Varying outcomes of triple-negative breast cancer in different age groups-prognostic value of clinical features and proliferation.

PURPOSE: Triple-negative breast cancer (TNBC) is an aggressive disease lacking specific biomarkers to guide treatment decisions. We evaluated the combined prognostic impact of clinical features and novel biomarkers of cell cycle-progression in age-dependent subgroups of TNBC patients. METHODS: One hundred forty seven TNBC patients with complete clinical data and up to 18 year follow-up were collected from Turku University Hospital, Finland. Eight biomarkers for cell division were immunohistochemically detected to evaluate their clinical applicability in relation to patient and tumor characteristics. RESULTS: Age at diagnosis was the decisive factor predicting disease-specific mortality in TNBC (p = 0.002). The established prognostic features, nodal status and Ki-67, predicted survival only when combined with age. The outcome and prognostic features differed significantly between age groups, middle-aged patients showing the most favorable outcome. Among young patients, only lack of basal differentiation predicted disease outcome, indicating 4.5-fold mortality risk (p = 0.03). Among patients aged > 57, the established prognostic features predicted disease outcome with up to 3.0-fold mortality risk for tumor size ≥ 2 cm (p = 0.001). Concerning cell proliferation, Ki-67 alone was a significant prognosticator among patients aged > 57 years (p = 0.009). Among the studied cell cycle-specific biomarkers, only geminin predicted disease outcome, indicating up to 6.2-fold increased risk of mortality for tumor size < 2 cm (p = 0.03). CONCLUSION: Traditional clinical features do not provide optimal prognostic characterization for all TNBC patients. Young age should be considered as an additional adverse prognostic feature in therapeutic considerations. Increased proliferation, as evaluated using Ki-67 or geminin immunohistochemistry, showed potential in detecting survival differences in subgroups of TNBC.

Tumor-infiltrating lymphocytes and CD8+ T cells predict survival of triple-negative breast cancer.

PURPOSE: Tumor inflammatory response was evaluated as a prognostic feature in triple-negative breast cancer (TNBC) and compared with the clinical prognosticators of breast cancer and selected biomarkers of cancer cell proliferation. METHODS: TNBC patients (n = 179) with complete clinical data and up to 18-year follow-up were obtained from Auria biobank, Turku University Hospital, Turku, Finland. Tumor-infiltrating lymphocytes (TILs) and several subtypes of inflammatory cells detected with immunohistochemistry were evaluated in different tumor compartments in full tissue sections and tissue microarrays. RESULTS: Deficiency of stromal TILs and low number of CD8+ T cells independently predicted mortality in TNBC (HR 2.4, p 0.02 and HR 2.1, p 0.02, respectively). Each 10% decrease in stromal TILs resulted in 20% increased risk of mortality. An average of 13.2-year survival difference was observed between the majority (> 75%) of patients with low (< 14% of TILs) vs high (≥ 14% of TILs) frequency of CD8+ T cells. The prognostic value of TILs and CD8+ T cells varied when evaluated in different tumor compartments. TILs and CD8+ T cells were significantly associated with Securin and Separase, essential regulators of metaphase-anaphase transition of the cell cycle. DISCUSSION: TILs and CD8+ T cells provide additional prognostic value to the established clinical prognostic markers in TNBC. However, possible clinical applications would still benefit from systematic guidelines for evaluating tumor inflammatory response. Increasing understanding on the interactions between the regulation of cancer cell proliferation and inflammatory response may in future advance treatment of TNBC.

Prognostic implications of securin expression and sub-cellular localization in human breast cancer.

PURPOSE: Securin belongs to a class of cell cycle regulators that prevent metaphase-to-anaphase transition until sister chromatid separation is complete. Evidence is accumulating that securin has a prognostic impact on a variety of malignancies but, thus far, the role and regulation of securin expression and its sub-cellular localization have not been systematically addressed in breast cancer. METHODS: In total 470 breast cancer specimens with follow-up data for up to 22 years were included. Immunohistochemical staining and immunofluorescence double-staining were performed for securin and its regulating proteins PTTG1IP, CDC20 and BUBR1. Prognostic associations were evaluated between the expression patterns of these proteins and established prognosticators of invasive breast cancer and patient survival. RESULTS: We found that a high fraction of securin expressing cancer cells predicted an unfavorable clinical outcome of the breast cancer patients (p < 0.001). Also in multivariate analyses, the fraction of securin expressing cancer cells served as an independent prognosticator of a poor survival (p < 0.0001). We also found that the sub-cellular localization of securin exhibited prognostic power, since cytoplasmic securin expression in the cancer cells appeared to be associated with aggressive breast cancer subtypes and high breast cancer-associated mortality rates (p = 0.003). Through immunofluorescence double-staining, we found that PTTG1IP, CDC20 and BUBR1 exhibited distinct patterns of co-expression with securin, suggesting a regulatory role in the metaphase-to-anaphase transition in human breast cancer cells. We also noted that a subgroup of triple-negative breast carcinomas exhibited deviant expression patterns for the proteins studied. CONCLUSIONS: Our data indicate that securin expression may serve as a strong and independent prognosticator of breast cancer outcome and that a cytoplasmic localization of the protein may provide additional prognostic information, particularly in the biologically and clinically challenging subgroup of triple-negative breast carcinomas. The sub-cellular localization of securin appears to reflect the expression of PTTG1IP, CDC20 and BUBR1, which may participate in the regulation of securin activity and, ultimately, in the survival of breast cancer patients.

Altered expression of p120catenin predicts poor outcome in invasive breast cancer.

PURPOSE: The study focuses on p120catenin, a regulator of cell adhesion, which has previously been described in many malignancies and suggested with a role in invasion and metastatic behaviour. In this study, we investigate the role of altered immunoexpression of p120catenin isoforms in the prognosis of invasive breast cancer (n = 351). METHODS: We used cDNA microarrays to screen differences in gene expression in invasive breast cancer in general, and between local and metastasized disease particularly. On this basis, we performed p120catenin immunohistochemistry in order to confirm the prognostic value of p120catenin isoforms on tissue microarrays comprising 341 patients from the era of mammographic screening, directed to modern surgical and oncological treatments, and followed-up for maximum of 20 years. RESULTS: In cDNA microarray analysis, p120catenin was discovered down-regulated along with E-cadherin and alpha-catenin. In addition, p120catenin distinguished metastasized breast cancer from local disease. Immunohistochemistry confirmed the value of p120catenin as an independent prognosticator of breast cancer survival. In our results, p120catenin was associated with 3.7-fold risk of breast cancer death in multivariate Cox's regression analyses adjusted for the established prognosticators of breast cancer (p = 0.039). Particularly, the long isoform of p120catenin predicted metastatic disease (p = 0.029). CONCLUSION: The present paper is the first report on p120catenin in invasive breast cancer based on a well-characterized patient material with long-term follow-up. We observed altered expression of p120catenin isoforms in invasive breast cancer and, in our material, the decrease in p120 immunoexpression was significantly associated with poor outcome of disease.

Securin promotes the identification of favourable outcome in invasive breast cancer.

BACKGROUND: Securin is a recently recognised oncogene with multiple known functions in initiation, progression and cell cycle regulation in several malignant diseases, including breast carcinoma. METHODS: In this paper, the prognostic value of securin is evaluated by immunohistochemistry in 310 patients diagnosed with invasive breast cancer during a mammographic screening programme in Central Finland. All patients were directed to modern surgical and oncological treatments and were followed up for a maximum of 20 years. RESULTS: Our results suggest that securin immunopositivity is an independent prognosticator of invasive breast cancer. In our study, securin predicted breast cancer-specific survival among all cases of invasive breast cancer and subgroups divided according to histological type, Ki-67 proliferation status and tumour size. Especially in a multivariate analysis standardised for axillary lymph node status, patient's age and tumour size at the time of diagnosis, securin immunopositivity indicated a 13.1-fold risk of breast cancer death (P=0.024) among invasive ductal breast carcinomas with low Ki-67 positivity. CONCLUSION: Our present and previous results suggest that securin could be useful in clinical pathology to intensify the power of the established prognosticators of invasive breast cancer and, especially, to assist in identifying patients with a more favourable outcome than that indicated by Ki-67 alone.

Expression of carbonic anhydrase IX suggests poor outcome in rectal cancer.

The aim of the study is to assess the value of carbonic anhydrase isozyme IX (CA IX) expression as a predictor of disease-free survival (DFS) and disease-specific survival (DSS) in rectal cancer treated by preoperative radio- or chemoradiotherapy or surgery only. Archival tumour samples from 166 patients were analysed for CA IX expression by three different evaluations: positive/negative, proportion of positivity and staining intensity. The results of immunohistochemical analysis were confirmed by demonstrating CA IX protein in western blotting analysis. Forty-four percent of the operative samples were CA IX positive, of these 34% had weak and 66% moderate/strong staining intensity. In univariate survival analysis, intensity of CA IX expression was a predictor of DFS (P=0.003) and DSS (P=0.034), both being markedly longer in tumours with negative or weakly positive staining. In multivariate Cox model, number of metastatic lymph nodes and CA IX intensity were the only independent predictors of DFS. Carbonic anhydrase isozyme IX intensity was the only independent predictor of DSS, with HR=9.2 for dying of disease with moderate-intense CA IX expression as compared with CA IX-negative/weak cases. Negative/weak CA IX staining intensity is an independent predictor of longer DFS and DSS in rectal cancer.

Proliferation marker securin identifies favourable outcome in invasive ductal breast cancer.

We introduce a new proliferation marker, securin (pituitary tumour-transforming 1 (PTTG1)), analysed in invasive ductal breast carcinomas by cDNA microarrays and immunohistochemistry. In cDNA microarray of a total of 4000 probes of genes, securin was revealed with a significant change in expression among the several proliferation-related genes studied. The value of securin as a proliferation marker was verified immunohistochemically (n=44) in invasive ductal breast cancer. In follow-up analyses of the sample of patients, the prognostic value of securin was compared with the established markers of breast cancer proliferation, Ki-67 and mitotic activity index (MAI). Our results of a small sample of patients suggest that low securin expression identifies a distinct subgroup of more favourable outcome among patients with high Ki-67 immunoexpression or high MAI. In univariate analysis of Cox's regression, 10-unit increment of securin immunopositivity was associated with a 2.3-fold overall risk of death due to breast cancer and a 7.1-fold risk of death due to breast cancer in the sample of patients stratified according to the cutoff points of 10 and 20% of securin immunopositivity. We suggest that securin immunostaining is a promising and clinically applicable proliferation marker. The finding urges further prognostic studies with a large sample of patients.

Zaspopathy in a large classic late-onset distal myopathy family.

Distal myopathies have been associated with mutations in titin, dysferlin, GNE, desmin and myosin. Of these, only titin mutations were previously known to cause dominant late-onset distal myopathy. Recent findings, however, have indicated that patients affected with myofibrillar myopathy have a more distal than proximal muscle phenotype and a proportion of these may have mutations in myotilin, ZASP or filamin C, besides previously known desmin and alphaB-crystallin. Here we report that the disorder in one of the well-characterized autosomal dominant distal myopathy families, the Markesbery et al. family, first reported in 1974, is caused by ZASP mutation A165V. Previous linkage to the titin locus 2q31 proved incorrect. ZASP expression by immunoblotting shows normal presence of the main 32 and 78 kDa bands and immunohistochemistry in patients reveals normal Z-disc localization except for moderate accumulations together with myotilin, desmin alphaB-crystallin and alpha-actinin. Muscle imaging reveals involvement in both the posterior and anterior compartments of the lower leg and considerable affection of proximal leg muscles at later stages. Haplotype studies in this family and in five other unrelated families with European ancestry carrying the identical A165V mutation share common markers at the locus suggesting the existence of a founder mutation.

Expression of group II phospholipase A2 in the liver in acute pancreatitis.

BACKGROUND: A number of distinct secretory phospholipases A2 (PLA2) have been characterized in the human. Elevated group II PLA2 serum levels are associated with inflammatory diseases such as infections, septic shock, rheumatoid arthritis, multiple organ failure and acute pancreatitis. The cellular source of circulating group II PLA2 has not been defined unequivocally. The possible role of the liver as a source of circulating group II PLA2 in acute pancreatitis was studied using liver biopsies from five patients operated on for necrotizing acute pancreatitis and from two control liver samples. METHODS: Reverse transcription polymerase chain reaction (RT PCR), northern hybridization and in situ hybridization were used to study the expression of group II PLA2. Immunohistochemistry was used to study the localization of the group II PLA2 protein in liver cells and time-resolved fluoroimmunoassay to measure the plasma group II PLA2 content. RESULTS: Expression of group II PLA2 was found in the livers of patients with acute pancreatitis by RT PCR and confirmed by northern hybridization. Group II PLA2 mRNA was localized in hepatocytes by in situ hybridization. Faint immunopositivity was found in Kupffer cells. Time-resolved fluoroimmunoassay revealed elevated concentration of group II PLA2 in plasma samples. Only low levels of expression were found in the control livers. CONCLUSIONS: Group II PLA2 is expressed in the livers of patients suffering from acute pancreatitis but not in the livers of patients without pancreatic disease. The current results support the idea that hepatocytes are an important source of circulating group II PLA2 in inflammatory diseases.

Baseline diene conjugation in LDL lipids as a direct measure of in vivo LDL oxidation.

OBJECTIVES: To additionally test validity of the recently developed method (LDL baseline diene conjugation, LDL-BDC) for determination of circulating oxidized LDL. DESIGN AND METHODS: A detailed comparison between the ultracentrifugation and heparin precipitation methods for LDL isolation was performed to test suitability of the fast precipitation method. Validity of LDL-BDC as an indicator of circulating oxidized LDL was tested by comparing LDL-BDC to results obtained by the immunological autoantibody method. RESULTS: BDC values in LDL isolated by heparin precipitation did not differ from those isolated by sequential ultracentrifugation. While highest amount of diene conjugation was found in LDL (40% of that in serum), substantial amounts were also found in VLDL (31%) and HDL (25%). When analyzed in the same samples, assays for the titer of autoantibodies against oxidized LDL and LDL-BDC were found to show good correlation (r = 0.57, p = 0.001, n = 29). CONCLUSIONS: These results, together with thus far conducted studies on clinical applicability of the method, indicate that LDL-BDC is a promising candidate in search for a method for estimation of LDL oxidation in vivo.