Gene Expression Profiling for the Diagnosis of Male Breast Cancer

doi:10.21203/rs.3.rs-4668835/v1

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Gene Expression Profiling for the Diagnosis of Male Breast Cancer

https://doi.org/10.21203/rs.3.rs-4668835/v1

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Background:

Male breast cancer (MBC) is a rare malignancy, but its global incidence has shown a notable increase in recent decades. Factors such as limited health literacy, insufficient health education, and reluctance to seek medical attention contribute to most MBC patients being diagnosed at an advanced stage. Consequently, there is an urgent need for a highly specific and sensitive diagnostic approach to MBC.

Methods:

This retrospective study enrolled 20 patients with 30 surgical or biopsy MBC specimens from August 2020 to August 2023. The 90-gene expression assay was performed to determine the tissue of origin. Predicted tumor types were then compared to the reference diagnosis for accuracy calculation. The differentially expressed genes were identified between male and female breast cancer.

Result:

The 90-gene expression assay demonstrated an overall accuracy of 96.7% (29/30) when compared with the pathological diagnosis. Subgroup analysis revealed accuracies of 100% (15/15) for primary tumors, 90.9% (10/11) for lymph node metastatic tumors, and 100% (4/4) for distant metastatic tumors. Five genes (RPS4Y1, PI15, AZGP1, PRRX1, and AGR2) were up-regulated, and six (XIST, PIGR, SFRP1, PLA2G2A, S100A2, andCHI3L1) were down-regulated in MBC.

Conclusion:

Our findings highlight the promising performance of the 90-gene expression assay in accurately identifying the tumor origin in MBC. Incorporating this assay into pathological diagnoses has the potential to empower oncologists with precision treatment options, ultimately enhancing the care and outcomes for patients with MBC.

male breast cancer

gene expression profile

tissue of origin

Male breast cancer (MBC) is a rare malignancy, accounting for less than 1% of all breast cancer cases but its global incidence has increased over recent decades^{[1, 2]}. The etiology and pathogenesis of MBC remain elusive. Studies have shown that risk factors for MBC include a family history of breast cancer, older age, carrying a predisposition germline genetic mutation (eg, BRCA 1/2), hormonal imbalance, and exposure to radiation^[3]. Owing to factors such as illiteracy, inadequate health education, and reluctance to report symptoms to the clinical physician, most MBC patients present at an advanced stage^[4]. A study conducted at a single institution in Italy reported distant metastasis in 32% of MBC patients, primarily affecting the lungs and bones^[5]. Consequently, precise diagnosis of MBC is essential for selecting appropriate treatment and improving prognosis.

Male breast tissue is typically less developed than its female counterpart, rendering physical examination and imaging interpretation more challenging. Both mammography and magnetic resonance imaging have limited diagnostic value in MBC^{[6, 7]}. Histopathology is considered the gold standard for diagnosing MBC metastases. Various immunohistochemical markers including androgen receptor (AR), GATA3, mammaglobin (MGB), and gross cystic disease fluid protein-15 (GCDFP15), play a crucial role in diagnosing the majority of metastatic MBC^[1]. However, due to its rarity and lack of awareness among healthcare providers and patients, misdiagnosis or delayed diagnosis of MBC is prevalent^[8]. Thus, identifying the tissue of origin of metastatic MBC, especially in patients without a prior history of breast cancer, is challenging. Furthermore, survivors of MBC face elevated risks of developing second primary cancers^[9]. Accurate diagnosis of second primary cancers or MBC metastasis is crucial for selecting appropriate therapy.

Previously, our research group developed a 90-gene expression assay (Canhelp-Origin Test) to identify 21 common tumor types using real-time PCR methods with total RNA isolated from formalin-fixed, paraffin-embedded (FFPE) tumor tissue^[10]. In a large-scale multicenter study comprising 1417 samples, the 90-gene expression assay demonstrated an accuracy of 94.4% and a specificity exceeding 99%^[11]. A randomized phase III trial demonstrated that site-specific treatment guided by the 90-gene-expression assay resulted in more therapy options, significantly improved progression free survival and favorable overall survival compared with empirical chemotherapy in patients with cancer of unknown primary^[12]. In the present study, we conducted a retrospective study to assess the performance of the 90-gene expression assay and explore its potential diagnostic utility for MBC.

2.1 Patient enrollment and specimen acquisition

The study protocol was approved by the institutional review board of Fudan University Shanghai Cancer Center (FUSCC, Shanghai, China). Surgical or biopsy specimens were enrolled in the present study and associated clinicopathologic data were collect. Biopsy samples were obtained by needle core biopsy (NCB) or fine-needle aspiration (FNA), using either transabdominal ultrasound or computed tomography (CT) guidance. The inclusion criteria for all specimens were the following: (1) FFPE tumor tissues, (2) the primary tumor sites were verified through evaluation of available medical records, pathological, and imaging information. The reference diagnosis of the primary tumor was male breast cancer (3) at least 60% tumor cell content, and (4) less than 40% necrosis based on the Hematoxylin and Eosin (H&E) staining evaluation.

2.2 Clinicopathological characteristics

Patient demographics including age, tumor site, tumor dimensions, lymph node involvement, and surgical approach were meticulously documented from medical records. Tumor staging followed the eighth edition of the American Joint Committee on Cancer (AJCC) staging system^[13]. Diagnoses were confirmed through an independent review of all case slides by two pathologists (QF W and J L). Histological types were characterized according to the World Health Organization (WHO) guidelines^[14], while histological grading for invasive cancers was defined using a modified Bloom and Richardson scoring scheme^[15]. Immunohistochemical analyses were performed on 4 µm sections of FFPE tissues, with a detailed list of all antibodies utilized provided in Table S1.

2.3 Sample preparation and RNA isolation

Five to fifteen 5µm unstained sections were freshly cut for gene expression analysis. The FFPE tissue samples were centralized and the H&E-stained slide of each case had been reviewed for evaluation of the percentage of tumor cells and necrotic areas by two pathologists from FUSCC (QF W and J L). The regions of tumor tissue were marked on the H&E-stained slides and macro-dissected manually for tumor cell enrichment. Total RNA isolation and gene expression profiling were performed at the Canhelp Genomics Reference Laboratory (Hangzhou, China). Total RNA was extracted using the FFPE total RNA Isolation Kit (Canhelp Genomics Co., Ltd., Hangzhou, China) according to the protocols. Briefly, FFPE tissue was deparaffinized, followed by digestion, DNase treatment and total RNA elution. The concentration of total RNA was measured by spectrophotometer at 260 nm absorbance, and the purity was quantified using the A260/A280 ratio. RNA samples with A260/ A280 ratios between 1.7 and 2.1 were enrolled in this study.

2.4 Gene expression profiling analysis

The 90-gene expression assay (Canhelp Genomics Co., Ltd) was carried out as previously described^[10]. In brief, the reverse transcription was performed on isolated total RNA. Next, the RT-PCR reaction was applied with a 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, United States) to perform tumor-specific gene expression profiling. The internal control (IC) gene was used to assess the sample quality, while a weak RT-PCR signal (cycle threshold [Ct] value of the IC, greater than 38) was excluded. Additionally, no template control (NTC) was used to evaluate the potential PCR reaction contamination. The sample was excluded when the Ct of the NTC was less than 38. For each case, the 90-gene classifier analyzed the gene expression pattern of the 90 tumor-specific genes and generated similarity scores for each primary tumor type based on the degree of similarities of the test specimen to the gene expression database. The range of similarity scores was 0 (low similarity) to 100 (high similarity) for each tumor type, and the sum of similarity scores across 21 tumor types was 100. The tumor type with the highest similarity score is considered as the predicted tumor type.

2.5 Statistical analysis

All statistical analysis was performed using the R software (version 3.6.1) and packages from the Bioconductor project (version 3.9). The hierarchical clustering of clinical specimens based on the gene expression pattern was performed using the “pheatmap” package (version 1.0.12). The differentially expressed genes between MBC and FBC were identified with Benjamini-Hochberg (B-H) p-value < 0.01 using the “limma” package (version 3.54.2).

3.1 Patients and samples

Initially, 20 patients archived from August 2020 and August 2023 were enrolled in this study, yielding 30 FFPE specimens, comprising 15 primary breast tumors (PBT), 11 lymph node metastases (LNM), and 4 distant organ metastases (DOM). Table 1 displays the clinicopathological characteristics of 20 patients. The median age at diagnosis was 65.5 years for all patients. Among the PBT cases, 10 occurred in the left breast and 5 in the right. Eight tumors measured less than 2 cm, and seven ranged between 2 and 5 cm. All histological subtypes were identified as invasive ductal carcinoma, with two cases exhibiting invasive ductal carcinoma coexisting with invasive micropapillary carcinoma. Surgical interventions comprised modified radical mastectomy (N = 11), simple mastectomy (N = 3), and simple mastectomy with sentinel lymph node biopsy (N = 1). Histologically, 12 (80%) PBTs were classified as Grade 2, and 3 (20%) were classified as Grade 3. Immunohistochemistry (IHC) results indicated positive staining for ER (18/19, 94.7%), PR (17/20, 85.0%), GATA3 (20/20, 100%), HER2 (3/20, 15.0%), AR (17/19, 89.5%), GCDFP15 (16/18, 88.9%), TRPS1 (13/13, 100%), and MGB (2/3, 66.7%) (Table 2).

Table 1

Patient's general characteristics included in this study
Characteristic	Primary tumor/paired lymph node metastasis	Distant organ metastasis
No. patients	15/11	4
Age (years)	65.5 (27 ~ 86)
Pathological type
IDC	13/10	4
IDC with invasive micropapillary	2/1	0
Tumor site
Left	10/7	/
Right	5/4	/
Tumor size (cm)
≤ 2	8	/
2–5	7	/
Surgical approach
Modified mastectomy	11
Simple mastectomy with SLN	1
Simple mastectomy	3
Histological grade
1	0	/
2	12	/
3	3	/
Metastatic sites
Lung	/	2
Bone	/	1
Thoracic wall	/	1
IDC: Invasive ductal carcinoma; SLN: Sentinel lymph node; /: Censored value.

Table 2

Clinical information and immunohistochemistry staining features of male breast cancer
no.	Type	Age	Size (cm)	pT	pN	M	ER	PR	HER2 (IHC)	HER2 (FISH)	GATA3	AR	GCDFP15	TRPS1	MGB
1/2	PBT/LNM	27	5	2	1a	0	+	+	2 +	-	+	+	+	+	/
3/4	PBT/LNM	67	2.5	2	1a	0	+	+	1 +	/	+	+	+	+	/
5/6	PBT/LNM	68	1	1b	2a	0	+	+	1 +	/	+	+	+	+	/
7/8	PBT/LNM	70	3	2	1a	0	+	+	1+	/	+	-	+	+	/
9/10	PBT/LNM	73	2	1c	1a	0	+	+	1+	-	+	+	+	+	/
11/12	PBT/LNM	44	1.1	1c	1a	0	+	+	1+	/	+	+	+	+	/
13/14	PBT/LNM	63	2.5	2	3a	0	+	+	2+	+	+	+	+	/	/
15/16	PBT/LNM	69	1.8	1c	2a	0	+	+	0	/	+	-	+	/	-
17/18	PBT/LNM	57	3	2	2a	0	-	-	3+	/	+	+	+	/	/
19/20	PBT/LNM	86	2	1c	3a	0	+	+	2+	-	+	+	+	/	/
21	PBT	52	3.2	2	0	0	+	+	1+	/	+	+	-	+	/
22	PBT	60	1.3	1c	0	0	+	+	1+	/	+	+	-	+	/
23	PBT	70	3	2	0	0	+	+	2+	-	+	+	+	+	/
24	PBT	67	2	1c	0	0	+	+	0	/	+	+	+	+	/
25	PBT	67	1.5	1c	0	0	+	+	1+	-	+	+	+	+	/
26	LNM	74	/	/	/	/	+	+	1+	/	+	+	+	+	+
27	DOM	44	/	/	/	/	+	+	0	/	+	/	/	+	/
28	DOM	62	/	/	/	/	+	+	1+	/	+	+	/	/	/
29	DOM	64	/	/	/	/	+	-	1+	/	+	+	+	/	-
30	DOM	53	/	/	/	/	/	-	3+	/	+	+	+	/	+
PBT: Primary tumor; LNM: Lymph node metastasis; DOM: Distant organ metastasis.

3.2 Performance of the 90-gene expression assay in male breast cancer

Total RNA was isolated from FFPE sections of 30 samples. Concentrations ranged from 9.7 to 671.1 ng/µL, with a median concentration of 224.5 ng/µL. The A260/A280 ratio ranged from 1.84 to 1.95. All specimens met all quality control criteria and were successfully analyzed using the 90-gene expression assay.

The results showed an overall concordance rate of 96.7% (29/30) with the reference diagnoses. Specifically, the consistency rates were 100% (15/15) for PBT cases, 90.9% (10/11) for LNM cases, and 100% (4/4) for DOM cases (Table 3). The median similarity scores generated by the 90-gene expression assay were 97.2 (range 63.3 ~ 98.8) for PBT, 92.3 (range 57.5 ~ 98.4) for LNM, and 86.4 (range 79.3 ~ 87.5) for DOM (Fig. 1A). Differences in the similarity scores of paired specimens are shown in Fig. 1B.

Table 3

Performance of 90-gene expression assay in male breast cancer
Tumor type	n	Agreement	Accuracy (%)
Primary male breast cancer	15	15	100
Lymph node metastasis	11	10	90.9
Distant organ metastasis	4	4	100
Total accuracy	30	29	96.7

3.3 Comparison of the gene expression profiling between male and female breast cancer

This study further included 54 cases of female breast cancer (FBC) for comparison of gene expression profiling with MBC. These specimens included 19 cases of metastatic breast cancer from the study by Wang et al^[16] and 35 cases of primary breast cancer from the study by Sun et al^[11]. The distribution of similarity scores for MBC (median 92.7, range 35.5–98.8) and FBC (median 90.9, range 48.4–98.6) is shown in Fig. 1C. Hierarchical clustering based on the 90 genes revealed a distinct separation between 30 cases of MBC and 54 cases of FBC (Fig. 2A).

Furthermore, differentially expressed genes were identified from the 90-gene panel comparing MBC and FBC, with B-H adjusted p-value below 0.01. Among them, five genes (RPS4Y1, PI15, AZGP1, PRRX1, and AGR2) were up-regulated, and six genes (XIST, PIGR, SFRP1, PLA2G2A, S100A2, and CHI3L1) were down-regulated in MBC (Fig. 2B). Further details about these genes are described in Table 4.

Table 4

Desription of 5 up-regulated and 6 down-regulated genes in male breast cancer
Symbol	Description	Cytoband	P-value	adjusted P-value	Regulation (MBC/FBC)
RPS4Y1	Ribosomal Protein S4 Y-Linked 1	Yp11.2	3.09E-49	2.78E-47	Up
PI15	Peptidase Inhibitor 15	8q21.13	1.73E-08	5.18E-07	Up
AZGP1	Alpha-2-glycoprotein 1, zinc-binding	7q22.1	1.96E-07	4.41E-06	Up
PRRX1	Paired Related Homeobox 1	1q24.2	2.73E-04	3.51E-03	Up
AGR2	Anterior Gradient 2	7p21.1	7.44E-04	6.09E-03	Up
XIST	X Inactive Specific Transcript	Xq13.2	1.04E-42	4.66E-41	Down
PIGR	Polymeric immunoglobulin receptor	1q31-q41	4.30E-07	7.73E-06	Down
SFRP1	Secreted Frizzled Related Protein 1	8p11.21	6.64E-07	9.95E-06	Down
PLA2G2A	Phospholipase A2 Group IIA	1p36.13	4.72E-04	4.54E-03	Down
S100A2	S100 Calcium Binding Protein A2	1q21.3	4.88E-04	4.54E-03	Down
CHI3L1	Chitinase 3 Like 1	1q32.1	5.05E-04	4.54E-03	Down

3.4 Specific Case

Among all cases, only one lymph node metastasis specimen (Sample 16) was misclassified by the 90-gene expression assay. The top two results were neuroendocrine tumor with a similarity score of 54.6 and breast cancer with a similarity score of 35.5. The predicted result indicated it was a neuroendocrine tumor, which was inconsistent with the reference diagnosis.

We further reviewed the clinicopathological information and performed additional IHC staining for neuroendocrine markers. This patient is a 69-year-old male who underwent seven months of tamoxifen neoadjuvant therapy followed by modified radical mastectomy. Microscopically, we observed eosinophilic change in tumor cell cytoplasm, tumor stroma fibrosis, and morphological features of locally metastatic lymph nodes showing neuroendocrine differentiation, along with post-treatment response. IHC markers including synaptophysin (Syn), chromogranin A (CgA), and INSM1 were all positive, consistent with the findings of the 90-gene expression assay. Finally, this case is diagnosed as male breast cancer with neuroendocrine differentiation. The histological morphology and IHC results of this case are depicted in Figure S1.

This study evaluated the potential diagnostic utility of the 90-gene expression assay for diagnosing MBC. The overall agreement of 96.7% (29/30) indicates the excellent performance of the 90-gene expression assay in identifying the tumor of origin in primary or metastatic MBC. Subgroup analysis shows consistency rates of 100% for PBT and DOM cases and 90.9% for LNM cases.

In our previous study, we conducted two multicenter validation studies to evaluate the performance of the 90-gene expression assay in FBC. The results demonstrated accuracies of 97.8% (44/45) and 98.4% (121/123), respectively^{[10, 11]}. Additionally, Qifeng et al. also validated the 90-gene expression assay in primary and metastatic triple-negative breast cancer among females, showing an overall accuracy of 97.4% (112/115)^[17]. Therefore, the results of all these studies in females are consistent with the present study’s findings in males.

We further found that 11 out of 90 genes exhibited significantly differential expression between MBC and FBC. Specifically, five genes were up-regulated, and six genes were down-regulated in MBC. The RPS4Y1 and XIST genes are located on sex chromosomes. AZGP1 has been recognized as a crucial promoter in cancer metastasis and lipid metabolism^[18]. Handong et al. observed higher expression of AZGP1 in T cells from MBC than in FBC, suggesting the immunological and metabolic differences between MBC and FBC^[19]. Several studies have noted correlations between PRRX1, AGR2, PIGR, SFRP1, S100A2, and CHI3L1 genes and breast cancer, but have not elucidated their differential expression in MBC and FBC^[20–25]. Due to the rarity of MBC, research on its molecular aspects is currently limited. Our study may offer valuable insights for future investigations.

Additionally, this study also had several limitations. Firstly, cases of MBC with neuroendocrine differentiation might be misclassified as a neuroendocrine tumor raher than breast cancer. Secondly, the comparison of gene expression profiling is limited to only 90 genes. Further transcriptomic sequencing is necessary to comprehensively analyze the difference in gene expression profiling between MBC and FBC.

In conclusion, the 90-gene expression assay demonstrates high accuracy in diagnosing both primary and metastatic MBC, indicating its significant potential as a supplementary diagnostic tool for MBC. Incorporating this assay into pathological diagnoses holds the potential to empower oncologists with precision treatment options, ultimately enhancing the care and outcomes for patients with MBC. Furthermore, several molecular biomarkers may help elucidate the distinct mechanisms underlying MBC and FBC.

DOM: Distant organ metastase

FBC: Female breast cancer

FFPE: Formalin-fixed, paraffin-embedded

FNA: Fine-needle aspiration

FUSCC: Fudan University Shanghai Cancer Center

LNM: Lymph node metastase

MBC: Male breast cancer

NCB: Needle core biopsy

PBT: Primary breast tumor

Ethics approval and consent to participate

Ethics approval and consent to participate in the study were obtained from the Clinical Research Ethics Committee of Fudan University Shanghai Cancer Center (FUSCC, Shanghai, China).

Consent for publication

Not applicable.

Availability of data and materials

Not applicable

Competing interests

Author YF S, YX W and QH X were employed by the company Canhelp Genomics Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Funding

This work was supported by research funding from the National Natural Science Foundation of China grants 81,972,728 (QF W) and 82,272,626 (QF W) and Canhelp Genomics Co., Ltd.

Acknowledgments

Not applicable.

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figures1.tif
Figure S1. The histologic features and immunohistochemical profiles of Sample 16. (A) Neuroendocrine differentiation of cells (200X). The tumor cells were (B) positive for CgA (200X). (C) Diffusely and strongly positive for Syn (200X). (D) Weakly positive for INSM1 (200X).
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Gene Expression Profiling for the Diagnosis of Male Breast Cancer

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Abstract

Figures

1. Background

2. Material and methods

2.1 Patient enrollment and specimen acquisition

2.2 Clinicopathological characteristics

2.3 Sample preparation and RNA isolation

2.4 Gene expression profiling analysis

2.5 Statistical analysis

3. Results

3.1 Patients and samples

3.2 Performance of the 90-gene expression assay in male breast cancer

3.3 Comparison of the gene expression profiling between male and female breast cancer

3.4 Specific Case

4. Discussion

5. Conclusions

Abbreviations

Declarations

References

Supplementary Files

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