The absence of aldehyde dehydrogenase 1 A1-positive cells in benign mammary stroma is associated with risk factors for breast cancer

Figures & data

Table 1 Antibodies used

Antigen/antibody (product code) and host species	Target species	Clonality (type)	Working dilution	Source
ALDH1 A1 (611194), mouse	Antihuman	Monoclonal IgG1	1:100	BD Biosciences, San Jose, CA, USA
CD24 (MA5-11833), mouse	Antihuman	Monoclonal IgM	1:100	Thermo Fisher Scientific, Waltham, MA, USA
CD44 (PA5-21419), rabbit	Antihuman	Polyclonal IgG	1:100	Thermo Fischer Scientific
AF488 (A11029), goat	Antimouse	Fab2 IgG H+L	1:150	Thermo Fisher Scientific
AF568 (A21043), goat	Antimouse	IFab2 gMµ	1:200	Thermo Fisher Scientific
AF647 (A21245), goat	Antirabbit	Fab2 IgG (H + L)	1:200	Thermo Fisher Scientific

Abbreviations: IgG, immunoglobulin G; IgM, immunoglobulin M.

Figure 1 Confocal microscopy images of triple immunofluorescence-labeled histologically normal breast tissue.

Notes: (A)–(E) Images of single optical sections (0.6 µm thick) from a stromal area with a duct visible in the upper left part of the field (* in lumen), demonstrating immunofluorescence multilabeling and DAPI (nuclear stain) in separate channels. (A) ALDH1, green, indicates the different shaped ALDH1⁺ cell types in stroma (round/oval cells [short arrows] and spindle-shaped/polygonal cells [long arrows]). (B) CD24, red, indicates the scarcity of CD24⁺ cells. (C) CD44, white, indicates the relatively large number of CD44⁺ cells, yet many ALDH1⁺ CD44⁻ spindle-shaped cells. (D) DAPI, blue. (E) All four channels merged indicate that many ALDH1⁺ cells are also CD44⁺ but not CD24⁺. Arrows indicate cell types as in (A). (F) Confocal image (merged channels are color coded as in A–E) from a different tissue area showing multilabeled breast tissue containing the stromal cell types as described in A–E, as well as ductules and numerous adjacent ALDH1⁺ CD24⁻ cells with weak membranous CD44 reactivity. Scale bars = 10 µm.
Abbreviation: DAPI, 4′,6-diamidino-2-phenylindole.

Table 2 Genetic and hormonal characteristics, and cancer status of the patients

Patient characteristics	All patients (n=40)	Patients with a BRCA1 mutation (n=17)	Patients with no BRCA1/2 mutation (n=23)
Age in years, median (range)a	28 (20–50)	34 (23–43)	24 (20–50)
Parous
Yes	18	12	6
No	20	3	17
Unknown	2	2	0
Contraceptive pill at the time of surgery
Yes	13	5	8
No	25	10	15
Unknown	2	2	0
Family history of breast cancer
Yes	21	17	4
No	19	0	19
Unknown	0	0	0
Synchronous breast cancer
Yes	7	7	0
No	21	10	23
Unknown	0	0	0

Note:

a All patients were premenopausal.

Figure 2 ALDH1⁺ cells in TDLUs.

Notes: Fluorescence microscope images of a triple-labeled TDLU from benign breast tissue with the following color coding: (A) DAPI nuclear staining (blue); (B) ALDH1 (green); (C) CD44 (white); (D) CD24 (red). (E) A digitally composed image showing all channels merged. Most of the cells located basally in ductules are ALDH⁻ CD44⁺, whereas some adluminal cells are ALDH1⁺ CD44⁺ and none are CD24⁺. In stroma, elongated ALDH1⁺ cells are seen between the ductules, and many such cells are present in the junction between TDLU stroma and generic connective tissue of the breast (arrows). Scale bars = 20 µm.
Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; TDLUs, terminal duct-lobular units.

Figure 3 ALDH1+ spindle-shaped/polygonal cells in TDLU stroma (arrows).

Notes: DAPI nuclear staining (blue), ALDH1 (green), CD44 (white), and CD24 (red). Scale bar = 20 µm.
Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; TDLU, terminal duct-lobular unit.

Figure 4 ALDH1⁺ round/oval-shaped cells in TDLU stroma.

Notes: DAPI nuclear staining (blue) and labeling of ALDH1 (green), CD44 (white), and CD24 (red). (A) A single-channel image depicting ALDH1+ round/oval cells (arrows). (B) A composite image with all channels demonstrating that the ALDH1⁺ round/oval cells are positive for CD44 but not for CD24 (arrows). Scale bars = 20 µm.
Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; TDLU, terminal duct-lobular unit.

Figure 5 CD24⁺ cells in TDLU stroma.

Notes: DAPI nuclear staining (blue) and labeling of ALDH1 (green), CD44 (white), and CD24 (red). The image depicts a CD24⁺ cell (arrow) exhibiting no CD44 or ALDH1 positivity. Scale bar = 20 µm.
Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; TDLU, terminal duct-lobular unit.

Table 3 Immunohistological results for the patient groups

Immunophenotype	Spindle-shaped/polygonal cells		Round/oval cells
	ALDH1^+ CD44^+ CD24^−	ALDH1^+ CD44^− CD24^−	ALDH1^+ CD44^+ CD24^−	ALDH1^+ CD44^− CD24^−
Family history (n=21)	19 (90%)	16 (76%)	6 (29%)	2 (11%)
No family history (n=19)	18 (95%)	14 (74%)	12 (63%)	2 (10%)
BRCA1 mutation (n=17)	15 (88%)	12 (71%)	6 (35%)	2 (12%)
No BRCA1 mutation (n=23)	22 (96%)	18 (78%)	12 (52%)	2 (9%)
Nulliparous (n=20)	18 (90%)	12 (60%)	10 (50%)	1 (5%)
Parous (n=18)	17 (94%)	17 (94%)	6 (33%)	3 (17%)
Age <28 years (n=19)	17 (89%)	14 (74%)	9 (47%)	2 (11%)
Age ≥28 years (n=21)	20 (95%)	16 (76%)	9 (43%)	2 (11%)

Note: The table presents the number of patients in each clinical category whose sample contained “any” cells exhibiting the investigated immunophenotypes.

Figure 6 Round/oval cells according to family history.

Notes: DAPI nuclear staining (blue) and labeling of ALDH1 (green), CD44 (white), and CD24 (red). (A)–(B) Images showing the absence of ALDH1⁺ CD44⁺ CD24⁻ round/oval cells in a TDLU from a woman with a family history of breast cancer. Arrows indicate ALDH1⁻ CD44⁺ CD24⁻ cells. (C)–(D) Images illustrate an ALDH1⁺ CD44⁺ CD24⁻ round/oval-shaped cell (arrow) in a TDLU from a woman with no family history of breast cancer. (A) and (C) show only the ALDH1⁺ channel, and (B) and (D) show all four channels merged. Scale bars = 20 µm.
Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; TDLU, terminal duct-lobular unit.