NKX3.1 Expression in Male Breast Carcinoma: The Phantom Menace

Abstract

Background. Male breast carcinoma is a rare, hormonally driven neoplasm constituting 0.1% of all malignancies. Molecular studies have identified 1000 genes differentially expressed between male breast carcinoma and female breast cancer with an up-regulation of androgen receptor (AR) related genes and X chromosome gain in male breast cancer. NKX3.1, an androgen-regulated homeobox gene, also has a stem cell signature. In prostate carcinoma, NKX3.1 expression correlates with AR. Although male breast cancer has greater AR expression, the expression profile of NKX3.1, as well as its correlation with AR, remains to be elucidated. Methods. This was a retrospective study conducted in a North Indian tertiary-care oncology institute. A clinicopathological review of registered male breast carcinoma patients from January 2020 to December 2024 was conducted. AR and NKX3.1 immunohistochemistry were attempted. Results. There were 57 patients of male breast cancer constituting 1.2% (57/4600) of all the breast carcinoma patients. The median age of diagnosis was 61 years. AR expression was observed in 83% of tumors (n = 44/53). NKX3.1 expression was observed in 23% of tumors (n = 12/53), which were predominantly grade 3 (n = 10/12, 83%), luminal B tumors. All NKX3.1 positive tumors expressed AR suggesting biological association. AR-expressing tumors had a longer duration of symptoms (P = .044) and had a significant association with the molecular subtype. Conclusion. NKX3.1 expression in male breast cancer is intriguing and needs to be explored in terms of tumor stemness. To the best of our knowledge, this is the first study evaluating AR and NKX3.1 expression in a single cohort of male breast carcinoma.

Keywords

male breast carcinoma NKX3.1 male breast androgen receptor histopathology

Introduction

The adult male breast consists primarily of adipose tissue, with few developed ducts and negligible lobular structures.¹ Occurrence of breast carcinoma is rare in males, accounting for 0.1% of all malignancies.² Male breast carcinoma is a hormonally driven neoplasm with more frequent expression of estrogen receptor (ER) and progesterone receptors (PRs), accompanied by a lower rate of HER-2 positivity.³ The androgen receptor (AR), a steroid nuclear receptor, crucial for sexual differentiation and growth, is expressed in 50% to 80% of malignant breast tissue.⁴ There is an X chromosome gain in male breast carcinoma with consequent AR overexpression.⁵ NKX3.1 is an androgen-regulated gene, acting as a transcription factor in the development of testes and prostate and as a tumor suppressor gene in oncogenesis. In prostate carcinoma, NKX3.1 co-localizes with AR across the cancer genome, and NKX3.1 correlates with AR expression.^6,7 Given its specific association with prostatic tissue, the potential expression of NKX3.1 in male breast carcinoma could pose a phantom menace in diagnosis, particularly in distinguishing male breast carcinoma from metastatic prostatic carcinoma. Notably, while male breast carcinoma has greater AR expression, similar to prostatic carcinoma, the expression profile of NKX3.1, and its correlation with AR, remains to be elucidated. In this retrospective study, we evaluated AR and NKX3.1 expression in male breast carcinoma, aiming to clarify their diagnostic significance and potential implications for patient management. To the best of our knowledge, this is the first study evaluating AR and NKX3.1 expression in a single cohort of male breast carcinoma.

Material and Methods

This was a retrospective study conducted in a North Indian tertiary-care oncology institute and approved by the Institutional Ethics Committee (IEC Project no. 14000084). A clinicopathological review of registered male breast carcinoma patients from January 2020 to December 2024 was conducted. Hematoxylin and eosin-stained slides and immunohistochemistry slides were reviewed. Clinical data included age, site, symptoms, duration, treatment history, smoking history, family history, and follow-up, including recurrence, time to recurrence, distant metastasis, sites of metastasis, and death due to disease. Clinical, treatment, and follow-up details were recorded from the hospital's electronic records (EMRs). Pathological data included tumor size, tumor quadrant, gross findings, surgery type, tumor type, lymphovascular emboli, perineural invasion, lymph node status (total number and metastatic nodes), margin status, and pTNM stage (AJCC/TNM8th Edition). Tumor size was determined based on available imaging data or macroscopic examination findings.

Immunohistochemistry

Estrogen receptor expression was evaluated by using CONFIRM anti-ER rabbit monoclonal antibody, SP1 clone (Ventana Medical Systems, Tucson, AZ). Progesterone receptor expression was evaluated by using a CONFIRM anti-PR rabbit monoclonal antibody 1E2 clone (Ventana Medical Systems, Tucson, AZ). HER2 evaluation was done by using PATHWAY anti-HER-2/neu rabbit monoclonal antibody 4B5 clone (Ventana Medical Systems, Tucson, AZ). For Ki-67 evaluation, Ki-67 8D5 clone mouse monoclonal antibody (Cell Signaling Technology) was used in 1:1600 dilution. All the immunohistochemistry tests were performed on Ventana Benchmark GX automated immunostainer.

Molecular classification of tumors into luminal A, luminal B, HER2 enriched, and triple-negative breast carcinoma was done by using IHC surrogates.⁸ Tumors were classified into luminal A (ER+, PR±, HER2–, and low Ki67), luminal B (ER+, PR±, HER2±, and high Ki67), HER2 enriched (ER–, PR–, and HER2+) and triple-negative breast carcinoma (TNBC, ER–, PR–, and HER2–). IHC evaluation was done in accordance with American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) guidelines.⁹ Ki67 evaluation was done in the hotspots using digitally scanned slides. Tumors with expression of ER, PR with or without HER2 overexpression, and a Ki67 labeling of >20% were classified as luminal B.¹⁰ For AR evaluation, ready-to-use mouse monoclonal antibody clone AR441 (Biocare) was employed. Normal prostate was used as a control on every test slide. ER, PR, and AR expressions were given an Allred score. The Allred score was obtained by adding the proportion score with the intensity score. The proportion score categorized the percentage of positively stained tumor cells as follows: 1 for < 1%, 2 for 1% to 10%, 3 for 11% to 33%, 4 for 34% to 66%, and 5 for > 66%. The intensity score graded the staining intensity as 1 for weak, 2 for moderate, and 3 for strong. The combined Allred scores ranging from 3 to 8 were considered positive.⁹

NKX3.1 immunohistochemical evaluation was done using a ready-to-use rabbit monoclonal antibody, EP356 clone (Cell Marque). Normal prostate was used as a control on every test slide. Since there is no standardized method for assessing NKX3.1 expression in male breast cancer, we constructed our own semiquantitative intensity-reactivity scoring system to evaluate NKX3.1 levels. Tumors were scored according to the intensity and reactivity of NKX3.1 immunostaining. Staining intensity (SI) was classified as negative (= 0), weak (= 1), moderate (= 2), or strong staining (= 3). Percentage tumor reactivity was scored: 1% to 10% positive (= 1), 11% to 30% (= 2), 31% to 60% (= 3), 60% to 100% (= 4). The intensity reactivity score (IRS) was calculated by multiplying the SI score with the percentage tumor reactivity score. IRS categorized NKX3.1 as weak expression (IRS: 1), moderate expression (IRS: 2-6), and strong expression (IRS: 8-12).

Statistical Analysis

Statistical Methods

Data was presented as mean ± (SD), median (range), and frequency (percentage). Continuous variables were analyzed using an independent t-test or Mann-Whitney test as per the distribution. Categorical data were analyzed using the chi-square test or Fisher exact test (for binary data). Survival plots were calculated using Kaplan-Meier and compared between groups using the log-rank test. P-value <.05 was considered statistically significant. IBM SPSS 24.0 (Armonk, NY) was used for analysis.

Results

There were 57 patients with male breast carcinoma, constituting 1.2% (57/4600) of all the breast carcinoma patients registered in our tertiary care oncology center from January 2020 to December 2024. The median age of diagnosis was 63 years (mean: 61 years, range 29-86 years). The average duration of symptoms was 14.1 months, with a median of 6 months. Most of the patients presented at an advanced age, with skin involvement occurring in 35/57 patients (61%) and distant metastasis occurring in 17/57 patients (30%) at the time of presentation. Family history of malignancy was documented in 8/57 patients (14%). One patient had a family history of breast cancer in their father, while another patient had a history of brain tumor in their father. Additionally, 2 patients had a family history of breast cancer in their mother, and one patient had a history of breast cancer in their sibling. Furthermore, one patient had a history of prostate cancer in their father, and another patient had a history of soft tissue tumor in their father.

The tumor predominantly occurred in the retro-areolar region of the left breast. Neoadjuvant chemotherapy was offered to 36/57 (63%) patients with locally advanced breast carcinoma. Surgical intervention was done in 29 patients (51%). The tumor size varied from 1.5 to 9.8 cm (mean: 3.7 cm and median: 3.4 cm). The clinical T stage was determinable in 49 patients, with the distribution as follows: T1c in 1 patient (2%), T2 in 14 patients (29%), T3 in 3 patients (6%), and T4 in 31 patients (63%).

A histological review was conducted on the full sample of 57 patients. However, one patient had a biopsy specimen solely from a metastatic lesion, and was, therefore, excluded from the analysis of tumor grade, DCIS, lymphovascular invasion, perineural invasion, molecular subtype, AR, and NKX3.1. Sufficient histological material for immunohistochemical testing was present in 53 patients of primary breast carcinoma. Consequently, AR and NKX3.1 were evaluated exclusively in these 53 patients. Histopathological examination revealed Invasive breast carcinoma, no special type (NST), grade 3, and luminal B as the predominant tumor type. Lymphovascular invasion was seen in 15/56 (27%), and perineural invasion in 11/56 (20%) specimens. Nodal metastases were documented in 20/29 resections (69%). Surgical margins were negative in all resection specimens. The results have been summarized in Table 1. There was a trend towards younger age in TNBC. Patients with luminal breast carcinoma had a mean age of 62.5 years (median 63 years) whereas the TNBC had a mean age of 49.5 years. This difference was not statistically significant (P = .139).

Table 1.

Clinicopathological Features of Male Breast Carcinoma.

Clinicopathological features of male breast carcinoma
Age	Mean	61 years
	Median	63 years
	Range	29-86 years
Tumor type	No special type (NST)	56 (98%)
	Mucinous carcinoma	1 (2%)
Tumor grade	3	43 (77%)
	2	13 (23%)
	1	0
Ductal carcinoma in situ (DCIS)	Present	9/56 (16%)
Quadrant	Retroareolar	44 (83%)
	Upper outer quadrant	5 (9%)
	Upper inner quadrant	2 (4%)
	Lower inner quadrant	1 (2%)
	Axilla	1 (2%)
Laterality	Left breast	36 (63%)
	Left axilla	1 (2%)
	Right breast	20 (35%)
Tumor size	Mean	3.7 cm
	Median	3.4 cm
	Range	1.5-9.8 cm
Skin involvement	Involved	35/57 (61%)
Nodal metastases		19/29 (66%)
Immunohistochemistry	Estrogen receptor (ER)	55/57 (97%)
	Progesterone receptor (PR)	51/57 (89%)
	HER2 positive (Score 3)	5/57 (9%)
	HER2 equivocal (Score 2)	10/57 (18%)
	HER2 negative (Score 1)	27/57 (47%)
	HER2 negative (Score 0)	15/57 (26%)
	Androgen receptor	45/53 (84%)
	NKX3.1	12/53 (22%)
Molecular subtype	Luminal A	12/56 (21%)
	Luminal B	42/56 (75%)
	Triple-negative breast carcinoma	2/56 (4%)
Metastasis at presentation		17/57 (30%)
NKX3.1 Expression in Male Breast Carcinoma
Grade	3	10 (83%)
	2	2 (17%)
Expression	Weak expression (IRS:1)	4 (33%)
	Moderate expression (IRS 2-6)	7 (58%)
	Strong expression (IRS 8-12)	1 (9%)
Molecular subtype	Luminal B	10 (83%)
	Luminal A	2 (17%)
AR Expression	Positive	12/12 (100%)

Of the 57 breast cancer samples examined, estrogen receptor expression was observed in 55/57 (97%) samples, Progesterone receptor expression was observed in 51/57 samples (89%), 42 samples (73%) were categorized as HER2 negative including 15 samples (26%) classified as HER2 negative (score 0), 27 samples were (47%) HER2 negative (score 1), 10 (18%) were identified as equivocal (score 2), and 5 (9%) were classified as HER2 positive (score 3). Notably, all samples that had been initially reported as equivocal were subsequently determined to be nonamplified upon further FISH analysis.

AR and NKX3.1 Evaluation

AR expression was observed in 84% of specimens (n = 45/53). NKX3.1 expression was observed in 23% of specimens (n = 12/53). The NKX3.1 expression was strong in 1 sample (9%) (Figure 1A and B) weak in 4 samples (33%) (Figure 1C and D), and moderate expression in 7 samples (58%) (Figure 1E to H). Tumors exhibiting NKX3.1 were predominantly grade 3 (n = 10/12, 83%), and luminal B tumors exhibited diffuse AR expression. Patients with AR-positive tumors had a longer median duration of symptoms of 6 months compared to negative tumors with a median duration of symptoms of 3 months. The P value by the Mann-Whitney U-test was .044 suggesting statistical significance (Figure 2A). Though NKX3.1 positive tumors had a trend towards longer duration of symptoms (median duration 12 months) compared to the NKX3.1 negative tumors (median duration 5 months), this difference was not statistically significant (P = .082) (Figure 2B). A statistically significant (0.040) association was found between AR-positive tumors and molecular subtypes (Figure 2C). No statistical significance was observed between other categorical parameters.

Follow-up was assessed in 52 patients in which AR and NKX3.1 testing was done and follow-up details were available. The median follow-up time was 8.4 duration months. At the time of the last follow-up, 24 patients were alive without disease, 27 patients were alive with disease and 1 patient died due to disease progression. Since there was a single death, overall survival could not be calculated meaningfully and event-free survival was estimated. The event-free survival probability at 6 months was 68%, at 12 months was 60.4%, and at 24 months was 54.1% (Figure 2D). There was no statistical difference in the event-free survival probability between AR and NKX3.1 positive and negative groups. The ability to identify meaningful prognostic factors was limited by the sample size, follow-up time, and event distribution. A larger cohort with longer follow-up would be needed to detect potential survival differences between subgroups.

Discussion

Breast carcinoma, a rare neoplasm of elderly men, is genetically distinct from female breast carcinoma. There is not only a higher prevalence of hormone receptor positivity but also X chromosomal gains that lead to AR overexpression.^5,11 Despite having the predominance of luminal A and luminal B neoplasms, male breast carcinoma seldom shows loss of chromosome 16q, which is often observed in the luminal neoplasms of the female breast.¹² Genetic expression profiling studies have also identified 1000 genes differentially expressed between male breast carcinoma and female breast carcinoma with an upregulation of AR-regulated genes.¹¹ In male breast carcinoma, the AR clusters with ER alpha, whereas in female breast cancer, the ER alpha clusters with PR.¹³ Concurrent with existing literature, the present study found a higher frequency of hormone receptor-positive disease accompanied by AR overexpression. AR is being regarded as one of the driver genes in male breast carcinogenesis and is being tested in different clinical trials.^12,14

AR expression in breast carcinomas is associated with improved clinical outcomes regardless of ER status; diminished AR expression is observed in advanced breast carcinoma metastases; and the loss of AR labeling predicts earlier recurrences in triple-negative breast carcinomas.^15–17 The AR-positive tumors in our cohort were predominantly grade 3, luminal B. Compared to the AR-negative tumors, we also found that the AR-positive tumors presented with a longer duration of symptoms, suggesting potentially slower growth or delayed presentation. There was also a statistically significant association between AR expression and molecular subtypes, suggesting a potential biological relationship between AR signaling and male breast cancer molecular classification. There was a predominance of the luminal B molecular subtype.

The central theme of our study was the evaluation of NKX3.1, an androgen-dependent homeobox gene that plays a crucial role in the development of the prostate and testes.¹⁸ NKX3.1 regulates the transcription of AR in prostatic carcinoma and also acts as a tumor suppressor with diminished expression with disease progression.^7,19 NKX3.1 expression has also been found in castration-resistant cells implicating its role in stemness.²⁰ It has also been found to replace OCT 3/4 (POU5F1) in cellular reprogramming, reiterating its role in stemness.²¹ Although NKX3.1 expression is characteristic of prostatic tissue and carcinoma, it has also been recognized in a subset of female ductal carcinoma and lobular carcinoma.²² It was observed in 23% of male breast carcinoma in our study. All NKX3.1 positive tumors expressed AR suggesting biological association. The expression was prevalent in the higher-grade tumors. With AR overexpression, the expression of NKX3.1 is particularly intriguing in male breast carcinoma and requires further exploration in context with tumor stemness.

Our study also establishes that NKX3.1 is not a reliable marker for distinguishing male breast carcinoma from metastatic prostatic carcinoma. Although exceedingly rare, prostatic carcinoma can metastasize to the breast. The expression of NKX3.1 in this context could pose a phantom menace in diagnosis, given the distinct clinical and therapeutic implications of breast and prostate cancer. Metastases from a prostatic primary are histologically indistinguishable from a primary breast carcinoma. Hormonal treatment for prostate cancer results in increased blood supply to the breast resulting in gynecomastia and rarely metastasis.²³ NKX3.1 expression needs to be considered with a grain of salt in such a clinical setting. It needs to be supplemented with other markers to support a diagnosis of metastases over a primary breast lesion.

Male breast carcinoma has been observed to exhibit a more aggressive clinical course compared to its female counterpart. One potential contributing factor may be the relatively small size of the male breast, which could potentially enable earlier and/or easier infiltration of the tumor cells into the chest wall.²⁴ Given the infrequent occurrence of male breast carcinoma, the therapeutic approach is extrapolated from the treatment protocols established for female breast carcinoma.²⁵ Therapeutic modalities depend upon the stage of the disease and include surgery, radiation therapy, and systemic therapy. Patients often present late due to a lack of awareness. In the present study, ∼30% of patients presented with systemic metastases, and 61% of patients presented with skin involvement. Increased awareness and recognition of the condition are necessary to facilitate earlier diagnosis and implementation of appropriate treatment strategies for patients.

Survival analysis was not able to achieve statistical significance in our study due to the small sample size and limited follow-up. The observed trends suggest potential biological differences that warrant further investigation in larger cohorts. The lack of statistical significance should be interpreted in the context of the study's limitation rather than as definitive evidence of no association. Future studies with larger, more balanced groups and longer follow-up periods may be better positioned to detect statistically significant differences in survival outcomes based on AR and NKX3.1 expression status.

Conclusion

The expression of NKX3.1 in male breast carcinoma is intriguing and needs to be explored in terms of tumor stemness. This is a prototype study evaluating AR and NKX3.1 expression in male breast carcinoma that will set the stage for future research to investigate. Further studies are required to understand the clinical and biological significance of these biomarkers in this rare malignancy.

Footnotes

Availability of Data and Material

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

Ethical Approval

Not applicable, because this article does not contain any studies with human or animal subjects.

Informed Consent

Not applicable, because this article does not contain any studies with human or animal subjects.

Consent for Publication

Not applicable, because this article does not contain any clinical trials.

Trial Registration

Not applicable, because this article does not contain any clinical trials.

ORCID iDs

Aishwarya Sharma

Puneet Kaur Somal

Nishtha Ahuja

Ankur Dwivedi

Sankalp Mahendra Sancheti

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