinoma: an immunohistochemical study of 279 ovarian tumours.
Transcriptomic mRNA-based studies of molecular AC demonstrated that although AR as well as the FOXA1 signatures were seen in 100% of cases studied (n = 58), a compatible IHC signature (ER-, AR+, FOXA1+) was seen in only 57% of cases.
Consistently, our analysis of previously published transcriptomic data (GSE1561) and TCGA showed that AMACR is overexpressed in apocrine breast carcinoma in comparison to basal and luminal subtype, and the expression in AC is correlated with expression of putative AC biomarkers – AR, GCDFP, and FOXA1.
However, the expression of FoxA1 mRNA was increased also in luminal subtype. Moreover, Lehman et al found that while all molecular apocrine tumors in their study (n = 58) displayed FOXA1 mRNA expression, only 58% of them were positive for FOXA1 by immunohistochemical analysis.
These conflicting data warrant further investigation to determine the biologic and clinical significance of FOXA1 in these tumors, as well as the precise relationship, if any, between FOXA1 and AMACR in these tumors. Importantly, there was a negative correlation between AMACR expression and progression free and distant metastasis free survival in ER/PR negative breast carcinoma. The association of AMACR expression with worse clinical prognosis was much stronger in ER/PR negative, HER2 amplified carcinomas, which according to Lehman et al could be classified as molecular apocrine breast carcinoma, indicating diagnostic as well as potential therapeutic value of AMACR.
Furthermore, we analyzed the association of AMACR expression and therapy outcome in our institutional cohort, as well as in the TCGA database. Our institutional cohort contained 7 AC cases which had undergone neoadjuvant therapy; of these, all received platinum-based chemotherapy, and 4 received additional HER2-targeted therapy (trastuzumab and/or pertuzumab; Supplementary Table 2). Of these 7 (diagnosed between 2011 and 2021), only one had a complete pathologic response, and all but one were alive without evidence of disease. AMACR expression in these cases ranged from 0% to 70% of tumor cells demonstrating positive staining. Interestingly, the one case with complete pathologic response, displayed 0% AMACR expression (Supplementary Table 2). The single case identified who died of disease, survived for 19.4 months after diagnosis, and had 15%, weak intensity staining of AMACR in invasive apocrine cells. The pathologic stage was pT3 N2a, a residual cancer burden calculation was not performed on the post-treatment specimen. Because of the low number of cases, the study was under-powered, and we could not reach any meaningful statistically relevant conclusions. However, the analysis of TCGA datasets showed a clear negative association of AMACR expression and therapy outcome. We acknowledge these are preliminary data and further study with larger cohorts need to be performed to determine the effect of AMACR expression on therapy outcome.
Further study is also required to assess the precise characteristics of AMACR positive and AMACR negative AC cases, as well as potential utility of the lack of AMACR expression in apocrine LCIS cases, to distinguish from apocrine DCIS. We acknowledge additional limitations of the study, which, due to the rarity of pure invasive apocrine carcinoma, include a relatively small case volume from a single institution. – This likely led to an underpowered study that may limit detection of significant interactions in multivariate analyses. Future studies are needed using a larger cohort of cases in a multi-institutional setting. Lack of AMACR in benign breast tissue, similar to prostate, colon, gastric, and bladder tissue, is suggestive on an oncogenic role of this molecule in pathogenesis of these cancers. Although AMACR expression was first investigated in a case of cutaneous apocrine carcinoma, we could not compare our AC cohort with these cases due to their rarity. Given recent interest in treatment response in TNBC tumors with low Ki67 index, future analyses of our cohort could include Ki67 investigation.
In summary, we have evaluated the expression of AMACR as a potential diagnostic marker in malignant apocrine lesions and compared them to benign and other breast cancer types. Further collaboration and published literature on AMACR in apocrine breast cancers will be important to assess the utility of AMACR as a diagnostic marker in both routine clinical practice as a stand-alone marker for apocrine lesions or part of a panel, as well as in research settings.
Clinical Practice Points
1)
Carcinomas with apocrine differentiation are a rare special subtype of breast cancer with distinctive morphologic, immunohistochemical and molecular features.
2)
Apocrine carcinomas are ER and PR negative and generally AR positive. While some apocrine carcinomas are triple negative others express HER2/neu making patients eligible for targeted therapies.
3)
Few recent studies have shown that AMACR expression is found in apocrine lesions of the breast. AMACR expression in apocrine breast lesions is a novel marker that is still investigational.
4)
AMACR may play a role in breast cancer progression as AMACR is not expressed in benign breast tissue, similar to prostate cancers.
5)
AMACR could be a promising new diagnostic and prognostic marker in carcinomas with apocrine differentiation.
The authors declare that they have no relevant financial interests to disclose.
Acknowledgments
The authors would like to gratefully acknowledge Lori Charette in Yale Pathology Tissue Services for her immense technical assistance in TMA construction and apocrine case collection, Drs Yuanxin Liang and Uma Krishnamurthy for apocrine case contributions, and Dr Deepika Kumar for review of manuscript figures. MH: Yale Department of Pathology research funds; RJ: National Institute for Cancer Research (reg. No. LX22NPO5102); European Union - Next Generation EU, Programme EXCELES; PRIMUS/22/MED/007 - Charles University.
Apocrine lesions of the breast: part 2 of a two-part review. Invasive apocrine carcinoma, the molecular apocrine signature and utility of immunohistochemistry in the diagnosis of apocrine lesions of the breast.
Characteristics and prognosis of 17 special histologic subtypes of invasive breast cancers according to World Health Organization classification: comparative analysis to invasive carcinoma of no special type.
Clinicopathological characteristics and prognosis of breast cancer with special histological types: a surveillance, epidemiology, and end results database analysis.
Decreased alpha-methylacyl CoA racemase expression in localized prostate cancer is associated with an increased rate of biochemical recurrence and cancer-specific death.
Cancer Epidemiol Biomarkers Prev.2005; 14: 1424-1432
Comparative analysis of Napsin A, alpha-methylacyl-coenzyme A racemase (AMACR, P504S), and hepatocyte nuclear factor 1 beta as diagnostic markers of ovarian clear cell carcinoma: an immunohistochemical study of 279 ovarian tumours.
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