The human complement inhibitor Sushi Domain-Containing Protein 4 (SUSD4) expression in tumor cells and infiltrating T cells is associated with better prognosis of breast cancer patients.

BACKGROUND: The human Sushi Domain-Containing Protein 4 (SUSD4) was recently shown to function as a novel inhibitor of the complement system, but its role in tumor progression is unknown. METHODS: Using immunohistochemistry and quantitative PCR, we investigated SUSD4 expression in breast cancer tissue samples from two cohorts. The effect of SUSD4 expression on cell migration and invasion was studied in vitro using two human breast cancer cell lines overexpressing SUSD4. RESULTS: Tissue stainings revealed that both tumor cells and tumor-infiltrating cells expressed SUSD4. The highest SUSD4 expression was detected in differentiated tumors with decreased rate of metastasis, and SUSD4 expression was associated with improved survival of the patients. Moreover, forced SUSD4 expression in human breast cancer cells attenuated their migratory and invasive traits in culture. SUSD4 expression also inhibited colony formation of human breast cancer cells cultured on carcinoma-associated fibroblasts. Furthermore, large numbers of SUSD4-expressing T cells in the tumor stroma associated with better overall survival of the breast cancer patients. CONCLUSION: Our findings indicate that SUSD4 expression in both breast cancer cells and T cells infiltrating the tumor-associated stroma is useful to predict better prognosis of breast cancer patients.

Local expression of complement factor I in breast cancer cells correlates with poor survival and recurrence.

Tumor cells often evade killing by the complement system by overexpressing membrane-bound complement inhibitors. However, production of soluble complement inhibitors in cells other than hepatocytes was rarely reported. We screened several breast cancer cell lines for expression of soluble complement inhibitor, complement factor I (FI). We also analyzed local production of FI in tissue microarrays with tumors from 130 breast cancer patients by in situ hybridization and immunohistochemistry. We found expression of FI in breast adenocarcinoma cell line MDA-MB-468 and confirmed its functional activity. Expression of FI at mRNA and protein levels was also confirmed in tumor cells and tumor stroma, both in fibroblasts and infiltrating immune cells. Multivariate Cox regression analyses revealed that high expression of FI protein in tumor cells was correlated with significantly shorter cancer-specific survival (HR 2.8; 95 % CI 1.0-7.5; p = 0.048) and recurrence-free survival (HR 3.4; 95 % CI 1.5-7.4; p = 0.002). High FI expression was positively correlated with tumor size (p < 0.001), and Nottingham histological grade (p = 0.015) and associated with estrogen and progesterone receptor status (p = 0.03 and p = 0.009, respectively). Our data show that FI is expressed in breast cancer and is associated with unfavorable clinical outcome.

Breastfeeding in relation to risk of different breast cancer characteristics.

BACKGROUND: The aim of this present study was to examine duration of breastfeeding in relation to the risk of different subgroups of breast cancer. A prospective cohort, The Malmö Diet and Cancer study, including 14092 parous women, were followed during a mean of 10.2 years and a total of 424 incident breast cancers were diagnosed. METHODS: Tumours were classified regarding invasiveness, tumour size, axillary lymph node status, Nottingham grade, tumour proliferation (Ki67), HER2, cyclin D1 and p27, WHO histological type and hormone receptor status. Duration of breastfeeding was measured using total time of breastfeeding, categorized in quartiles using the lowest as the reference group (<4.0, ≥4.0- < 8.0, ≥8.0- < 13.0 and ≥13.0 months). Average duration of breastfeeding per child and breastfeeding duration of the first child were also used as exposures in separate analyses. Relative risks, with 95% confidence intervals, were obtained using a Cox's proportional hazards analysis adjusted for potential confounders. RESULTS: Overall risk for breast cancer was similar in all quartiles of breastfeeding. No strong results regarding breastfeeding duration and breast cancer subgroups were seen. A few results indicated an association between a relatively long duration of breastfeeding and tumours with high proliferation (Ki67) and grade III histological grade. CONCLUSIONS: Breastfeeding duration was not associated with breast cancer risk and no strong results were seen with regard to breast cancer subgroups.

Cyclin A1 modulates the expression of vascular endothelial growth factor and promotes hormone-dependent growth and angiogenesis of breast cancer.

Alterations in cellular pathways related to both endocrine and vascular endothelial growth factors (VEGF) may contribute to breast cancer progression. Inhibition of the elevated levels of these pathways is associated with clinical benefits. However, molecular mechanisms by which endocrine-related pathways and VEGF signalling cooperatively promote breast cancer progression remain poorly understood. In the present study, we show that the A-type cyclin, cyclin A1, known for its important role in the initiation of leukemia and prostate cancer metastasis, is highly expressed in primary breast cancer specimens and metastatic lesions, in contrasting to its barely detectable expression in normal human breast tissues. There is a statistically significant correlation between cyclin A1 and VEGF expression in breast cancer specimens from two patient cohorts (p<0.01). Induction of cyclin A1 overexpression in breast cancer cell line MCF-7 results in an enhanced invasiveness and a concomitant increase in VEGF expression. In addition, there is a formation of protein-protein complexes between cyclin A1 and estrogen receptor ER-α cyclin A1 overexpression increases ER-α expression in MCF-7 and T47D cells. In mouse tumor xenograft models in which mice were implanted with MCF-7 cells that overexpressed cyclin A1 or control vector, cyclin A1 overexpression results in an increase in tumor growth and angiogenesis, which is coincident with an enhanced expression of VEGF, VEGFR1 and ER-α Our findings unravel a novel role for cyclin A1 in growth and progression of breast cancer, and suggest that multiple cellular pathways, including cell cycle regulators, angiogenesis and estrogen receptor signalling, may cooperatively contribute to breast cancer progression.

Glomus tumours in the long finger and in the thumb of a young patient with neurofibromatosis-1 (Nf-1).

Glomus tumours are rare in adults, and such tumours may be associated with neurofibromatosis-1 (Nf-1). Here we describe successful treatment of two glomus tumours initially in the long finger and, one year later, in the thumb in a 17-year young girl with Nf-1.

CDK1 interacts with RARγ and plays an important role in treatment response of acute myeloid leukemia.

Alterations in cell cycle pathways and retinoic acid signaling are implicated in leukemogenesis. However, little is known about the roles of cyclin-dependent kinases (CDKs) in treatment response of leukemia. In this study, we observed that CDK1 expression was significantly higher in bone marrow from 42 patients with acute myeloid leukemia (AML) at recurrence than that at first diagnosis (p = 0.04). AML patients had higher level of nuclear CDK1 in their leukemic blasts tended to have poorer clinical outcome compared with those with lower levels. We showed that CDK1 function is required for all-trans retinoic acid (ATRA) to achieve the optimal effect in U-937 human leukemic cells. CDK1 modulates the levels of P27(kip) and AKT phosphorylation in response to ATRA treatment. Further, we show, for the first time, that RARγ in concert with ATRA regulates protein levels of CDK1 and its subcellular localization. The regulation of the subcellular content of CDK1 and RARγ by ATRA is an important process for achieving an effective response in treatment of leukemia. RARγ and CDK1 form a reciprocal regulatory circuit in the nucleus and influence the function and protein stability of each other and the level of P27(kip) protein. In addition, expression of wee1 kinase and Cdc25A/C phosphatases also coincide with CDK1 expression and its subcellular localization in response to ATRA treatment. Our study reveals a novel mechanism by which CDK1 and RARγ coordinate with ATRA to influence cell cycle progression and cellular differentiation.

Expression and localization of serotonin receptors in human breast cancer.

The aim of this study was to examine the expression of serotonin receptors in patients with breast cancer and to explore their utility as diagnostic and prognostic markers. Immunohistochemical analysis was performed to examine the expression of serotonin (5-HT) receptor subtypes 1A, 1B, 2B and 4 in a tissue microarray containing tumor specimens from 102 patients. Statistical analysis was performed to correlate the expression of these proteins with regard to clinical parameters. We found that all four serotonin receptors (5-HTRs) exhibited different expression patterns in breast cancer specimens. In general strong staining for 5-HTR1A was observed on the membrane of cancer cells but it was detected only in the cytoplasm of non-malignant cells. 5-HTR1B and 5-HTR2B were predominantly expressed in the cytoplasm of breast cancer cells, while 5-HTR4 was exclusively found in the nucleus of malignant and non-malignant cells. Correlation analysis revealed a significant correlation of 5-HTR2B with estrogen receptor-α (ER-α) and 5-HTR4 with ER-α and progesterone (PR). In conclusion, the different expression patterns and subcellular localization of 5-HTRs in breast cancer may reflect their role in breast cancer progression.

Automated image analysis of cyclin D1 protein expression in invasive lobular breast carcinoma provides independent prognostic information.

The emergence of automated image analysis algorithms has aided the enumeration, quantification, and immunohistochemical analyses of tumor cells in both whole section and tissue microarray samples. To date, the focus of such algorithms in the breast cancer setting has been on traditional markers in the common invasive ductal carcinoma subtype. Here, we aimed to optimize and validate an automated analysis of the cell cycle regulator cyclin D1 in a large collection of invasive lobular carcinoma and relate its expression to clinicopathologic data. The image analysis algorithm was trained to optimally match manual scoring of cyclin D1 protein expression in a subset of invasive lobular carcinoma tissue microarray cores. The algorithm was capable of distinguishing cyclin D1-positive cells and illustrated high correlation with traditional manual scoring (κ=0.63). It was then applied to our entire cohort of 483 patients, with subsequent statistical comparisons to clinical data. We found no correlation between cyclin D1 expression and tumor size, grade, and lymph node status. However, overexpression of the protein was associated with reduced recurrence-free survival (P=.029), as was positive nodal status (P<.001) in patients with invasive lobular carcinoma. Finally, high cyclin D1 expression was associated with increased hazard ratio in multivariate analysis (hazard ratio, 1.75; 95% confidence interval, 1.05-2.89). In conclusion, we describe an image analysis algorithm capable of reliably analyzing cyclin D1 staining in invasive lobular carcinoma and have linked overexpression of the protein to increased recurrence risk. Our findings support the use of cyclin D1 as a clinically informative biomarker for invasive lobular breast cancer.

Expression of VEGF and VEGF receptors in childhood precursor B-cell acute lymphoblastic leukemia evaluated by immunohistochemistry.

Perturbation in the expression and signaling pathways of vascular endothelial growth factor (VEGF) has been linked to pathogenesis of hematologic malignancies. We investigated the expression and clinical importance of VEGF and two of its receptors, VEGFR-1 and VEGFR-2, in childhood precursor B-cell acute lymphoblastic leukemia (pre-B ALL) by using immunohistochemistry. These angiogenic proteins were expressed in the majority of leukemic bone marrow samples. Notably, pre-B ALL patients had significantly increased expression of VEGFR-1 compared with no expression in the nonmalignant group, indicating a link between VEGFR-1 protein expression and pre-B ALL. These novel findings suggest that VEGFR-1 may have clinical importance in childhood pre-B ALL.

Parity and age at first childbirth in relation to the risk of different breast cancer subgroups.

The aim of the present study was to examine parity and age at first childbirth, in relation to the risk of specific breast cancer subgroups. A prospective cohort, The Malmö Diet and Cancer Study, including 17,035 women were followed with linkage to Swedish Cancer Registry until December 31, 2004. A total of 622 incident breast cancers were diagnosed during follow-up and were evaluated regarding invasiveness, tumour size, axillary lymph node status, Nottingham grade, tumour proliferation (Ki67), HER2, cyclin D1 and p27. The tumours were also examined for WHO type and hormone receptor status. Nulliparity was associated with an overall increased risk of breast cancer, although not statistically significant (the relative risk was 1.39 with a 95% confidence interval of 0.92-2.08). Nulliparity was also associated with large tumours (>20 mm) (1.89: 0.91-3.91), high Ki67 levels (1.95: 0.93-4.10), high cyclin D1 levels (2.15: 0.88-5.27), grade III (2.93: 1.29-6.64) and HER2 positive tumours (3.24: 1.02-10.25). High parity was not statistically significantly associated with any specific breast cancer subgroup. Older age at first childbirth (>30) was associated with a slightly increased risk of breast cancer (1.39: 0.94-2.07). There was a statistically significant association between late first childbirth and lobular type (2.51: 1.01-6.28), grade III tumours (2.67: 1.19-6.02), high levels of cyclin D1 (2.69: 1.18-6.12) and low levels of p27 (2.23: 1.15-4.35). We conclude that nulliparity and late first childbirth are associated with relatively more aggressive breast cancer subgroups.

Serum calcium and tumour aggressiveness in breast cancer: a prospective study of 7847 women.

Experimental, epidemiological and clinical studies suggest that calcium and/or its regulating hormones affect breast cancer risk. There has been no prospective cohort study investigating serum calcium levels and breast cancer aggressiveness, as determined by tumour histology and stage. Dichotomized prediagnostic serum calcium levels were investigated in relation to breast cancer aggressiveness as determined by grade (mitotic frequency, tubule formation, nuclear atypia) and stage (tumour size and axillary lymph node status). Cox's proportional hazards analysis and heterogeneity analysis were used to investigate the associations between low/high calcium and grade/stage in a prospective cohort study of 7847 women, out of whom 462 women were diagnosed with incident breast cancer during a mean follow-up of 17.2 years. All analyses were stratified for body mass index and menopausal status. Prediagnostic serum calcium levels in premenopausal women were positively associated with increased tumour aggressiveness as determined by a higher risk of nodal metastasis; relative risk (RR) for calcium above median as compared with calcium below median was 1.88 with a 95% confidence interval (CI) of 1.04-3.38. In overweight women, prediagnostic serum calcium levels were also associated with tumour aggressiveness, as determined by both a higher risk of nodal metastasis [RR (95% CI) 1.69 (0.95-3.02)] and severe nuclear atypia [RR (95% CI) 2.06 (1.10-3.86)]. Results also indicate that, in overweight women, calcium is positively associated with worse grade as determined by tubule formation and mitotic frequency. In conclusion, prediagnostic serum calcium levels are positively associated with increased tumour aggressiveness in premenopausal and/or overweight women.

Anthropometric factors in relation to different tumor biological subgroups of postmenopausal breast cancer.

Overweight and obesity is associated with an increased risk of postmenopausal breast cancer. However, less is known about the impact of anthropometric factors on tumor pathology and biology. A Swedish population-based prospective cohort study of 9,685 postmenopausal women not using hormonal replacement therapy (HRT) were followed for an average of 10.3 years during which 305 incident breast cancer cases were diagnosed. Invasive and sufficient tumor material was available in 248 cases. Pathological reevaluation of histological type and grade was conducted. Using a tissue microarray (TMA), the tumor expression of Ki67, HER2, ERalpha, ERbeta, PgR, cyclin D1 and p27 was evaluated. Six anthropometric factors: height, weight, body mass index (BMI), waist- and hip circumference and body fat percentage were categorized by quartiles of baseline anthropometric measurements, and relative risks were calculated using multivariate Cox regression models. Invasive breast cancer incidence was increased for women in the higher quartiles of all anthropometric measurements. Height was positively associated with Grade I and ERalpha-positive tumors. Women in the highest quartiles of weight, BMI, waist- and hip circumference and body fat percentage were all associated with tumors of ductal type, Grade II, low Ki67 index, HER2 negativity and low expression of the oncogene cyclin D1. Obesity was further associated with tumors expressing ERalpha and PgR but interestingly not ERbeta. This study confirmed previously described associations between overweight/obesity and increased risk of postmenopausal breast cancer. Furthermore, obesity was associated with tumors expressing several markers corresponding with low malignancy.

Immunohistochemical analyses of phosphatases in childhood B-cell lymphoma: lower expression of PTEN and HePTP and higher number of positive cells for nuclear SHP2 in B-cell lymphoma cases compared to controls.

Although many pediatric B-cell lymphoma patients are being cured today, much is still unknown about the pathogenesis of this disease. Protein tyrosine phosphatases are involved in the control of survival, growth, and differentiation of cells. The authors have analyzed 26 pediatric B-cell lymphoma cases for the expression of a panel of phosphatases and report a statistically significant lower expression intensity of PTEN and HePTP and higher nuclear SHP2 expression in B-cell lymphoma cases compared to lymphoid tissue. Knowledge about the expression of key regulatory proteins in pediatric B-cell lymphomas is necessary for revealing the complex molecular background of this disease.

HMG-CoA reductase expression in breast cancer is associated with a less aggressive phenotype and influenced by anthropometric factors.

Although several studies have reported on the anti-tumoural properties exerted by 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoAR) inhibitors (statins), the in vivo expression of HMG-CoAR in human cancer has been considerably less investigated. In our study, we examined the immunohistochemical expression of HMG-CoAR in 511 incident breast cancers within the Malmö Diet and Cancer Study in order to explore its relationship to established clinicopathological and tumour biological parameters. Furthermore, the potential influence of estrogen exposure on HMG-CoAR expression was assessed by performing Cox's proportional hazards analyses of the relationship between the use of hormone replacement therapy (HRT), obesity (waist circumference) and tumour-cell specific HMG-CoAR expression. We found that HMG-CoAR was present in various fractions and intensities in the cytoplasm, sometimes with a membranous pattern, but not in the tumour cell nuclei. The expression of HMG-CoAR was associated with a smaller tumour size (p = 0.02), low histological grade (p = 0.001), low Ki67 index (p = 0.004), ERalpha+ (p = 0.02), ERbeta+ (p = 0.005), and high p27 expression (p = <0.001). The incidence of tumours with a high HMG-CoAR-expression was increased among HRT-users, although this was not statistically significant in a heterogeneity analysis. Obesity was significantly associated with a high HMG-CoAR expression assessed both as a high (>50%) fraction of positive cells (relative risk: 2.06; 95% confidence interval: 1.20-3.51), and a strong staining intensity (2.33: 1.08-5.02). In summary, we demonstrate that HMG-CoAR is differentially expressed in breast cancer and that a high expression is associated with prognostically favourable tumour parameters. Moreover, estrogen related life-style and anthropometric factors might indeed regulate HMG-CoAR expression.

Protein expression and cellular localization in two prognostic subgroups of diffuse large B-cell lymphoma: higher expression of ZAP70 and PKC-beta II in the non-germinal center group and poor survival in patients deficient in nuclear PTEN.

Patients diagnosed with diffuse large B-cell lymphoma (DLBCL) show varying responses to conventional therapy, and this might be contributed to the differentiation stage of the tumor B-cells. The aim of the current study was to evaluate a panel of kinases (ZAP70, PKC-beta I and II and phosphorylated PKB/Akt) and phosphatases (PTEN, SHP1 and SHP2) known to be frequently deregulated in lymphoid malignancies. De novo DLBCL cases were divided into two subgroups, the germinal center (GC) group (14/28) and the non-germinal center (non-GC) or activated B-cell (ABC) group (14/28). ZAP70 and PKC-beta II were expressed in a significantly higher percentage of tumor cells in the clinically more aggressive non-GC group compared with the prognostically favourable GC group. Also, the subcellular localization of PKC-beta I and II differed in DLBCL cells, with the PKC-beta I isoform being expressed in both the cytoplasm and nucleus, while PKC-beta II was found exclusively in the cytoplasm. Loss of nuclear PTEN correlated with poor survival in cases from both subgroups. In addition, five cell lines of DLBCL origin were analyzed for protein expression and for mRNA levels of PTEN and SHP1. For the first time, we show that ZAP70 is expressed in a higher percentage of tumor cells in the aggressive non-GC subgroup of DLBCL and that PKC-beta I and II are differently distributed in the two prognostic subgroups of de novo DLBCL.

Breast tumours following combined hormone replacement therapy express favourable prognostic factors.

The aim of the present study was to evaluate the association between different types of hormone replacement therapy (HRT) and risk of specific breast cancer subgroups. A population-based prospective cohort study including 12,583 peri- or postmenopausal women were followed using record-linkage with national cancer registries. During an average follow-up of 4.5 years, 332 cases of invasive breast cancer were diagnosed. Tumour samples were available from 283 cases. These tumours were re-evaluated according to histological type, grade, and mitotic index. Evaluation of tumours included estrogen and progesterone receptor status (ERalpha, ERbeta and PgR), as well as expression of Ki67, HER2, cyclin D1 and p27. The incidence of breast cancer in current users of combined HRT (CHRT) was significantly higher than in non-users. The difference corresponded to an adjusted relative risk (95% confidence interval) of 3.01 (2.35-3.84) as obtained using a Cox's proportional hazards analysis. CHRT was associated with lobular tumours (3.48:1.99-6.10), grade 1 tumours (4.46:2.79-7.13) and tumours with a low mitotic index (4.35:2.99-6.34). CHRT was not related to any specific subgroup in terms of ERalpha-, ERbeta- or PgR-expression. CHRT was associated with low proliferating tumours, defined by the Ki67 index (3.58:2.60-4.93), HER2 amplified tumours (4.40:1.93-10.06), low expression of the oncogene cyclin D1 (3.14:2.32-4.23) and high expression of the tumour suppressor gene p27 (3.47:2.40-5.01). Use of estrogen-alone HRT (ERT) was not associated with any statistically significant risk of breast cancer. We conclude that the use of CHRT is associated with an increased incidence of breast tumours with comparatively favourable prognostic factors.

Diet and body constitution in relation to subgroups of breast cancer defined by tumour grade, proliferation and key cell cycle regulators.

BACKGROUND: The general lack of clear associations between diet and breast cancer in epidemiological studies may partly be explained by the fact that breast cancer is a heterogeneous disease that may have disparate genetic associations and different aetiological bases. METHOD: A total of 346 incident breast cancers in a prospective cohort of 17,035 women enrolled in the Malmö Diet and Cancer study (Sweden) were subcategorized according to conventional pathology parameters, proliferation and expression of key cell cycle regulators. Subcategories were compared with prediagnostic diet and body measurements using analysis of variance. RESULTS: A large hip circumference and high body mass index were associated with high grade tumours (P = 0.03 and 0.009, respectively), whereas low energy and unadjusted fat intakes were associated with high proliferation (P = 0.03 and 0.004, respectively). Low intakes of saturated, monounsaturated and polyunsaturated fatty acids were also associated with high proliferation (P = 0.02, 0.004 and 0.003, respectively). Low energy and unadjusted fat intakes were associated with cyclin D1 overexpression (P = 0.02 and 0.007, respectively), whereas cyclin E overexpression was positively correlated with fat intake. Oestrogen receptor status and expression of the tumour suppressor gene p27 were not associated with either diet or body constitution. CONCLUSION: Low energy and low total fat (polyunsaturated fatty acids in particular) intakes, and high body mass index were associated with relatively more malignant breast tumours. Dietary behaviours and body constitution may be associated with specific types of breast cancer defined by conventional pathology parameters and cyclin D1 and cyclin E expression. Further studies including healthy control individuals are needed to confirm our results.

Cyclin A1 expression and associations with disease characteristics in childhood acute lymphoblastic leukemia.

A critical cell cycle regulatory protein, cyclin A1, has been implicated in the development of acute myeloid leukemia (AML). Here, we have examined the expression and clinical significance of cyclin A1 in childhood acute lymphoblastic leukemia (ALL). Cyclin A1 was highly expressed in lymphoblastic leukemic cell lines and in 22 of 30 ALL patients (73%). Cyclin A1 expression correlated with patient age (P=0.006), but not with cytogenetic abnormalities. Patients with high levels of cyclin A1 had poorer event-free survival (57.9%) compared to patients with lower levels (75%).

Expression of cyclin A1 and cell cycle proteins in hematopoietic cells and acute myeloid leukemia and links to patient outcome.

Abnormal expression of several key regulators essential for G1/S transitions has been implicated in tumorigenesis. A critical role of cyclin A1 in the development of acute myeloid leukemia (AML) has previously been demonstrated in transgenic mice. Our present study focused on the expression and prognostic significance of cyclin A1 and a panel of cell cycle regulatory proteins including cyclin A2, cyclin B1, cyclin E, CDK1, CDK2, p21 and p27 in bone marrow samples from 40 patients with AML. Freshly isolated CD34+ hematopoietic cells and bone marrow samples from 10 healthy donors were also assessed for cell type- and subcellular-specific expression of the cell cycle regulatory proteins. The level of cyclin A1 expression was the only factor that showed a significant correlation with patient outcome. In log-rank test stratified by levels of cyclin A1 expression, patients with high levels of cyclin A1 had significantly worse overall survival (OS) (P = 0.012) compared to those with low levels. Further, patients with high levels of cyclin A1 had significantly lower disease-free survival (DFS) (P = 0.028). Multivariate analysis indicated that cyclin A1 protein expression was an independent prognostic factor for predicting DFS (P = 0.035) and OS (P = 0.045). No correlation between cyclin A1 expression and age was found. However, expression of cyclin A2, cyclin B1, cyclin E, CDK1, CDK2, p21 and p27 did not show prognostic significance in these AML patients.