Characterisation of the immune microenvironment of primary breast cancer and brain metastasis reveals depleted T-cell response associated to ARG2 expression.

BACKGROUND: Immune checkpoint inhibition is an established treatment in programmed death-ligand 1 (PD-L1)-positive metastatic triple-negative (TN) breast cancer (BC). However, the immune landscape of breast cancer brain metastasis (BCBM) remains poorly defined. MATERIALS AND METHODS: The tumour-infiltrating lymphocytes (TILs) and the messenger RNA (mRNA) levels of 770 immune-related genes (NanoString™, nCounter™ Immuno-oncology IO360) were assessed in primary BCs and BCBMs. The prognostic role of ARG2 transcripts and protein expression in primary BCs and its association with outcome was determined. RESULTS: There was a significant reduction of TILs in the BCBMs in comparison to primary BCs. 11.5% of BCs presented a high immune infiltrate (hot), 46.2% were altered (immunosuppressed/excluded) and 34.6% were cold (no/low immune infiltrate). 3.8% of BCBMs were hot, 23.1% altered and 73.1% cold. One hundred and twelve immune-related genes including PD-L1 and CTLA4 were decreased in BCBM compared to the primary BCs (false discovery rate <0.01, log2 fold-change >1.5). These genes are involved in matrix remodelling and metastasis, cytokine-chemokine signalling, lymphoid compartment, antigen presentation and immune cell adhesion and migration. Immuno-modulators such as PD-L1 (CD274), CTLA4, TIGIT and CD276 (B7H3) were decreased in BCBMs. However, PD-L1 and CTLA4 expression was significantly higher in TN BCBMs (P = 0.01), with CTLA4 expression also high in human epidermal growth factor receptor 2-positive (P < 0.01) compared to estrogen receptor-positive BCBMs. ARG2 was one of four genes up-regulated in BCBMs. High ARG2 mRNA expression in primary BCs was associated with worse distant metastasis-free survival (P = 0.038), while ARG2 protein expression was associated with worse breast-brain metastasis-free (P = 0.027) and overall survival (P = 0.019). High transcript levels of ARG2 correlated to low levels of cytotoxic and T cells in both BC and BCBM (P < 0.01). CONCLUSION: This study highlights the immunological differences between primary BCs and BCBMs and the potential importance of ARG2 expression in T-cell depletion and clinical outcome.

Aberrant expression of metastasis-inducing proteins in ectopic and matched eutopic endometrium of women with endometriosis: implications for the pathogenesis of endometriosis.

BACKGROUND: Endometriosis is a metastatic disease without obvious tumorigenesis. Expression of S100P, S100A4, osteopontin (OPN) or anterior gradient homologue 2 (AGR2) proteins can induce metastasis but fail to induce tumorigenesis per se. We now explore whether this group of metastasis-inducing proteins (MIPs) are associated with the pathogenesis of endometriosis. METHODS: Eutopic endometrial biopsies were taken from 73 women (35 fertile women without endometriosis and 38 women with surgically diagnosed endometriosis). Ectopic endometriotic lesions were collected from eight of the women with endometriosis. The expression of MIPs at the cellular level was evaluated by immunohistochemistry and the presence of these proteins in the endometrial tissues was verified by western blotting and their gene expression was confirmed by RT-PCR. RESULTS: All four MIPs were immunolocated in the endometrium of control women and S100P, AGR2 and OPN showed a cyclical variation. Proliferative phase eutopic endometrium of both groups showed a similar staining pattern for all MIPs, whereas secretory phase endometrium showed a differential expression between controls and cases. The secretory phase endometrial immunostaining of controls showed weak stromal and perivascular AGR2, and decreased stromal and glandular S100P. In contrast, immunostaining for all MIPs was increased in the late secretory endometrial samples of women with endometriosis and intense immunostaining was seen for S100A4 in the stroma (P< 0.05) and for S100P (P< 0.001) and AGR2 (P< 0.0001) in both glands and stroma (P< 0.001). All active peritoneal endometriotic lesions showed strong immunostaining for each of the MIPs studied. CONCLUSIONS: We propose that these MIPs enhance endometrial cell invasiveness and contribute to the establishment of ectopic endometriotic deposits after retrograde menstruation.

Significance of the metastasis-inducing protein AGR2 for outcome in hormonally treated breast cancer patients.

The anterior gradient protein-2 (AGR2) is inducible by oestrogen and itself can induce metastasis in a rat model for breast cancer. Here, a rabbit antibody to recombinant human AGR2 was used to assess its prognostic significance in a retrospective cohort of 351 breast cancer patients treated by adjuvant hormonal therapy. The antibody stains 66% of breast carcinomas to varying degrees. The percentage of positive carcinoma cells in tumours directly correlates with the level of AGR2 mRNA (Spearman's rank correlation, P = 0.0007) and protein (linear regression analysis r2 = 0.95, P = 0.0002). There is a significant association of staining of carcinomas for AGR2 with oestrogen receptor alpha (ERalpha) staining and with low histological grade (both Fisher's Exact test P<0.0001). In the ERalpha-positive cases, but not the ERalpha-negative cases, when subdivided into the separate staining classes for AGR2, there is a significantly progressive decrease in patient survival with increased staining (log rank test, P = 0.006). The significant association of staining for AGR2 with patient death over a 10-year period (log rank test P = 0.007, hazard ratio = 3) only becomes significant at 6 years of follow-up. This may be due to the cessation of adjuvant hormonal therapy at an earlier time, resulting in adverse re-expression of the metastasis-inducing protein AGR2.

Expression of calcium-binding protein S100A2 in breast lesions.

A suppression subtraction cDNA library representing mRNAs expressed at a higher level in a benign breast tumour-derived cell line relative to the malignant MCF-7A cell line contained cDNAs corresponding to mRNAs for plasminogen activator inhibitor I, annexin VIII and the EF-hand protein S100A2. S100A2 protein has previously been shown to be expressed in normal human breast epithelium, but not in human breast carcinoma cell lines. Using a PCR-based assay and in situ hybridization on histological sections of human breast specimens, the mRNA for S100A2 was shown to be present in all benign breast lesions examined as well as in normal epithelium. S100A2 mRNA was detectable in 37% of specimens of carcinoma in situ, but in less than 15% of carcinoma specimens. The results suggest that the loss of S100A2 is associated with the development of malignant cells and is not associated with early tumour development.

Variant estrogen receptor alpha mRNAs in human breast cancer specimens.

A panel of human breast cancer specimens was examined for single base change mutations by DNA sequencing and for larger deletions using a PCR-based assay. In the cancer specimens examined, no sequencing variants were detected other than a previously characterized polymorphism. Although most of the specimens contained estrogen receptor (ER) variants at a low level, 2 of 118 specimens exhibited variants which, after amplification, constituted most of the amplified ER cDNA. One specimen contained a single variant form, and there was little evidence of the wild-type ER mRNA by PCR, Northern blotting or immunocytochemistry. The second specimen, despite the presence of a normal-sized mRNA by Northern blotting and normal immunocytochemical staining for ER, contained at least 5 different variant forms as well as the wild-type ER. All but 1 of the variant forms were processing variants, and 3 of these processing variants have not been described before. One variant, although lacking exons 2-4, has break points in exons 1 and 5 that do not correspond to intron-exon boundaries. This variant might reflect more widespread damage to the genome in this breast cancer specimen. The low level of occurrence of variants suggests that ER variant forms, at least in the coding region, do not contribute generally to the progression of breast cancer.

Prognostic significance of the metastasis-inducing protein S100A4 (p9Ka) in human breast cancer.

The calcium-binding protein S100A4 is capable of inducing metastasis in rodent models for breast cancer. We now show that rabbit antibodies to recombinant rat S100A4 recognize specifically human S100A4 using Western blotting techniques and use them to assess the prognostic significance of S100A4 in primary tumors from a group of 349 patients treated between 1976 and 1982 for stage I and stage II breast cancer. The antibody stains normal breast tissue heterogeneously, but stains positively 41% of the carcinomas, leaving the remaining 59% as negatively stained. In addition to the carcinoma cells, some host stromal cells and lymphocytes are also stained, but these have been discounted in subsequent analyses. There is an association of staining of carcinomas for S100A4 with some tumor variables considered to be associated with poor prognosis for patients: tumor present in axillary lymph nodes (borderline P = 0.058), staining for c-erbB-3 (P = 0.002), cathepsin D (P = 0.024), and c-erbB-2 (P = 0.048). The association of staining for S100A4 with patient survival has been evaluated using life tables and analyzed using generalized Wilcoxon statistics. Eighty percent of the S100A4-negative patients but only 11% of the S100A4-positive patients are alive after 19 years of follow-up, and this association is highly significant (P < 0.0001); the former have a median survival of >228 months and the latter 47 months. The other tumor variables that show significant association with survival time are nodal status (P < 0.0001), tumor size (P = 0.0035), histological grade (P = 0.013), staining for c-erbB-2 (P = 0.0015), estrogen receptor (P = 0.028), and p53 (P = 0.032). Analysis of the association of patients with carcinomas staining for S100A4 and their survival in subgroups defined by these other tumor variables shows that in each subgroup, staining for S100A4 is associated with poorer survival. Patients whose tumors stain for S100A4 and possess involved lymph nodes (P < 0.0001), which are fixed to the chest wall (P = 0.015) or which stain for c-erbB-2 (P = 0.050), show a significant reduction in survival times over those with only S100A4-staining tumors. Patients with involved lymph nodes, or staining for c-erbB-2 in the S100A4-negative group fail to show any significant reduction in survival times. Multivariate regression analysis for 137 patients shows that staining for S100A4 is most highly correlated with patient deaths (P < 0.0001), but involved lymph nodes (P = 0.001), fixed tumors (P = 0.0002), and high histological grade (P = 0.022) are also significant independent prognostic variables. These results suggest that in this group of patients, the metastasis-inducing protein S100A4 is most tightly correlated with patient demise.

Comparison of the metastasis-inducing protein S100A4 (p9ka) with other prognostic markers in human breast cancer.

Our aim was to compare the occurrence and prognostic significance over 14-20 years of immunocytochemically detected S100A4 and other tumour variables in primary tumours from 349 patients with operable breast cancer. For a cut-off of 1% staining of the malignant cells, the antibody to S100A4 stains positively 56% of the carcinomas. There was a significant association of staining for S100A4 with tumours fixed to the chest wall, staining for c-erbB-2, c-erbB-3, pS2, cathepsin D and, inversely, at borderline levels with staining for estrogen receptor. Using Wilcoxon statistics in univariate analyses, staining for S100A4, nodal status, tumour class, histological grade and staining for c-erbB-2, p53 were associated negatively and staining for estrogen receptor, progesterone receptor were associated positively with patient survival times. The survival times of patients with S100A4-negative carcinomas with or without one of the other tumour variables showed no significant differences, whilst those of patients with S100A4-positive carcinomas showed significant differences in a negative or a positive way. Multivariate regression analysis for 137 patients showed that staining for S100A4 is most highly correlated with patient deaths, but involved lymph nodes, fixed tumours, high histological grade and staining for progesterone receptor were also significant independent prognostic variables. Our results suggest that in this set of patients, the tumour variable most tightly correlated with patient death is S100A4.

Development of hyperplasias, preneoplasias, and mammary tumors in MMTV-c-erbB-2 and MMTV-TGFalpha transgenic rats.

Human cDNAs corresponding to two epidermal growth factor-related products that are overexpressed in human breast cancers, that for c-erbB-2 (HER-2) and for transforming growth factor alpha (TGFalpha), have been cloned downstream of the mouse mammary tumor virus (MMTV) long terminal repeat promoter and injected into the pronucleus of fertilized oocytes of Sprague-Dawley rats to produce transgenic offspring. Expression of the transgenic mRNAs is not detectable in mammary tissue from virgin transgenic rats but is detected in mammary tissue from certain lines of mid-pregnant transgenic rats. When two such lines of either type of transgenic rat are subjected to repeated cycles of pregnancy and lactation, they produce, primarily in the mammary glands, extensive pathologies, whereas virgin transgenic rats produce no such abnormalities. Multiparous transgenic female offspring from c-erbB-2-expressing lines develop a variety of focal hyperplastic and benign lesions that resemble lesions commonly found in human breasts. These lesions include lobular and ductal hyperplasia, fibroadenoma, cystic expansions, and papillary adenomas. More malignant lesions, including ductal carcinoma in situ and carcinoma, also develop stochastically at low frequency. The mammary glands of transgenic females invariably fail to involute fully after lactation. Similar phenotypes are observed in female MMTV-TGFalpha transgenic rats. In addition, multiparous TGFalpha-expressing female transgenics frequently develop severe pregnancy-dependent lactating hyperplasias as well as residual lobules of hyperplastic secretory epithelium and genuine lactating adenomas after weaning. These transgenic rat models confirm the conclusions reached in transgenic mice that overexpression of the c-erbB-2 and TGFalpha genes predisposes the mammary gland to stochastic tumor development.

Human S100A4 (p9Ka) induces the metastatic phenotype upon benign tumour cells.

The rodent S100-related calcium-binding protein, S100A4 induces metastasis in non-metastatic rat and mouse benign mammary cells and co-operates with benign-tumour-inducing changes in two transgenic mouse models, to yield metastatic mammary tumours. Co-transfection of the human gene for S100A4 with pSV2neo into the benign rat mammary cell line, Rama 37, yielded cells which expressed a low level of the endogenous S100A4 mRNA, and either high or undetectable levels of human S100A4 mRNA. The cells which expressed a high level of human S100A4 mRNA induced metastasis in the benign rat mammary cell line Rama 37 in an in vivo assay, whereas the cells which expressed an undetectable level of human S100A4 did not induce any detectable metastases. The primary tumours arising from the S100A4-expressing cells contained high levels of immunocytochemically-detected S100A4 and this high level of S100A4 and the metastatic potential were maintained when cells from a metastasis were re-injected into syngeneic rats. The results show that the human S100A4 possesses metastasis-inducing capabilities.

Cytoplasmic staining of c-erbB-2 is not associated with the presence of detectable c-erbB-2 mRNA in breast cancer specimens.

The cell-surface receptor tyrosine kinase protein c-erbB-2 is immunocytochemically detected as membrane staining on the surface of cancer cells in 20-30% of cases of breast cancer, and its presence has been associated with poor prognosis for the patient. However, there have been numerous reports of immunocytochemical staining for c-erbB-2 solely in the cytoplasm of some normal and tumour specimens with frequently used anti-sera, and the presence of such staining has been difficult to interpret. It is not known for certain that cytoplasmic c-erbB-2 staining is an artefact of the immunocytochemical procedures used. Thus, mRNA for c-erbB-2 has been quantified in tumours exhibiting only cytoplasmic staining or varying levels of membrane staining using a sensitive, competitive PCR method. Whereas abundant levels of c-erbB-2 mRNA are found in tumours exhibiting membrane staining for c-erbB-2 and these levels correlate with the percentage of tumour cells showing membranous staining for c-erbB-2, the level of c-erbB-2 mRNA in tumours displaying only cytoplasmic staining is no higher than in c-erbB-2-negative specimens.

Immunocytochemical distribution of the calcium-binding protein p9Ka in normal rat tissues: variation in the cellular location in different tissues.

The family of S-100-related proteins consists of a number of small potential calcium-binding proteins of unknown function. Elevated expression of one of these proteins, p9Ka, or of its mRNA, correlates with the metastatic potential of cultured mammary epithelial cells from rat or mouse. Over-expression of p9Ka by transfection of benign rat mammary epithelial tumor cells with the gene for p9Ka induces the metastatic phenotype. At present there is little information on the occurrence of p9Ka in normal rat tissues. A specific antiserum immunocytochemically detects p9Ka intracellularly in most normal adult rat tissues studied, including smooth muscle, brown adipose tissue, and liver. In other tissues, p9Ka is localized specifically to some absorptive and keratinized epithelia, the acid-secreting parietal cells of the stomach, the neuronal cells within plexuses of the autonomic nervous system, and a proportion of cells of the immune system in spleen, lymph nodes, bone marrow, and blood. p9Ka is found widely in both arteries and veins, particularly in the smooth muscle and in the endothelium of smaller veins. In mammary gland, the pattern of staining suggests that p9Ka is extracellularly located in a region surrounding the ducts.

Immunolocalization of alpha-transforming growth factor in the developing rat mammary gland in vivo, rat mammary cells in vitro and in human breast diseases.

Immunoreactive alpha-transforming growth factor (alpha-TGF) was shown by immunocytochemistry to be present in the rat mammary gland at various stages of development, the staining being most intense in mature myoepithelial cells. Alpha-TGF was also detected in the secretions of the mammary glands of pregnant and lactating rats. alpha-TGF in the extracts of rat mammary glands at each stage of development, and in several rat mammary cell lines and in culture medium in which they had been grown, was shown by Western blotting to consist primarily of a protein of molecular weight 50 kDa. The amount of this protein was greater in the mammary gland of the lactating rat than in resting or involuting glands. alpha-TGF was also found in some, but not all, human breast carcinomas, and in benign hyperplastic breast diseases.

Immunocytochemical identification of basic fibroblast growth factor in the developing rat mammary gland: variations in location are dependent on glandular structure and differentiation.

We raised antiserum to human recombinant basic fibroblast growth factor (rbFGF) in rabbits. With this affinity-purified antiserum, other antisera to rbFGF, and commercial antiserum to bovine pituitary bFGF, we undertook immunocytochemical detection of bFGF in histological sections of rat mammary glands at different developmental stages. In non-growing ducts, anti-bFGF serum stains the basement membrane/myoepithelial cells, whereas in serial sections most of this stain is observed to be associated with anti-laminin-staining basement membranes rather than with anti-callus-keratin-staining myoepithelial cells. The weak staining of the myoepithelial cells is enhanced when NiCl2 is included in the detection system, but little staining for bFGF is observed in the epithelial cells. In growing neonatal ducts from 1-day-old rats, in growing terminal end buds (TEBs) and, to a lesser extent, in growing alveolar buds (ABs) in prepubescent (21-day) and pubescent (50-day) rats, both their inner and outer cells are stained moderately by anti-bFGF sera. In non-growing ducts from rats aged 6 days, in non-growing ABs of rats aged 60 days and more, and in alveoli from pregnant and lactating rats, only the basement membrane/myoepithelial cell area is stained by anti-bFGF sera; the epithelial cells are unstained. Staining of the myoepithelial cells is enhanced by mixtures of rbFGF and anti-bFGF sera in non-growing ducts, but there is little change in the staining of growing TEBs. All staining by anti-bFGF sera is abolished with heparin in the reactions. We suggest that the immunoreactive bFGF is present mainly bound to heparan sulfate glycosaminoglycans in the basement membrane of resting structures, but that immunoreactive bFGF becomes associated with proliferating cells, particularly those intermediate in characteristics between epithelial and myoepithelial cells in growing structures such as TEBs.

Prognostic significance of cathepsin-D in patients with breast cancer.

The expression of the protease cathepsin-D has been evaluated using an immunohistochemical technique with a polyclonal antibody in paraffin-embedded tissue from 359 patients treated between the years 1975-1981 for Stage I and II breast cancer. One hundred and twenty seven patients (35%) have strongly positive, granular staining, 138 (38%) are intermediately stained in the cytoplasm, and in 94 (26%) no staining is observed. There is a strong positive association between expression of cathepsin-D and the presence of tumour in axillary lymph nodes (P < 0.006). Expression of the protease is associated with significantly poorer survival of patients in univariate analysis (P = 0.025); however, this is not independent of other tumour variables.

The long term prognostic significance of c-erbB-2 in primary breast cancer.

The expression of the c-erbB-2 oncogene has been evaluated using an immunohistochemical technique with the 21N polyclonal antibody in paraffin embedded tissue from 465 patients treated between the years 1975-1981 for Stage I and II breast cancer. One hundred and four (22%) patients exhibited positive staining. This was not associated with any other variables. Expression of the oncogene was associated with significantly poorer survival which was independent of other tumour variables.

Morphogenetic behavior of simian virus 40-transformed human mammary epithelial stem cell lines on collagen gels.

Transformation of primary cultures of human breast cells with simian virus 40 and clonal selection has yielded single-cell-cloned, epithelial cell lines, as well as myoepithelial-related cell lines. When grown on floating collagen gels, the epithelial cell lines give rise to branching rays of cells, thick fingerlike protrusions, saclike structures, and degenerating areas. The myoepithelial-related cell lines give rise only to the branching rays. Epidermal growth factor stimulates the production of the thick protrusions, whereas cholera toxin stimulates the production of the degenerating areas. Immunocytochemical staining of these cultures using reagents directed against the cell surface-extracellular matrix or the cellular cytoskeleton confirms the epithelial and myoepithelial nature of the cells, and demonstrates that the degenerating areas are undergoing squamous metaplasia. The fingerlike protrusions consist of cords of cells composed of inner, epithelial and outer, myoepithelial-related cells sometimes surrounding a central lumen reminiscent of ducts. The saclike structures resemble alveoli. Ultrastructural analysis confirms the identification of the basic cell types and also identifies indeterminate cells possessing features of both epithelial and myoepithelial cells. It is suggested that the epithelial cell lines represent human mammary stem cells that can undergo processes of morphogenesis and differentiation in vitro to form many of the three-dimensional structures found within the breast.