Kirsi-Maria Haapasaari

Systemic therapy with estrogen or estrogen with progestin has no effect on skin collagen in postmenopausal women

OBJECTIVESnTo investigate the effect of estrogen alone or combined with progestin on the amount and synthesis of skin collagen in postmenopausal women.nnnMETHODSnForty-three early postmenopausal women were enrolled into this open, non-randomized parallel-groups study. Fifteen women received a continuous oral dose of 2 mg of 17 beta-estradiol and 1 mg of norethisterone acetate daily and 14 women an oral dose of 2 mg estradiol valerate daily. Fourteen subjects served as controls. The histology and type I and III procollagen immunohistochemistry of the skin, skin thickness, the amount of total collagen determined by a colorimetric method and the synthesis of type I and III collagens determined by analysing procollagen propeptides in the suction blister fluid were studied before the treatment and at 6 and 12 months. The proportional area of elastic fibers and the thickness of the epidermis were assessed from the sections obtained before the treatment and at 12 months, with computerized image analysis.nnnRESULTSnSkin thickness, the amount and rate of collagen synthesis, the proportional area of elastic fibers and the thickness of the epidermis were not affected by either 17 beta-estradiol and 1 mg of norethisterone acetate or 2 mg of estradiol valerate. No histological or immunohistological changes were detected in the skin specimens during the 12-month treatment period compared to the baseline or to the skin specimens of the control group.nnnCONCLUSIONSnA 1-year treatment with systemic estrogen alone or combined with progestin does not change the amount of collagen or the rate of collagen synthesis in postmenopausal women.

The prognostic significance and value of cyclin D1, CDK4 and p16 in human breast cancer.

IntroductionLoss of the retinoblastoma protein tumor suppressor gene (RB) coding for a nuclear phosphoprotein that regulates the cell cycle is found in many human cancers and probably leads to disruption of the p16-cyclin D1-CDK4/6-RB pathway. Cyclin D1 is known to activate CDK4, which then phosphorylates the RB protein, leading to cell cycle progression. p16 inhibits CDK4, keeping RB hypophosphorylated and preventing cell cycle progression. The significance of these three markers, cyclin D1, CDK4 and p16, for breast cancer and carcinogenesis is nevertheless still controversial.MethodsThe material consisted of 102 formalin-fixed human breast cancer samples, in which cyclin D1, CDK4 and p16 expression was evaluated immunohistochemically. The amounts of cyclin D1 mRNA present were analyzed by quantitative real time PCR.ResultsHigh cyclin D1 expression statistically significantly correlated with lower tumor grade, estrogen and progesterone receptor positivity and lower proliferation activity in breast tumors and increased breast cancer-specific survival and overall survival. Tumors with high cyclin D1 protein had 1.8 times higher expression of cyclin D1 mRNA. CDK4 expression did not correlate with cyclin D1 expression or the survival data. p16 expression was associated with Human Epidermal Growth Factor Receptor 2 (HER2) negativity and increased breast cancer-specific survival and disease-free survival. No statistical correlations between cyclin D1, CDK4 and p16 were found.ConclusionsCyclin D1 was associated with a good breast cancer prognosis but functioned independently of CDK4. High cyclin D1 expression may be partially due to increased CCND1 transcription. p16 correlated with a better prognosis and may function without CDK4. In conclusion, it appears that cyclin D1, CDK4 and p16 function independently in human breast cancer.

Recovery of human skin collagen synthesis after short-term topical corticosteroid treatment and comparison between young and old subjects

Summary In the present study, the recovery of the collagen synthesis rate after topical potent glucocorticoid treatment in the human skin in vivo was investigated. In the first experiment, two age groups were compared: young subjects with an age range of 21–26 years (mean 23), and old subjects, aged 55–70 years (mean 64). Twenty healthy male volunteers applied betamethasone‐17‐valerate to their abdominal skin for 3 days twice a day. Suction blisters were induced on the treated areas, and on the opposite side (healthy non‐treated skin), of the abdominal skin on the day following the discontinuation of the treatment, and on the second and seventh day. In another experiment, suction blisters were induced after the treatment and 2 weeks later on the treated area and on healthy skin, in eight male volunteers. In both experiments, the aminoterminal propeptides of type I and III collagens (PINP and PIIINP, respectively) were measured radioimmunologically from the suction blister fluid. Corticosteroid treatment decreased the collagen synthesis in both age groups after a 3‐day treatment period, and essentially no recovery in the collagen synthesis could be seen during a 1‐week corticoid‐free period. The inhibition and downregulation of collagen synthesis in the corticoid‐treated skin was similar in both young and old subjects, up to 7 days after the treatment. During the 2‐week corticoid‐free period, collagen synthesis was recovered to about 50% of the level seen in the non‐treated skin. Indicating that collagen synthesis is not completely normalized in the human skin even during a 2‐week corticoid‐free period.

Nuclear Nrf2 expression is related to a poor survival in pancreatic adenocarcinoma

The aim of this study was to investigate the expression of Nrf2, sulfiredoxin and DJ1 in pancreatic cancer. The expression of Nrf2, sulfiredoxin and DJ1 was studied immunohistochemically in a large set of pancreatic adenocarcinomas consisting of 103 cases. Eighty six percent of the cases showed cytoplasmic Nrf2 and 24% nuclear Nrf2 positivity. Sulfiredoxin positivity was observed in 54% and DJ1 positivity in all cases. Nuclear Nrf2 positivity had an association with sulfiredoxin (p=0.019) and was associated with a poor survival (p=0.010). Stage IV tumors tended to have a more nuclear Nrf2 expression (p=0.080). DJ1 expression was more often found in well-differentiated tumors (p=0.012), and DJ1 expression was associated with better survival (p=0.020). According to the results, nuclear Nrf2 expression predicts a worse survival in pancreatic adenocarcinoma, which is in keeping with its protection of cells against oxidative or xenobiotic stress. In accordance with Nrf2s regulation of the synthesis of sulfiredoxin, there was an association between them (p=0.019). DJ1 had no association with Nrf2, and its expression predicted a better survival of patients.

Expressions of individual PHDs associate with good prognostic factors and increased proliferation in breast cancer patients

Tumor hypoxia-inducible transcription factor (HIF) overexpression often associates with a poor prognosis. Stability of the HIF-α subunits is regulated by HIF prolyl 4-hydroxylases, PHD1–3. We assessed here immunohistochemical expression of PHD1–3 and HIF-1α and 2α in patients with invasive ductal breast carcinoma (nxa0=xa0102) and correlated their expression levels with main clinical prognostic factors and survival. PHD1 expression correlated with high proliferation, and these tumors were mainly estrogen receptor-negative. PHD3 expression declined in tumors of large size, poor differentiation, and high proliferation. There was a tendency for increased breast cancer-specific survival and longer disease-free survival among patients with high tumor PHD2 expression. Surprisingly, PHD1–3 expression did not correlate with HIF-1α or HIF-2α downregulation. However, HIF-2α expression correlated independently with low tumor stage and HIF-1α expression had a tendency for decreased breast cancer-specific survival. PHD1 and 3 appear to be HIF-independent factors in breast cancer. Not all PHD1 associated proliferation is estrogen-dependent and it is associated with a poor prognosis of cancer. PHD3 may be an important regulator of apoptosis and it is mainly found in tumors with good prognosis. PHD2 expression is likely to be involved in increased survival.

Expression and prognostic evaluation of oxidative stress markers in an immunohistochemical study of B-cell derived lymphomas

Abstract Although oxidative stress plays an important role in the biology of solid malignant tumors, little is known about oxidative stress in hematological malignancies. In this study, we evaluated the immunohistochemical expression and clinical correlations of oxidative stress markers and several essential antioxidant enzymes in B-cell lymphomas. Paraffin-embedded diagnostic tissue samples from 18 diffuse large B-cell lymphomas (DLBCL), 18 follicular lymphomas (FL), 19 Hodgkin lymphomas (HL), 7 chronic lymphocytic leukemias (CLL), 7 mantle cell lymphomas (MCL) and 7 mucosa-associated lymphoid tissue (MALT) lymphomas, together with samples from 6 reactive lymph nodes were stained for oxidative stress markers 8-hydroxydeoxyguanosine (8-OHdG) and nitrotyrosine and antioxidant enzymes manganese superoxide dismutase (MnSOD), thioredoxin (Trx) and γ-glutamyl cysteine synthetase (γ-GCS). There was increased 8-OHdG reactivity in DLBCL compared to more indolent lymphomas and reactive lymph nodes. Positivity for Trx was most intense in HL. In DLBCL, positivity for 8-OHdG and nitrotyrosine associated with shorter survival (p = 0.032 and p = 0.026, respectively). This study showed increasing expression of oxidative stress markers and antioxidant enzymes in a series of lymph node samples evolving from reactive lymph nodes to indolent and aggressive lymphomas. These markers seem to have strong prognostic value, but this has to be verified in larger studies.

Oxidative stress and redox state-regulating enzymes have prognostic relevance in diffuse large B-cell lymphoma

BackgroundOxidative stress and redox-regulating enzymes may have roles both in lymphomagenesis and resistance to lymphoma therapy. Previous studies from the pre-rituximab era suggest that antioxidant enzyme expression is related to prognosis in diffuse large B-cell lymphoma (DLBCL), although these results cannot be extrapolated to patient populations undergoing modern treatment modalities. In this study we assessed expression of the oxidative stress markers 8-hydroxydeoxyguanosine (8-OHdG) and nitrotyrosine and the antioxidant enzymes thioredoxin (Trx), manganese superoxide dismutase (MnSOD) and glutamate-cysteine ligase (GCL) via immunohistochemistry in 106 patients with DLBCL. All patients were treated with CHOP-like therapy combined with rituximab. Immunostaining results were correlated with progression-free survival, disease-specific survival and traditional prognostic factors of DLBCL.ResultsStrong 8-OHdG immunostaining intensity was associated with extranodal involvement (p = 0.00002), a high International Prognostic Index (p = 0.002) and strong Trx (p = 0.011) and GCL (p = 0.0003) expression. Strong Trx staining intensity was associated with poor progression-free survival (p = 0.046) and poor disease-specific survival (p = 0.015). Strong GCL immunostaining intensity predicted poor progression-free survival (p = 0.049). Patients with either strong Trx or strong nitrotyrosine expression showed significantly poorer progression-free survival (p = 0.003) and disease-specific survival (p = 0.031) compared with the other patients.ConclusionsThe redox state-regulating enzymes GCL and Trx are promising markers in the evaluation of DLBCL prognosis in the era of modern immunochemotherapy.

Tumor infiltrating CD8+ T lymphocyte count is independent of tumor TLR9 status in treatment naïve triple negative breast cancer and renal cell carcinoma

Toll-like receptor 9 (TLR9) is a cellular DNA-receptor of the innate immune system that is widely expressed in cancers. We demonstrated that low tumor TLR9 expression predicts poor disease-specific survival in triple negative breast cancer (TNBC) and renal cell carcinoma (RCC). We hypothesized that this is because TLR9 expression affects tumor immunophenotype. To begin to test this, we compared the number of tumor infiltrating CD8+ T lymphocytes with TLR9 expression in treatment naïve breast cancer (n = 197) and RCC (n = 94) cohorts with known TLR9 expression status. CD8+ T lymphocyte counts were assayed with image analysis after immunohistochemistry (IHC). Tumor TLR9 expression was not correlated with CD8+ T cell counts in breast cancer or RCC. CD8+ T cell counts were significantly associated with tumor proliferation index in TNBC, but not in non-TNBC. CD8+ T cell counts were also significantly associated with tumor grade in non-TNBC, but not in TNBC. In RCC, CD8+ T cell counts were significantly associated with tumor stage. CD8+ T cell counts were significantly associated with prognosis in TNBC and RCC, but the presence of CD8+ T cells in these tumors had opposite effects on disease-specific survival: High CD8+ counts were associated with better prognosis in TNBC and worse prognosis in RCC. Among TNBC patients, those with low tumor TLR9 and low CD8+ T cell counts had the poorest prognosis (log-rank p = 0.0002 vs. high tumor TLR9 and high CD8+ T cell count). In conclusion, pre-treatment tumor TLR9 status is not associated with tumor infiltrating CD8+ T lymphocytes in TNBC or RCC. The combination of TLR9 and CD8+ TIL count might be a novel composite prognostic marker in TNBC.

Bone morphogenetic proteins 4 and 2/7 induce osteogenic differentiation of mouse skin derived fibroblast and dermal papilla cells

Heterotopic ossification is a pathological condition in which bone forms outside the skeletal system. It can also occur in skin, which is the case in some genetic disorders. In addition to precursor cells and the appropriate tissue environment, heterotopic ossification requires inductive signals such as bone morphogenetic proteins (BMP). BMPs are growth and differentiation factors that have the ability to induce cartilage and bone formation in ectopic sites. The objective of this study is to explore the effect of the BMP-4 homodimer and BMP-2/7 heterodimer on the osteogenic differentiation of primary mouse skin fibroblasts and hair follicle dermal papilla (DP) cells. Osteogenic differentiation was induced by osteogenic induction medium (OS) containing 10 nM dexamethasone. The effect of BMP-4 and BMP-2/7 was studied using alkaline phosphatase (ALP) and calcium assays after 1.5, 3 and 5xa0weeks of differentiation. Fibroblasts and DP cells were able to differentiate into osteoblast-like matrix mineralizing cells. The first visible sign of differentiation was the change of morphology from rounded to more spindle-shaped cells. BMP-4 and BMP-2/7 exposure elevated ALP activity and calcium production significantly more than OS alone. The osteogenic response to BMP-4 and BMP-2/7 was similar in fibroblasts, whereas, in DP cells, BMP-2/7 was more potent than BMP-4. OS alone could not induce osteogenic differentiation in DP cells. Clear and consistent results show that dermal fibroblasts and stem cells from the dermal papilla were capable of osteogenic differentiation. The BMP-2/7 heterodimer was significantly more effective on hair follicular dermal stem cell differentiation.

Lack of prognostic value of MMP-9 expression and immunohistochemically defined germinal center phenotype in patients with diffuse large B-cell lymphoma treated with modern chemotherapy with or without CD20 antibody

Diffuse large B-cell lymphomas (DLBCLs) are a heterogeneous group of lymphomas, with no accepted biological prognostic markers in routine clinical practice. Previously, matrix metalloproteinase (MMP-9), tissue inhibitors of matrix metalloproteinase (TIMP)- 1 and non-germinal center (GC) phenotype have been shown to associate with poor prognosis in DLBCL patients. The aim of this study was to find out whether tissue expression of gelatinases (MMP-2 and MMP-9) or their tissue inhibitors (TIMP-1 and TIMP-2) or immunohistochemically defined GC phenotype could act as prognostic markers in patients treated with modern treatments. Additionally, correlations between these proteins and GC phenotype were investigated. GC phenotype and tissue expression of MMP-2, MMP-9, TIMP-1 and TIMP-2 were analyzed by immunohistochemistry in tissue samples from 114 DLBCL patients. In this study, in patients treated with modern lymphoma treatments (5-year cause-specific survival 69.8%) MMP-2, MMP-9, TIMP-1 or TIMP-2 expression or GC phenotype did not correlate with survival. International Prognostic Index (IPI) and stage were the only factors, which retained their prognostic significance in this patient material. Gelatinases or TIMPs did not correlate with GC phenotype, either. Prognostic markers are dependent on the lymphoma treatments used. In DLBCL patients treated with modern chemotherapy with or without rituximab, MMP-9, TIMP-1 and GC phenotype seem to have lost their prognostic value.