Impact of NOTCH1 expression in primary breast adenoid cystic carcinoma

Aims Adenoid cystic carcinoma (AdCC) originates from salivary-type like glands in the head and neck, lung, and breast. AdCC shows chromosomal translocation, resulting in MYB::NFIB fusion and overexpression of MYB. Recently, NOTCH1 pathway alteration has been recognised in a subset of patients with salivary gland AdCC and has been shown to be associated with poor survival. In this study, we investigated the correlation of NOTCH1 pathway alteration with the clinical outcome of patients with primary breast AdCC by examining NOTCH1 immunoreactivity in attempts to better predict clinical outcomes. Methods We identified 25 cases of breast AdCC, reviewed the clinical outcome and performed immunohistochemical (IHC) staining for NOTCH1 on FFPE sections. Results IHC evaluation of NOTCH1 expression in 25 cases of primary breast AdCCs revealed a positive correlation between NOTCH1 expression and primary tumour size. All cases with NOTCH1 expression were greater than 15 mm in size at presentation but only 50% of NOTCH1 negative tumours were greater than 15 mm. We demonstrated a positive correlation between NOTCH1 positive AdCCs and recurrence/metastases. 63.6% of NOTCH1 positive AdCCs had either metastases or recurrence. On the contrary, only 21.5% of NOTCH1 negative AdCCs had recurrence or metastases. AdCCs with NOTCH1 positivity correlated with inferior relapse free survival (median 33 vs 129 months). Conclusions Our study demonstrates that in patients with breast AdCC, overexpression of NOTCH1 ≥20% is associated with larger tumour size and aggressive clinical outcomes. Importantly, NOTCH1 inhibitors may have potential therapeutic effect in patients with breast AdCC.


INTRODUCTION
Salivary gland-type neoplasms of the breast are uncommon.These tumours are triple negative (<1% nuclear expression of oestrogen receptor/ progesterone receptor and negative for HER2 by immunohistochemical (IHC) or negative for HER2 gene amplification by FISH) and share some overlapping morphological, immunophenotypic and molecular similarities with salivary gland counterparts. 1Adenoid cystic carcinoma (AdCC) is a subtype of salivary gland-type neoplasms of the breast with a better prognosis compared with head and neck origin and is categorised into three subtypes including classic type (tubular and cribriform growth patterns), solid basaloid (SB-AdCC), and AdCC with high-grade transformation (AdCC-HGT). 2 Mixed type AdCC with varying proportions of the aforementioned histological patterns is often encountered in clinical practice.
The classic type of AdCC is characterised by two cell type proliferation of luminal and myoepithelial cells forming tubular and cribriform growth patterns with pseudo lumens filled with basophilic basement membrane material.The tumour cells exhibit low nuclear atypia, mitotic activity and lack necrosis. 3The luminal cells are immunoreactive for low molecular weight cytokeratin (CK7, CK8/18) and c-KIT (CD117) and myoepithelial cells are immunoreactive for high molecular weight cytokeratin (CK5/6) and p63.AdCC shows characteristic chromosomal translocation resulting in MYB::NFIB fusion gene and overexpression of MYB. 4 The solid type of AdCC is associated with an aggressive clinical course including perineural invasion and axillary node metastases and is characterised by solid growth pattern with a basaloid appearance and infiltrating borders.Basaloid neoplastic cells are medium to large and round to oval with scant cytoplasm, hyperchromatic nuclei and moderately increased mitotic activity. 5AdCC mixed-type shows both cribriform and solid histological features.Recently, AdCC-HGT was described which is characterised by sheet-like growth pattern with micropapillary, squamous or small cell differentiation, with loss of biphasic morphology and absence of myoepithelial cell layer.The neoplastic cells have enlarged nuclei with irregular contours, scant cytoplasm, increased mitotic activity and prominent necrosis.AdCC-HGT shows a high Ki-67 proliferative index and strong p53 expression. 6Only rare

Original research
cases with high-grade transformation have been reported in the breast, especially cases with multiple differentiation.Noske et al described the first report on such an unusual case of breast carcinoma, presenting as an admixture of an AdCC and malignant melanoma in a 51-year-old female patient.The resection specimen revealed a multi-directional tumour differentiation consisting predominantly a poorly differentiated basaloid epithelial cell type, consistent with an AdCC and areas of a spindle cell carcinoma with a melanocytic differentiation. 7OTCH1 mutations are detected in a subset of patients with salivary gland AdCC and are associated with shorter relapse free and overall survival (OS). 8 9Previous studies have shown that NOTCH1 signalling has both tumour suppression and oncogenic effects in cell proliferation and angiogenesis. 10here is a need to develop markers to help in the stratification of patient outcomes with mammary AdCC.The purpose of this study was to explore the effects of the NOTCH1 pathway on the clinical behaviour of patients with primary breast AdCC by examining NOTCH1 immunoreactivity and comparing it with histology and patient outcomes.

Human tumour samples
We reviewed 25 cases of breast AdCC in patients diagnosed at a single institution from 2015 to 2023.Clinical outcomes were reviewed from electronic medical records under an institutional review board approved protocol.Follow-up data was available in all 25 patients.

IHC analysis
IHC staining for NOTCH1 was performed on FFPE sections from 25 mammary AdCCs.Whole tissue sections of the primary site tumour were used to analyse the expression of NOTCH1.IHC staining was performed in a Leica Bond-Max autostainer system following standard automated protocols, with a NOTCH1 rabbit monoclonal antibody (clone Val1744 (D3B8), Cell Signaling Technology, Danvers, MA).
IHC staining for anti-c-Myb was performed on FFPE sections from 12 mammary AdCCs.Whole tissue sections of the primary site tumour were used to analyse the expression of MYB IHC staining was performed in a Leica Bond-Max autostainer system following standard automated protocols, with a MYB rabbit monoclonal antibody (EP769Y) (ab45150) to c-Myb (phospho S11), ABCAM Co. 152 Grove Street, Waltham, MA.Only the nuclear expression of MYB was considered as positive.MYB positivity ranged from 20% to 100%, with the majority of cases examined showing strong and diffuse positivity for MYB.

Statistics
Patients follow-up data was determined from the electronic medical record.Kaplan-Meier curves were generated with GraphPad Prism 9 software.The significance of differences in survival between groups was assessed using the log-rank (Mantel-Cox) test.Recurrence free survival (RFS) was calculated from the date of primary diagnosis to the date of first occurrence or distant metastasis.The associations between NOTCH1 expression and clinical outcomes were analysed by Fisher's exact test.The level of significance was set at 0.05.
On average, patients with NOTCH1 positive AdCCs were younger than patients with negative NOTCH1 expression (61.7 vs 70).Average tumour size in NOTCH1 positive AdCCs was 3.19 cm and average tumour size in NOTCH1 negative AdCCs was 2.4 cm (table 1).
All cases with positive NOTCH1 expression measured greater than 15 mm in size to include seven cases with recurrence/ metastases.In contrast, only 50% (7 of 14) cases with negative NOTCH1 expression were greater than 15 mm to include two cases with metastases.The remaining 50% were 15 mm or smaller (Fisher's exact test 0.0078, p<0.05) (table 1).
Follow-up data showed that in 63.6% (7 of 11) of NOTCH1 positive AdCCs either distant metastases or recurrences were identified.On the contrary, only 21.4% (3 of 14) of NOTCH1 negative AdCCs had either recurrences or distant metastases (Fisher's exact test 0.0486, p<0.05) (table 1 and figure 1).We also evaluated the correlation between NOTCH1 expression and outcome by calculating relapse free survival rate (RFS).Kaplan-Meier analysis revealed that AdCCs with NOTCH1 positivity were associated with significantly worse outcomes than NOTCH1 negative (median relapses free survival of 33 months

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in NOTCH1 positive AdCCs vs 129 months in NOTCH1 negative AdCCs, p=0.0442) (figure 2).Two of total 25 patients with AdCC died of their disease.NOTCH1 was positive in one case (disease free survival rate of 57 months) and NOTCH1 was negative in the other (disease free survival rate of 23 months) (tables 2 and 3; online supplemental tables 1 and 2).The NOTCH1 negative deceased patient was stage 2, with a 3 cm in greatest dimension tumour which showed solid-basaloid histological features, and she developed metastasis to the liver.Conversely, the NOTCH1 positive deceased patient had stage 1 tumour, with a tumour size of 1.9 cm which showed mixed cribriform and solid histological features, with subsequent development of lung and bone metastases.
We also investigated the correlation between MYB and NOTCH1 expression.MYB IHC was performed in 12 cases and showed positive expression in all cases.In 50% (6 of 12 cases) of which, NOTCH1 was positive and in the remaining 50% (6 of 12 cases) NOTCH1 was negative (figure 3).

DISCUSSION
AdCC originates from salivary-type glands in head and neck, lung, and breast.Even though most often the tumours are indolent, patients with mammary AdCC can have an aggressive clinical course including recurrence and distant metastases. 11The treatment plan for patients with mammary AdCC is controversial.In most cases AdCC is treated with local excision and postoperative radiation may be recommended.These tumours most often show resistance to systemic chemotherapies. 11In patients with breast AdCC, overexpression of NOTCH1 is associated with larger tumour size and aggressive clinical outcomes and is a potential biomarker for this disease and an adjunct to histology and tumour size.
AdCC shows chromosomal translocation, t(6;9)(q22-23;p23-24) resulting in MYB::NFIB fusion gene and overexpression of MYB. 12 Recently, NOTCH1 pathway alteration was recognised in up to 20% of patients with AdCC of salivary gland origin. 11 13revious studies have shown an association between the NOTCH1 activation mutations and poor OS, disease recurrence, or metastasis in patients with head and neck AdCC. 11 14OTCH1 activation pathway is characterised by increased NOTCH intracellular domain (NICD) and transcriptional activator complex formation.In cases with gain-of-function NOTCH1 and NOTCH3 mutation, NOTCH1 pathway

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activation is associated with nuclear expression of NICD protein. 11 15Therefore, in addition to molecular studies IHC staining for NICD nuclear staining can be a useful method to detect NOTCH1 pathway activation.
NOTCH1 plays a significant role in cell growth, adhesion, differentiation and angiogenesis. 8Su et al showed NOTCH1 upregulation by IHC staining in AdCC tissues in comparison with normal tissues, and even more enhanced expression in tissues with metastases and recurrence when compared with tumour tissues without metastases. 16eeney et al showed patients with salivary gland origin AdCCs and NOTCH1 pathway activation had significantly shorter recurrence free survival (RFS) and OS.There was also a significant OS reduction from the time of disease recurrence/metastasis. 11Sajed et al studied AdCCs of salivary glands and head neck using IHC and NOTCH1 mutational analysis and revealed diffuse NOTCH1 staining expressed in AdCCs with solid growth pattern and was associated with gain-of-function mutation and worse clinical outcome.In addition, the result showed MYB gene rearrangements were common in AdCCs with diffuse NICD1 positivity. 17reast AdCC is a rare type of triple negative carcinoma.Similar to the salivary gland counterpart, breast AdCCs are characterised by the t(6;9)(q22-23;p23-24) chromosomal translocation, resulting in MYB::NFIB fusion gene. 3Solid-basaloid type of AdCC has morphological similarities with basaloid TNBC and shows more aggressive clinical manifestations with shorter disease free survival in comparison with conventional AdCCs. 18nly limited studies have investigated the NOTCH1 expression impact on breast AdCC.
Schwartz and colleagues studied the molecular alterations in breast AdCC by reviewing targeted sequencing and showed MYB rearrangements were significantly lower in solid-basaloid AdCCs versus conventional AdCCs and NOTCH1 mutations were identified only in the subset of SB-AdCCs. 18n additional study evaluated 17 cases of breast SB-AdCCs by both IHC and molecular sequencing and showed similar to head and neck counterparts, NOTCH activating mutation was associated with a strong diffuse nuclear NICD1 staining in 30% of SB-AdCCs.In addition, diffuse MYB expression by IHC was noted in the majority of solid AdCCs (82%), while MYB rearrangements were detected only (19%) of cases. 19he current study of 25 cases of mammary AdCC evaluated by IHC demonstrated that NOTCH1 was positive in 44% of cases, predominantly in mixed-type AdCCs (64%) with expression in tumour cells with both solid and tubular/cribriform growth patterns and classic-type AdCCs (27%).Only 12.5% (1 of 8 cases) of pure solid-basaloid AdCC was NOTCH1 positive.
Patients with NOTCH1 positive AdCCs were younger than patients with negative NOTCH1 tumour expression and had

Figure 1
Figure 1 NOTCH1 expression in breast adenoid cystic carcinoma.(A) Representative H&E micrograph of breast adenoid cystic carcinoma with recurrence.(B) Corresponding positive NOTCH1 nuclear immunostaining.(C) Representative H&E micrograph of breast adenoid cystic with distant metastasis.(D) Corresponding positive NOTCH1 nuclear immunostaining.(E) Representative H&E micrograph of breast adenoid cystic carcinoma with neither recurrence or metastasis.(F) Corresponding negative NOTCH1 nuclear immunostaining.

Table 1
Tumour characteristics

Table 2
Patients with NOTCH1 positive AdCC