Supplementary material 2: Expression of human growth hormone (GH) and the
receptor growth hormone (RGH) in pathological specimens of ductal invasive
carcinoma and benign fibroadenoma
1. Materials and Methods
1.1. Epithelial component analysis of ductal invasive breast carcinoma and breast
fibroadenoma
Ductal invasive breast carcinoma samples and breast fibroadenoma samples obtained
upon surgery, and confirmed by standard immunohistochemical procedures were
employed for this study. Tissue samples were obtained during regular surgery in the
Clinical hospital “Sisters of mercy”, Croatia. The study was performed by using 40
metastatic breast carcinomas (ductal invasive breast carcinomas) and 30 benign breast
tumors (fibroadenomas). The obtained results were analyzed according to standard
histological procedures on formalin-fixed, paraffin-embedded tissue sections (thickness 5
µm) . Standard hemalaun-eozine procedure has been employed. Deparaffinization and
immunohistochemical staining were carried out following microwave streptavidin
immunoperoxidase (MSIP) protocol and by use of labeled streptavidin biotin (LSAB)
method on a DAKO TechMateTM Horizon automated immunostainer. The reactions of
epithelial cellular component of ductal invasive carcinomas and fibroadenomas on the
growth hormone (GH) and growth hormone receptor (RGH) were monitored. Precisely,
the presence of GH and RGH in the cellular component of samples was determined.
The GH and RGH epithelial reaction of ductal invasive breast carcinoma and breast
fibroadenoma has been determined by immunohistochemistry on the position of strongest
reaction («hot spot») (magnification factor 400X) for a total of 1000 tumor cells. The
«hot spot» has been established upon whole section inspection (magnification of 40X).
The results are presented semi-quantitatively by determination of immunohistochemical
staining index (IIB) by taking into account the intensity of reaction and upon calculation
of cells with positive reaction. The intensity of the reaction (IR) has been scored as
followed: 0, for no reaction; 1, for a weak reaction; 2, for a moderate to strong reaction;
and 3, for a very strong reaction. The percentage of immunoreactive (positive) tumor
cells has been scored as follows: 0, for no reaction; 1, for a weak reaction (up to 20% of
positive cells); 2, for a moderate reaction (20%- 50% positive cells); and 3, for a strong
reaction (over 50% of positive cells).
The IIB value has been calculated by multiplication of the reaction intensity (staining) IR
and the percentage of positive cells. Upon determination of IIB, and by taking into
account the requirements for maximal resolution and statistically relevant numbers of
samples per group, the following groups have been formed: IIB 0, – no reaction; IIB 1, –
a weak reaction (IIB equals to 1, 2 or 3); IIB 2, – moderately to strong reaction (IIB
equals to 4 or 6); IIB 3, – extremely strong reaction (IIB equals 9).
1.2. Stromal component analysis of ductal invasive breast carcinoma and breast
fibroadenoma
The same procedure as described in the previous chapter (1.1.) was performed for
determination of stromal GH and RGH presence in ductal invasive breast carcinomas and
breast fibroadenomas.
The stromal reaction of ductal invasive breast carcinoma and breast fibroadenoma on GH
and RGH has been determined by immunohistochemistry on the position of the strongest
reaction («hot spot») and on four neighboring sight fields (magnification factor 400X)
for a total of 1000 tumor cells. The «hot spot» has been established upon whole section
inspection (magnification of 40X). The results are presented semi-quantitatively and are
scored as: 0, for no reaction; 1, for aweak reaction; 2, for a moderate to strong reaction;
and 3, for a very strong reaction.
2. Results
RGH has been detected in fibroadenoma tissue cells in 29 out of 30 samples (96,7%)
showing a very strong reaction in 8 samples (score 3), a moderate to strong reaction in
14 samples (score 2) and a weak reaction in 7 samples (score 1).
It was almost impossible to detect RGH in the fibroadenomas’ stroma. No reaction at all
has therefore, been established in 28 samples while a weak reaction has been determined
in 2 samples (score 1).
GH has been detected in tissue cells of fibroadenomas in all 30 samples (100%) showing
a very strong reaction in 7 samples (score 3), a moderate to strong reaction in 13 samples
(score 2) and a weak reaction in 9 samples (score 1). No reaction at all has been
established for one sample only (score 0).
GH has been detected in the fibroadenomas stroma for 29 (96,7%) out of 30 samples,
precisely a very strong reaction has been determined in 20 samples (score 3), a moderate
to strong reaction in 9 samples (score 2) and a weak reaction in one sample (score 1)
(supplementary figure 1, supplementary table 1).
Epithelial RGH has been detected in 26 ductal invasive carcinomas (65%) out of 40
tested samples showing a very strong reaction in 9 samples (score 3), a moderate to
strong reaction in 5 samples (score 2) and a weak reaction in 12 samples (score 1). No
reaction at all has been determined for 14 samples (score 0).
Stromal RGH has been detected in 30 ductal invasive carcinomas (75%) showing a very
strong reaction in 12 samples (score 3), a moderate to strong reaction in 10 samples
(score 2) and a weak reaction in 8 samples (score 1). No reaction at all has been
determined for 10 samples (score 0).
Epithelial GH has been detected in 22 ductal carcinomas (55%) showing a very strong
reaction in 5 samples (score 3), a moderate to strong reaction in 7 samples (score 2) and
a weak reaction in 10 samples (score 1). No reaction at all has been determined for 18
samples (score 0).
Stromal GH has been detected in 8 ductal carcinomas (20%) while no reaction at all has
been determined for a total of 32 samples (score 0). A moderate to strong reaction has
been determined in 3 samples (score 2), while a weak reaction has been determined only
in 5 samples (score 1) (supplementary table 2).
In addition, other clinical breast cancer parameters have been determined including the
tumor size, tumor grade, estrogen and progesterone receptors status, HER-2 status and
the TNM classification. No correlation between these parameters and cellular and/or
stromal GH and RGH reactions has been found out.
In conclusion, a significant difference has been observed between ductal invasive breast
carcinoma samples. Therefore, RGH reaction in the stroma has been established for 75%
of the total sample number while almost no RGH reaction in the stroma of fibroadenomas
has been observed. Oppositely, a lower GH reaction in the stroma of ductal invasive
breast carcinomas has been observed (positive reaction in 20% of the samples) in
comparison to GH reaction in the stroma of fibroadenoma samples (positive reaction in
96,7% of the samples).
3. Supplementary tables and figures
Supplementary table 1. Immunohistochemical analysis results of cell and stromal
components for fibroadenoma specimens. GH (growth hormone) and RGH (receptor
growth hormone) presence has been determined for a total of 30 samples as follows: 0, no
reaction; 1, weak reaction; 2, moderate reaction; 3, strong reaction.
RGH,
epithel
RGH,
stroma
GH,
epithel
GH,
stroma
Fibroadenoma mammae
3
0
3
1
Fibroadenoma mammae
3
0
2
2
Fibroadenoma mammae dextri
3
0
2
2
Fibroadenoma mammae dextri
2
0
3
3
Fibroadenoma mammae dextri
2
0
2
3
Fibroadenoma mammae sinistri
2
0
2
3
Fibroadenoma mammae
2
0
3
3
Fibroadenoma mammae sinistri
0
0
1
2
Fibroadenoma mammae dextri
1
1
1
3
Fibroadenoma mammae dextri
1
0
0
2
Fibroadenoma mammae dextri
2
0
2
2
Fibroadenoma mammae dextri
2
0
2
3
Fibroadenoma mammae sinistri
2
0
1
3
Fibroadenoma mammae dextri
2
0
2
3
Fibroadenoma mammae dextri
2
0
2
3
Fibroadenoma mammae
3
0
3
3
Fibroadenoma mammae
1
0
3
2
Fibroadenoma mammae dextri
2
0
2
3
Fibroadenoma mammae dextri
3
0
1
2
Fibroadenoma mammae
1
0
2
3
Fibroadenoma mammae dextri
3
0
2
2
Pathohistological diagnosis (PHD)
Fibroadenoma mammae sinistri
3
0
3
3
Fibroadenoma mammae sinistri
1
0
2
3
Fibroadenoma mammae sinistri
1
0
1
3
Fibroadenoma mammae dextri
2
1
3
3
Fibroadenoma mammae dextri
2
0
1
3
Fibroadenoma mammae dextri
1
0
2
3
Fibroadenoma mammae dextri
2
0
1
3
Fibroadenoma mammae
3
0
1
2
Fibroadenoma mammae
2
0
1
3
Supplementary figure 1. GH (growth hormone) and RGH (receptor growth hormone)
immunohistochemical reactions in fibroadenoma samples (for a total of 30 samples).
Supplementary figure 2. GH (growth hormone) and RGH (receptor growth hormone)
immunohistochemical reactions in ductal invasive breast carcinoma (for a total of 40
samples).