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Background: Carnitine palmitoyl transferase 1A (CPT1A), the key regulator of fatty acid oxidation, contributes to tumor metastasis and therapeutic resistance. We aimed to identify its clinical significance as a biomarker for the diagnosis and prediction of breast cancer., Methods: Western blot, ELISA and in silico analysis were used to confirm CPT1A levels in breast cancer cell lines, cell culture medium and breast cancer tissues. Four hundred thirty breast cancer patients, 200 patients with benign breast disease, and 400 healthy controls were enrolled and randomly divided into a training set and a test set with a 7:3 ratio. Training set was used to build diagnostic models and 10-fold cross validation was used to demonstrate the performance of the models. Then test set was aimed to validate the effectiveness of the diagnostic models. ELISA was conducted to detect individual serum CPT1A levels. Receiver operating characteristic (ROC) curves were generated, and binary logistic regression analyses were performed to evaluate the effectiveness of CPT1A as a biomarker in breast cancer diagnosis. CPT1A levels between post-operative and pre-operative samples were also compared., Results: CPT1A was overexpressed in breast cancer tissues, cell lines and cell culture medium. Serum CPT1A levels were higher in breast cancer patients than in controls and were significantly associated with metastasis, TNM stage, histological grading and molecular subtype. CPT1A levels were decreased in post-operative samples compared with paired pre-operative samples. Moreover, CPT1A exhibited a higher efficacy in differentiating breast cancer patients from healthy controls (training set: area under the curve, AUC, 0.892, 95% CI, 0.872-0.920; test set, AUC, 0.904, 95% CI, 0.869-0.939) than did CA15-3, CEA, or CA125., Conclusion: CPT1A is overexpressed in breast cancer and can be secreted out of breast cancer cell. Serum CPT1A is positively associated with breast cancer progression and could serve as an indicator for disease monitoring. Serum CPT1A displayed a remarkably high diagnostic efficiency for breast cancer and could be a novel biomarker for the diagnosis of breast cancer.

Originally described as a systemic self-limiting disease in infancy, the spectrum of ALK-positive histiocytosis has recently been broadened to include localized diseases in older children and young adults. Despite different manifestations, these tumors share histologic characteristics and a highly recurrent KIF5B-ALK fusion. ALK-positive histiocytosis is poorly characterized in the breast. In this study, we report 3 cases of ALK-positive histiocytosis of the breast. The patients were Asian women, aged 16 to 45 years. Two patients presented with an isolated breast mass, while 1 exhibited multiorgan involvement. The latter patient received ALK inhibitor after surgery, which led to complete remission. Histologically, well-circumscribed tumors displayed fascicular and storiform growth of uniform, nonatypical spindle cells admixed with lymphocytic infiltrates. Fewer conventional epithelioid histiocytes with lobulated or clefted nuclei were observed within the same breast tumors in 2 cases or within a concomitant brain tumor in the third case. Touton-type giant cells were focally present in 2 cases. Immunohistochemically, tumor spindle, and epithelioid cells were diffusely positive for CD163 and ALK in all cases and focally positive for S100 protein in 1 of the cases. CD1a and langerin were negative. Actin-positive myofibroblasts were admixed within the tumor in 2 cases, and their reactive nature was highlighted using double immunostaining. Break-apart fluorescence in situ hybridization assay demonstrated gene rearrangements involving KIF5B and ALK in all the 3 cases. ALK-positive histiocytosis rarely occurs as a spindle cell breast tumor, and should be distinguished from other diseases such as inflammatory myofibroblastic tumors and spindled histiocytic reaction., Competing Interests: Conflicts of Interest and Source of Funding: Supported in part by JSPS Grant-in-Aid for Young Scientists 18K15108 (A.Y.). K.I. received research grants and honoraria from Chugai Pharmaceutical Co. Ltd that are not related to this study. K.Y. served as an advisor for Chugai Pharmaceutical Co. Ltd, but not related to this study. For the remaining authors none were declared., (Copyright © 2020 Wolters Kluwer Health, Inc. All rights reserved.)

Matrix metalloproteinase-9 (MMP-9) and heparanase (HPSE) are thought to be involved in tumor progression and metastasis. However, up to now, there are no studies that simultaneously investigated the expression levels of MMP-9 and HPSE in tumor tissue and serum of breast cancer patients. Their correlation in breast cancer pathological processes is unknown. The purpose of this study was to investigate the expression profile of MMP-9 and HPSE in breast cancer and to assess their clinicopathological significance. We measured serum MMP-9 and HPSE by enzyme-linked immunosorbent assay in healthy women, and in patients with benign and malignant breast disease. We also evaluated the expression of MMP-9 and HPSE protein in paraffin-embedded tumor tissues by immunohistochemistry. We then correlated serum and tissue levels of MMP-9 and HPSE in breast cancer samples and their expression with patients' clinicopathologic characteristics. We found that serum levels of MMP-9 and HPSE were significantly higher in breast cancer patients than in benign breast disease and in healthy controls (P = 0.001). There was positive correlation between MMP-9 and HPSE in breast cancer patients. The tissue and serum levels of MMP-9 were associated with histology grade, lymph node status, pathological stage, and lymphovascular invasion (all P < 0.05). The tissue levels of MMP-9 were also associated with ER (P = 0.038) and Ki-67 (P = 0.032). The tissue and serum levels of HPSE expression were associated with tumor size, histology grade, lymph node status, and pathological stage (all P < 0.05). Our findings suggested that MMP-9 and HPSE might further be evaluated as biomarkers for predicting progression and prognosis of breast cancer.

Inflammatory myofibroblastic tumours (IMTs), also known as inflammatory pseudotumours, include a diverse group of lesions characterised by inflammatory cell infiltration and variable fibrotic responses. Their occurrence in the breast is unusual. We present a case of an IMT of the breast in a 46-year-old woman who complained of a breast mass with palpable axillary lymph node. The initial clinical diagnosis was breast cancer, and the patient underwent a conservative excision with apparently negative margins and an axillary lymph node excisional biopsy. A histopathological examination showed the presence of myofibroblastic spindle cells with mixed inflammatory infiltrates, and the pathological diagnosis was IMT. Significantly, the case we present here is unique in showing anaplastic lymphoma kinase 1 (ALK1) overexpression and ALK1 gene amplification in IMT of the breast. Therefore, our case suggests that ALK1 gene amplification in IMT of the breast has important diagnostic and therapeutic implications.

The phosphatidylinositol-3-kinase pathway is one of the most commonly mutated pathways in invasive breast carcinoma, with PIK3CA mutations in ∼25% of invasive carcinomas, and AKT1 mutations in up to 5%. Ductal carcinoma in situ, and benign papillomas harbor similar mutations. However, activating point mutations in breast columnar cell lesions have been infrequently studied. Twenty-three breast resection specimens containing columnar cell lesions were identified; 14 with associated invasive carcinoma or carcinoma in situ. DNA extracts were prepared from formalin-fixed paraffin-embedded tissue and screened for a panel of point mutations (321 mutations in 30 genes) using a multiplex PCR panel with mass-spectroscopy readout. PIK3CA mutations were identified in 13/24 columnar cell lesions (54%) and 3/8 invasive carcinomas (37%). The mutation status of columnar cell lesions and associated carcinoma was frequently discordant. Of the 14 cases, only 5 demonstrated the same genotype in matched samples of columnar cell lesions and carcinoma (4 wild type, 1 PIK3CA H1047R). Interestingly, five patients had mutations in columnar cell lesions with wild-type carcinoma; two patients had different point mutations in columnar cell lesions and carcinoma. Only three cases had wild-type columnar cell lesion and mutated carcinoma. The 50% PIK3CA mutation prevalence in columnar cell lesions is greater than reported in most studies of invasive breast cancer. Further, columnar cell lesions and carcinoma were frequently discordant for PIK3CA/AKT1 mutation status. These findings raise interesting questions about the role of PIK3CA/AKT pathway in breast carcinogenesis, and the biologic/precursor potential of columnar cell lesions.

It is well known that estrogens play important roles in the cell proliferation of breast carcinoma. Benign breast disease (BBD) contains a wide spectrum of diseases, and some are considered an important risk factor for subsequent breast carcinoma development. However, the significance of estrogens in BBD has remained largely unknown. Therefore, in this study, we examined tissue concentrations of estrogens and immunolocalization of estrogen-producing/metabolizing enzymes in BBD, and compared these findings with those in the normal breast and ductal carcinoma in situ (DCIS). Tissue concentration of estradiol in BBD (n = 9) was significantly (3.4-fold) higher than normal breast (n = 9) and nearly the same (0.7-fold) as in DCIS (n = 9). Immunoreactivity of estrogen sulfotransferase in BBD was significantly lower (n = 82) than normal breast (n = 28) but was not significantly different from DCIS (n = 28). Aromatase and steroid sulfatase immunoreactivities tended to be higher (P = 0.07) in BBD than in normal breast, and 17β-hydroxysteroid dehydrogenase type 1 immunoreactivity was significantly higher in BBD than normal breast in the postmenopausal tissues. Immunoreactivity of estrogen and progesterone receptors was also significantly higher in BBD than normal breast. These results suggest that tissue concentration of estradiol is increased in BBD at a level similar to DCIS, which is considered mainly due to loss of estrogen sulfotransferase expression. Increased local estradiol concentration in BBD due to aberrant expression of estrogen-producing/metabolizing enzymes may play important roles in the accumulation of estradiol-mediated growth and/or subsequent development of breast carcinoma., (© 2010 Japanese Cancer Association.)

The objective was to investigate the changes occurring in the activities of the enzymes lactate dehydrogenase (LDH), alkaline phosphatase (ALP) and aspartate aminotransferase (AST) in sheep and goat milk as a result of subclinical intramammary infections (IMI) and to evaluate the use of these enzymes for the diagnosis of subclinical IMI in dairy sheep and goats. A total of 206 samples of sheep milk and 162 samples of goat milk, obtained from equal udder halves, were used in the study. For each species they were divided into two groups: a no-infection group and a subclinical infection group. Activities of LDH, ALP and AST were significantly higher in the subclinical infection group than in the no-infection group (P<0.05) in both sheep (LDH: 350.42+/-11.25 v. 120.91+/-4.41; ALP: 2773.43+/-105.18 v. 2189+/-94.24; AST: 29.57+/-0.74 v. 17.32+/-0.46) and goats (LDH: 354.07+/-13.33 v. 103.79+/-3.75; ALP: 311.13+/-25.74 v. 137.24+/-19.62; AST: 27.59+/-6.42 v. 15.87+/-0.45). The activity of LDH was identified as indicator for subclinical IMI in both sheep and goats. The optimum cut-off values for LDH activity, offering the highest diagnostic sensitivity (DSn) and diagnostic specificity (DSp), determined by receiver operating characteristic (ROC) analysis, were at 197 U/l, 185 U/l and 197 U/l for sheep, goats and both species, respectively. DSn for sheep, goats and both species at these cut-off values was 92.8%, 98.2% and 94.0%, whereas DSp was 95.4%, 96.3% and 96.3%, respectively. It was concluded that the determination of LDH activity in milk serum is a sensitive and reliable method for the detection of subclinical IMI in dairy sheep and goats.

Cathepsin-D (CathD) is an aspartyl lysosomal protease expressed in all tissues that might play a role in antigen processing, cell proliferation and tissue renewal, and activation of different pro hormones. The aim of our study was to compare the expression of CathD in most common breast tumors and tumor-like breast lesions. The study includes 21 patients with histologically verified breast lesions (adenosis, ductal hyperplasia, fibroadenomas, and different types of invasive carcinoma). We investigated the cathepsin-D expression in these breast lesions using immunohistochemistry (IH; paraffin-embedded tissues). Cathepsin-D staining within each lesion was assessed by estimating the area of the objects and the medium pixel intensity per object, as the integrated optical density (IOD). The immunostaining was more obvious in breast invasive carcinomas and macrophages. The reaction in tumor tissue was heterogeneous with little variation of staining intensity in positive tumor cells. Adenosis had the maximum area/signal intensity from all studied breast benign lesions (p<0.001, Student t-test). The general tendency (all benign lesions, lobular carcinomas and G3 ductal invasive carcinoma) was a more prominent representation of the cellular compartment. In the G3 ductal invasive carcinoma-type, the group of patients with metastases had a stronger expression in the cellular compartment. These results suggest that CathD expression was strongest in malignant than in benign breast disease, the positivity being present in both epithelial neoplastic and stromal cells. We also conclude that our procedure in IOD measurement is prone to less subjective-related biases, and thus more accurate and constant than other methods employed by other authors.

Tumor progression and metastasis contribute to the great majority of breast cancer deaths. Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) are thought to be involved in tumor progression and metastasis. Thus, we determined whether the expression of MMP-9 and TIMP-1 is associated with prognosis in breast cancer patients. We measured serum MMP-9 and TIMP-1 by enzyme-linked immunosorbent assay in 60 breast cancer patients, 18 benign breast disease patients and 15 healthy controls. We also evaluated the expression of MMP-9 and TIMP-1 protein and mRNA in paraffin-embedded tumor tissues from the 60 breast cancer patients by immunohistochemistry and in situ hybridization. We then correlated serum and tissue levels of MMP-9 and TIMP-1 in breast cancer samples and their expression with patients' clinicopathologic characteristics. We found that serum levels of MMP-9 and TIMP-1 were significantly higher in breast cancer patients than in benign breast disease and in healthy controls. High serum levels of MMP-9 and TIMP-1 were associated with lymph node metastasis, higher tumor stage and lower relapse-free and overall survival (OS) rates. Compared to low expression, high tissue expression of MMP-9 protein was associated with lymph node metastasis and higher tumor stage; and high tissue expression of TIMP-1 was associated with a lower OS rate. Our findings suggest that MMP-9 and TIMP-1 may further be evaluated as biomarkers for predicting progression and prognosis of breast cancer., ((c) 2008 Wiley-Liss, Inc.)

Cytochrome P450 1B1 (CYP1B1) and catechol-O-methyltransferase (COMT) are important estrogen-metabolizing enzymes that may affect breast cancer risk. Few studies have directly measured the expression of CYP1B1 and COMT genes in breast tissue samples. The subjects in this study were a subgroup of participants of the Shanghai Breast Cancer Study including 64 patients diagnosed with breast cancer and 68 patients diagnosed with benign breast diseases (BBD) who provided samples of tumor tissue and adjacent nontumor tissue to the study. We compared CYP1B1 and COMT mRNA expression in tumor tissue and adjacent nontumor tissue in both breast cancer patients and BBD patients. High levels of CYP1B1 expression and low levels of COMT expression in adjacent nontumor tissue were associated with a significantly increased breast cancer risk in a nonlinear manner. Odds ratios and 95% confidence intervals (in parentheses) for the midpoints of the first, second, fourth, and fifth quintiles of gene expression levels compared with the overall median levels in BBD subjects were 0.21 (0.07-0.67), 0.81 (0.69-0.95), 1.20 (1.05-1.38), and 1.55 (1.12-2.15) for CYP1B1 and 1.72 (1.17-2.55), 1.19 (1.05-1.35), 0.83 (0.73-0.95), and 0.78 (0.65-0.93) for COMT, respectively. These results support the hypothesis that the formation and accumulation of catechol estrogens in breast tissue through increased CYP1B1 expression and reduced COMT expression may play a significant role in breast cancer risk.

Matrix metalloproteinase (MMP) 2 and 9 are involved in cancer invasion and metastasis, and increased levels occur in serum and plasma of breast cancer (BC) patients. It is, however, unclear whether changes in serum levels can be exploited for early detection or classification of patients into different risk/disease categories. In our study, we measured concentration and activity of MMP2/9 in sera of 345 donors classified as low risk (Gail score <1.7), high risk (HR) (Gail score > or =1.7), benign disease or BC. Kruskal-Wallis and Mann-Whitney nonparametric tests showed that total-MMP2 concentration is higher in HR compared to control (p = 0.012), benign (p = 0.001) and cancer (p = 0.007). Active MMP2 (aMMP2) concentration is higher in control than benign and cancer (p < 0.001, respectively). Total and aMMP9 concentrations are higher in cancer than benign (p < 0.001, p = 0.002, respectively). Total-MMP2 and total-MMP9 activities are lower in control than benign (p < 0.001, p = 0.002, respectively) and cancer (p < 0.001, respectively). Total-MMP2 and MMP9 activities are also higher in cancer than benign (p = 0.004, p < 0.001) and HR (p = 0.008, p = 0.007, respectively). These results were not affected by age or inclusion/exclusion of donors with noninvasive cancer or atypical hyperplasia. Linear discriminant analysis revealed that HR donors are characterized by lower total-MMP2 and higher aMMP2. Overall group classification accuracy was 64.5%. Independent validation based on the leave-one-out cross validation approach gave an overall classification of 63%. Our study provides evidence supporting the potential role of serum MMP2/9 as biomarkers for breast disease classification.

Mucin O-glycosylation is characterized in cancer by aberrant expression of immature carbohydrate structures (Tn, T, and sialyl-Tn antigens). The UDP-N-acetyl-D-galactosamine: polypeptide N-acetylgalactosaminyltransferases (ppGalNAc-T) family enzymes regulate the initial steps of mucin O-glycosylation and could be responsible for the altered glycosylation observed in cancer. Considering that we recently found the ppGalNAc-T6 mRNA expressed in breast carcinomas, we produced a highly specific monoclonal antibody (MAb T6.3) to assess the expression profile of ppGalNAc-T6 protein product in breast tissues. The expression of ppGalNAc-T6 by breast carcinoma cells was confirmed on MCF-7 and T47D cell lines. In formalin-fixed tissues, ppGalNAc-T6 expression was observed in 60/74 (81%) breast cancers, 21/23 (91.3%) adjacent ductal carcinoma in situ (DCIS), 4/20 benign breast lesions (2/2 sclerosing adenosis and 2/13 fibroadenoma), and in 0/5 normal breast samples. We observed a statistically significant association of ppGalNAc-T6 expression with T1 tumor stage. This fact, as well as the observation that ppGalNAc-T6 was strongly expressed in sclerosing adenosis and in most DCIS, suggests that ppGalNAc-T6 expression could be an early event during human breast carcinogenesis. Considering that an abnormal O-glycosylation greatly contributes to the phenotype and biology of breast cancer cells, ppGalNAc-T6 expression could provide new insights about breast cancer glycobiology.

Matrix metalloproteinases (MMPs) are involved in extracellular matrix modification and associated with invasive and metastatic behavior of human malignant tumors. Specifically, MMP2 and MMP9 are implicated in both early and late processes of tumor development. It is reported that MMPs occur as inactive precursors, active enzymes or enzyme inhibitor complexes in biological samples. However, there is limited knowledge on the role of each form in disease and/or the significance of changes in the plasma concentration and/or activity in breast cancer patients. The aim of this study was to determine if patients with breast cancer, benign disease and at risk for developing breast cancer display characteristic levels of active and/or total MMP2 and MMP9 in plasma. Concentration and activity of MMP2 and MMP9 were determined quantitatively in the plasma of 124 female volunteers diagnosed with breast cancer (n=31), benign disease (n=38), or determined by the Gail Model to be at high risk (n=31) or low risk (controls, n=24) of developing breast cancer. Data obtained was statistically analyzed to search for differences/patterns characteristic of each category. Concentration of total MMP2 was significantly lower in control individuals than benign, high risk (P<0.001 respectively) and breast cancer patients (P=0.002). Activity of total MMP2 was significantly lower in controls compared to cancer, benign and high risk patients (P<0.001 respectively). Attempts to build a predictive/descriptive model using canonical discriminant analysis (utilizing all eight features; concentrations and activity levels of active/total MMP2 and MMP9) enabled the distinction of the controls from the high risk, benign and cancer groups. Our results suggest that preoperative plasma concentration and activity of MMP2 and MMP9 may permit sub-classification of female patients with breast disorders.

Focal adhesion kinase (FAK) is a protein tyrosine kinase that is overexpressed in a subset of invasive breast cancers. FAK transmits signals that mediate several functions including tumor cell proliferation, migration, adhesion and survival. We used immunohistochemical techniques to assess FAK expression in patients with fibrocystic disease (FCD), atypical ductal hyperplasia (ADH), ductal carcinoma in situ (DCIS) and infiltrating ductal carcinoma (IDC). Formalin-fixed, paraffin-embedded (FFPE) tissue sections were obtained from 119 patients (12 FCD, 38 ADH, 51 DCIS and 18 IDC). The anti-FAK 4.47 monoclonal antibody was used to detect FAK expression. FAK expression was scored as high (3 or 4 intensity and > or =90% positive cells) or low. The DCIS tissue sections demonstrated high FAK expression in 34/51 (66%) of the sections. High FAK expression was demonstrated in 6/18 (33%) of the IDC tissue sections and 8/38 (21%) of the ADH tissue sections. None (0/12) of the FCD tissues sections stained high for FAK. The pattern of FAK expression in DCIS was significantly higher than ADH (p < 0.0001) and IDC (p = 0.02). We conclude that FAK overexpression in preinvasive, DCIS tumors precedes tumor cell invasion or metastasis, suggesting that FAK may function as a survival signal and be an early event in breast tumorigenesis.

Background & Objective: Telomerase is a ribonucleoprotein enzyme, which plays an important role in cell immortalization and carcinogenesis. Recent studies on the association of telomerase activity with prognostic factors of breast cancer were controversial due to different methods. This study was to establish a feasible assay of quantitative detection of telomerase activity based on silver staining, and investigate possible association between telomerase activity and clinicopathological prognostic factors in primary breast cancer., Methods: Highly sensitive silver-staining telomeric repeat amplification protocol assay (SS-TRAP) was used to quantify telomerase activity in 52 frozen human breast cancer samples and their adjacent breast tissues, 32 benign lesions, and 14 normal mammary gland lesions. The association between telomerase activity and clinicopathological data was analyzed., Results: Telomerase activity was detected in 47 of the 52 breast cancer samples (90.38%), and in 10 of the 32 benign lesions, whereas no activity was detected in 37 of 52 adjacent nonmalignant breast tissues, and all 14 normal mammary gland tissues. The telomerase activity levels were 36.91+/-15.35, 8.27+/-4.37, 14.10+/-5.28, 0 (unit: TPG) in breast cancer, adjacent tissue of cancer, benign lesion, and normal tissue, respectively. The difference of telomerase activity was significant between breast cancer and the other 3 groups by using ANVOA (all P< 0.01). A strong correlation was found between telomerase activity and pathological category, and differentiation degree by logistic regression analysis, i.e. with ongoing tumor progression, telomerase activity appeared to increase in primary breast cancer (P=0.003, and P=0.004). No correlation was seen between telomerase activity and disease course, age, and menopause status of patients (all P >0.05). Telomerase activity level of invasive non-special cancer was higher than that of invasive special cancer(P< 0.05). Telomerase activity level of moderately/poorly differentiated carcer was higher than that of highly differentiated cancer (P< 0.05), while no obvious difference was found between moderately and poorly differentiated cancer (P >0.05)., Conclusions: The activation of telomerase might occur early in breast cancer,and plays a critical role in carcinogenesis and tumor development. Telomerase may serve as a specific marker of early diagnosis and prognosis in mammary gland neoplasm.

Apoptosis is of important significance in the pathogenesis of cancer. Many methods are available for the measurement of apoptosis but the 'gold standard' is to identify apoptotic cells by their morphological features using microscopy. Caspase 3 is a cytosolic enzyme that is activated only in cells committed to undergo apoptosis. The activation of caspase 3 precedes the development of the classical morphological features of apoptosis. Using immunohistochemistry with an antibody against the active form of caspase 3, the apoptotic index (AI) was measured in 116 samples of human breast tissue (22 normal/benign and 94 invasive carcinomas). The AI obtained by measuring caspase activation has a strong correlation with the AI derived by morphological assessment (r = 0.736, P < 0.01). The AI is higher in the invasive group than in the benign group (P = 0.008), and in invasive cancer high AI is associated with high tumour grade (P = 0.013), positive node status (P < 0.001) and negative steroid receptor status (P = 0.001 for ER; P = 0.004 for PR). No significant association is observed between AI and tumour size. Measurement of apoptosis by immunohistochemistry using an antibody against the active form of caspase 3 is therefore reliable and correlates strongly with morphological assessment.

Telomerase plays an important role in maintaining the stability of chromosomes. This ribonucleoprotein prevents chromosome ends (telomeres) from gradual loss with each cell division. It enables tumor cells to maintain telomere length, allowing indefinite replicative capacity. Telomerase activity has been detected in the majority of tumor and germ cells and in immortalized cell lines. Quantitative telomerase PCR-ELISA (TeloTAGGG Telomerase PCR ELISA(PLUS)) was evaluated for distinguishing benign and malignant breast tissue. Activity of telomerase was determined in 27 samples of fibrocystic and dysplastic tissues, 28 fibroadenomas and phylloid tumors, and 154 breast cancer tissues; 59 specimens were analyzed retrospectively. Analytical precision and linearity of the assay was tested using breast carcinoma cell line ZR-75-1 and breast tumor tissue extracts. About 4% of tumor samples were excluded from analysis due to interferences in the PCR reaction. Relative telomerase activity differed significantly in the groups of dysplastic tissues, fibroadenomas and carcinomas. The highest activity was found in breast cancer tissue. This method can identify breast cancer tissue with 73% clinical sensitivity and 93% specificity as compared to benign breast tumors. We did not find a correlation between telomerase activity and the tissue levels of estrogen and progesterone receptors, HER-2/neu oncoprotein concentration, tumor size, and lymph node positivity. Probability of disease-free survival was significantly lower for patients with telomerase activity higher than median value. As the assay for telomerase activity has very high analytical sensitivity and high specificity for cancer cells, this routinely used method may prove useful for distinguishing malignant phenotype of breast tissues.

A centrosomal serine/threonine kinase, AIK1(3)/breast tumor amplified kinase/aurora2, which was recently identified as an oncogene, shows high amino acid identity with chromosome segregation kinases, fly Aurora, and yeast Ipl1. Immunohistochemical analyses of invasive ductal adenocarcinomas of the breast revealed that overexpression of AIK1 was observed in 94% of the cases, irrespective of the histopathological type, whereas the protein was not detected in normal ductal and lobular cells. Benign breast lesions including fibrocystic disease and fibroadenoma (epithelial components) displayed weakly detectable AIK1 expression in part of the lesions. This is the first immunohistochemical report of AIK1 expression in primary human breast carcinomas. Although the physiological function(s) of AIK1 kinase during cell division remains to be determined, the markedly high positivity of AIK1 staining in the cancer lesions suggested a possible involvement of its overexpression in the tumorigenesis of some of breast cancer cells.

The activity of thymidine kinase, thymidine phosphorylase, adenosine deaminase and 5'-nucleotidase of AMP was studied in tissues of 39 healthy females, as well as blood serum and lymphocytes of 60 healthy females, as well as in 50 patients with fibrocavernous mastopathy aged as 23-70. Comparative determination of adenosine metabolism enzymes activity in lymphocytes was carried out simultaneously with studying some immunological indexes in the organism of the same-aged healthy females and ones with mastopathy. It was revealed that age-related changes in the activity of thymidine kinase in blood serum reflected the analogous changes in enzyme activity in tissues of the healthy women. A direct correlation was established between thymidine kinase activity and age both in the healthy females and those with mastopathy. A significant decrease in activity of thymidine phosphorylase was demonstrated in blood serum of the patients with mastopathy in the age 46-60. Determined 4-fold increase in the activity of adenosine deaminase in serum was accompanied by decreased enzyme activity in lymphocytes and decreased Lymphocyte Blast Transformation Index in the same age range. Changes of immunological status are more expressed in T-system of immunity. The revealed metabolic changes in DNA-precursors metabolism in the patients with mastopathy aged as 46-60 might be one of the reasons of increased risk of oncological disease in this age group.

The aim of the present study was to assess serum levels of copper and zinc levels and erythrocytes Cu,Zn-SOD activity and to determine probable changes in gastric and colorectal precancerous diseases, benign breast diseases, gastric, colorectal and breast cancer. The study included 165 subjects with cancer, 348 subjects with precancerous (atrophic gastritis, gastric adenoma, colon adenoma, rectal adenoma) and/or benign diseases (weak dysplasia, severe dysplasia, fibroadenoma, cystic disease) and 161 randomly selected healthy controls. Our results suggest that while in gastric and colorectal cancer there were mostly increased copper levels, in breast cancer they were not changed. Zinc levels were weakly decreased in atrophic gastritis, gastric adenoma and breast cancer. There was a strong positive correlation between zinc levels and SOD activity in fibroadenoma and a weak positive correlation in colorectal adenoma and colorectal cancer without any correlation between SOD activity and copper in these groups. In gastric precancerous disease there was a positive correlation between SOD and copper. The results of this study suggest that serum trace element levels and activity of related enzymes might be different in various neoplastic processes. This variation in neoplastic processes might be influenced by other factors that have to be considered in complex relationships between the whole body and neoplastic cells.

For the cancer cells which have overcome the second mitotic clock (M2), activated telomerase is essential and used as another marker of immortality. Many trials had been initiated to target telomerase, which is known to be specific to tumors. To determine the best in vitro cell system for testing the efficacy of telomerase inhibitors, we evaluated the telomerase activity of various cancer cell lines and measured their telomere lengths. We also treated some cancer cell lines with adriamycin and measured the changes of telomerase activity. Telomerase activity was evaluated in various cell lines with the TRAP (telomeric repeat amplification protocol) assay. Telomerase activity was calculated and translated into arbitrary units by computer-assisted densitometry with the control of telomerase activity in the 293 control cell line. Also, terminal restriction fragment lengths were measured using Southern blotting. We also measured telomerase activity and telomere lengths in 11 benign breast tumor tissues and 19 paired stomach cancer and normal tissues. Cancer cell lines treated with adriamycin we evaluated for changes of telomerase activity and the cell proliferation by MTT assay and dye exclusion test. Telomerase activity of cell lines was 95.3 24.1 unit with a range of 27.6-129.6 unit, while the telomere lengths of those cell lines were variable from 5.0 to 10.4 kbp with a median of 6 kbp. In 11 cancer cell lines which were not yet firmly established, we could not detect any telomerase activity. Low telomerase activity was detected in only 2 benign tumor tissues of breast with a median telomere length of 8.8 (7-10.5) kbp. Among paired 19 gastric cancer and normal tissues, only 7 cancer tissues showed weak telomerase activity. After adriamycin treatment, telomerase activity in YCC-S-1, YCC-S-3, MCF-7 and MCF-7/ADR was decreased in accordance with the changes of the cell numbers. Telomerase is specific to cancer tissues and is expressed differently from organ to organ. Telomerase activity by TRAP assay could be used as a chemosensitivity assay.

The application of telomerase activity is expected to serve as a reliable indicator of biological malignancy in breast tumors. We performed a study to quantify telomerase activity, the results of which revealed that this activity tends to be unexpectedly low in scirrhous carcinoma having low histological differentiation. It is therefore felt that additional studies at the tissue level using in situ hybridization will be required in the future. Although we think that atypical cystic duct is a true pre-cancerous lesion in the sense of having a high possibility of progressing into breast cancer, it will be interesting to determine whether or not the tissue is composed of cells having telomerase activity.

Telomerase, a cellular reverse transcriptase, has been detected in the majority of human malignant tumors, where it provides an escape mechanism from proliferative limitations due to progressive telomere erosion with each cell division. In this study, we used a non-radioactive telomeric repeat amplification protocol (TRAP) with an internal telomerase assay standard for the detection and semiquantitative analysis of 98 single frozen sections of normal breast tissue and benign and malignant breast lesions on an automated laser-fluorescence sequencer. Telomerase activity was detected in 36 of 40 (90%) infiltrating breast carcinomas, whereas no activity was found in nonmalignant breast tissues including blunt duct adenosis, papilloma, ductal hyperplasia and atypical ductal hyperplasia. However, telomerase activity was detected in 59% of ductal in situ carcinomas, suggesting that telomerase reactivation is an early event in breast carcinogenesis. We found a positive correlation between telomerase activity levels and cell proliferation determined by MIB1 immunostaining. No correlation, however, could be demonstrated between telomerase activity and other known breast cancer prognostic indicators. Telomerase activity was also detected in 60% of fibroadenomas indicating that careful interpretation of analysis of telomerase activity in fine needle aspirates is required, since low telomerase activity may not necessarily be an indicator of malignancy in breast tissue.

GGT 129, a polyclonal antibody directed against gamma-glutamyl transpeptidase (GGT), was used to study GGT expression in formalin-fixed paraffin-embedded tissues from normal breast, 24 benign lesions, 27 in situ carcinomas or atypical hyperplasias, and 79 infiltrating mammary carcinomas. Epithelium of the ducts and ductules in normal breast tissue showed immunoreactivity along the apical surface. There was a strong correlation (P < 0.01) between the histologic classification of the tissue and GGT expression. All of the benign breast lesions stained positive for GGT. Among in situ carcinomas and atypical hyperplasias, 5/27 (19%) were negative for GGT while 22/27 were immunopositive. Infiltrating carcinomas showed the greatest deviation from normal tissue with 23/79 (29%) negative for GGT. GGT expression in benign and malignant breast tissue was not correlated with the age of the patient, suggesting that menopausal status does not influence expression of GGT. Correlation of GGT immunoreactivity with tubule formation, nuclear pleomorphism, mitoses, grade, size of tumor, lymph node status, and ER/PR status was performed for 69 cases of infiltrating ductal adenocarcinoma. There were no statistically significant relationships between the level of GGT immunoreactivity and any of the parameters. The loss of GGT in some of the cases is evidence that this enzyme is not required for mammary tumor development or maintenance. However, as GGT is a component of the pathways that metabolize glutathione and glutathione-conjugates, the difference in levels of the enzyme in invasive breast cancers may be one explanation for the variation in chemotherapy response that has been observed in patients treated for advanced mammary cancer.

Serum ferritin (SF) and lactate dehydrogenase (LDH) was estimated in 117 patients presenting with a breast lump and in 40 controls. Both pre and post treatment values were determined. Both the values were significantly higher in breast malignancies (p = 0.00) and also corresponded with the clinical stage and bulk of the tumour. The fall in post treatment values was proportional to the response to therapy. Persistent rise in values in the post treatment period was indicative of local recurrence of metastasis.

Glutathione S-transferase (GST) represents a multifunctional enzyme family consisting of four known cytosolic isoforms (alpha, mu, pi, and Phi) that detoxify a variety of xenobiotic chemicals and may confer resistance to both chemotherapeutic drugs and carcinogens in various experimental models. GST-pi has already been extensively studied in clinical specimens, including breast cancer. We studied the immuno-histochemical distribution and relative immunopositivity of GST-alpha and GST-mu, based on a grading system for immunointensity, in samples of 51 neoplastic and 46 normal breast samples and 12 lymph node metastases from patients treated with intensive chemotherapy and bone marrow transplant. In normal breast tissue, GST-alpha localized predominantly to the cytoplasm of scattered cells lining the luminal aspects of the ducts. Occasional cells showed both cytoplasmic and nuclear GST-alpha immunoreactivity. GST-mu was stained in myoepithelial cells preferentially as well as in occasional ductal cells (including apocrine epithelium), vascular smooth muscle, and plasma cells. GST-alpha and GST-mu were detected in 22 of 51 (43%) and 24 of 48 (50%) invasive cancers, respectively. In paired samples of normal and malignant tissue from the same patient, GST-alpha immunostaining in cancers was significantly less intense compared to that of normal breast tissue in 13 of 41 (32%) cases. No such trend was found for GST-mu in paired samples. Neither GST-alpha nor GST-mu immunopositivity in tumor or nonneoplastic breast was found to correlate with relapse-free or overall survival in this clinical context; however, the apparent decreased expression of GST-alpha in malignant versus normal breast epithelial cells could have important implications in breast carcinogenesis.

Our purpose was to determine whether the expression of cathepsin D, a proteolytic enzyme implicated in basement membrane degradation, is associated with dynamic magnetic resonance imaging (MRI) enhancement of breast lesions. Forty-five patients with 48 breast lesions underwent gadolinium-enhanced spoiled gradient recalled echo MRI followed by excisional biopsy and cathepsin D staining and semiquantitative measurement in the lesions. There was no significant difference in cathepsin D staining of 25 malignant and 23 benign breast lesions. A significant association was seen between high cathepsin D staining and positive axillary lymph nodes in invasive carcinomas. Nine of nine (100%) node-positive carcinomas had high cathepsin D, as compared to three of seven (43%) node-negative carcinomas (P = 0.02). No significant associations were observed between cathepsin D staining and MRI enhancement amplitude, rate, or washout. Cathepsin D has no effect upon MRI gadolinium enhancement of malignant and benign breast lesions but is associated with positive axillary lymph nodes in invasive carcinomas.

One hundred seventy two consecutive cases of female breast pathology have been intraoperatively processed by frozen sections (FS) in combination with aspecific alkaline phosphatase (AAF) histochemical evaluation. The corresponding histology on permanent embedded tissues (ET) was compared with both these techniques. The related theoretical and practical methodological aspects are presented and discussed. The consultations concerned the following histopathological types (WHO terminology): 136 malignant epithelial tumours(intraductal carcinoma: 11; invasive ductal carcinoma: 100; invasive lobular carcinoma: 8; medullary carcinoma: 3; mucinous carcinoma: 8; papillary carcinoma: 3; tubular carcinoma: 2); one cystosarcoma phyllodes; 19 benign tumours; 15 mammary dysplasias (fibrocystic disease); one tumour-like lesion(hamartoma); one fibromatous lesion. In terms of concordance/discordance between FS and ET diagnoses no false positive cases and only one false negative resulted. The diagnosis was deferred in 18 circumstances (10,5%). The AAF reaction in 64/153 (41,8%) cases (correctly interpreted on FS) displayed more histological evidence with special reference to the detection of the myoepithelial cells. The morphological implementation was particularly relevant as far as the differentiation between intraductal and microinvasive carcinoma, carcinomatous and papillomatous papillary proliferation, carcinomatous and non carcinomatous tubular structures, pseudocarcinomatous (florid and/or sclerosing) dysplasia and carcinoma is concerned. Analogously in 12/17 (70,6%) comparable deferred diagnoses as well as in the single false negative case the interpretation could have been correctly stated intraoperatively. The normal and pathological features as disclosed by the AAF reaction have been analytically examined. The AAF procedure seems to be a possible relevant tool in order to improve the classical FS performance also in respect to the increasingly systematic, precocious and conservative approach of the modern breast surgery.

The proteolytic processes are thought to be the critical point in tumor invasion and metastasis, mainly by matrix metalloproteinases (MMPs) and serine proteases. We measured the activity of MMP-2 from 28 normal, 12 benign and 126 breast cancer tissues using gelatin zymography. Inactive MMP-2 (72 kD) was expressed in 53.6% of the normal and 66.6% of the cancer tissues, respectively (P = 0.77), while active MMP-2 (62 kD) was expressed in 28.6% and 73.0%, respectively (P = 0.003). The enzymatic activity of active MMP-2 (62 kD) measured in the gel band area was 4.0 +/- 7.2 mm2 in normal breasts, 7.7 +/- 9.8 mm2 in benign breast diseases, 9.5 +/- 8.5 mm2 in ductal carcinoma in situ (DCIS), and 12.0 +/- 13.7 mm2 in invasive cancers. The MMP-2 activation ratio (62 kD/62 kD + 72 kD) was 0.12 +/- 0.18 in normal tissues, 0.10 +/- 0.20 in benign diseases, 0.61 +/- 0.22 in DCIS, and 0.50 +/- 0.28 in invasive cancers. In conclusion, MMP-2 activation was the main cause of the increased 62 kD MMP-2 activity during the early phase of breast cancer, while production of MMP-2 supplemented the increased 62 kD activity in the late phase. We suggest, therefore, that these differential expressions of MMP-2 activation and production during the different stages of breast cancer progression are potential therapeutic targets for biological or gene therapy under the concept of stage-oriented cancer treatment.

We analysed telomerase activity in normal, benign and malignant breast tissues and in fine needle aspirates by a PCR-based assay. The tissue samples we used in this assay consisted of 20 cryostat sections, 10 microns thick, from each breast biopsy. This method was used to obtain effective extraction from small samples and to confirm the histological identity of the specimen by microscopical examination of serial sections. Fifty-two of 71 breast carcinomas were positive for telomerase activity, and the intensity of this was strong in most cases, whereas all 6 samples of normal breast tissue and 17 of fibrocystic disease were negative and only 1 of 15 fibroadenomas was positive. Invasive ductal carcinomas were more frequently positive than invasive lobular carcinomas. There was no correlation of telomerase activity with tumour size or the occurrence of lymph node metastasis. Evaluation of our assay system showed that a signal of telomerase activity was detectable in extracts from single cryostat sections (< 1 mm2) of a cancer specimen and from as few as 4 cells of a human breast cancer cell line. On the basis of the above data, we applied this assay to fine needle aspirates of breast lesions. Ten of 15 aspirates which had been cytopathologically diagnosed as cancer were strongly positive, while 26 of 29 benign aspirates were totally negative and the remaining 3 showed only borderline activity. In 3 cases, the telomerase result could have helped establish a diagnosis when the cytological observations were inconclusive. Our results indicate that this sensitive assay could become a useful new modality for supplementing microscopic cytopathology in the detection of cancer cells in small tissue biopsies and fine needle aspirates.

Pyrimidine nucleoside phosphorylase (PyNPase) is considered to be important for producing antitumor function of 5'-DFUR or 5-FU inducing compounds, since this enzyme transforms 5'-DFUR into 5-FU and 5-FU into their activated forms. Additionally, PyNPase shows high activity in various malignant tumor tissues. In this investigation, the distributions of PyNPase were examined in the tissues of mastopathy, fibroadenoma, phyllodes tumor and mammary cancer using immunohistochemical procedures. The following results were obtained. In the tissues of mastopathy or fibroadenoma, immunoreactivity was generally very weak and only a few positive epithelial cells were observed. In the phyllodes tumor, immunoreactivity was also weak, but every epithelial cell showed positive immunoreactivity. On the contrary, apparent immunoreactivity was observed in the tissues of mammary cancer, and cancer cells revealed prominent immunoreactivity.

Background: The activity of the ribonucleoprotein enzyme telomerase is not detected in normal somatic cells; thus, with each cell division, the ends of chromosomes consisting of the telomeric repeats TTAGGG progressively erode. The current model gaining support is that telomerase activity in germline and immortal cells maintains telomere length and thus compensates for the "end-replication problem.", Purpose: Our objective was to determine when telomerase activity is reactivated in the progression to malignant breast cancer and if knowledge of telomerase activity may be an indicator for the diagnosis and potential treatment of breast cancer., Methods: Using a polymerase chain reaction-based telomerase activity assay, we examined telomerase activity in 140 breast cancer specimens (from 140 patients), four phyllodes tumors (from four patients), 38 noncancerous lesions (20 fibroadenomas, 17 fibrocystic diseases, one gynecomastia; from 38 patients), and 55 adjacent noncancerous mammary tissues (from 55 of the 140 breast cancer patients). In addition, 33 fine-needle-aspirated breast samples (from 33 patients) were analyzed., Results: Among surgically resected samples, telomerase activity was detected in 130 (93%) of 140 breast cancers. Telomerase activity was detected in 68% of stage I primary breast cancers, in 73% of cancers smaller than 20 mm, and in 81% of axillary lymph node-negative cancers. Moreover, the activity was detected in more than 95% of advanced stage tumors but in only two (4%) of 55 adjacent noncancerous tissues. While telomerase activity was not detected in any of 17 specimens of fibrocystic disease, surprisingly low levels of telomerase activity were detected in nine (45%) of 20 fibroadenomas. Among samples obtained by fine-needle aspiration, 14 (100%) of 14 patients whose fine-needle-aspirated specimen contained telomerase activity and who subsequently underwent surgery were confirmed to have breast cancer. Multivariate analysis of 125 specimens from patients for whom data were available on age at surgery, stage of disease, tumor size, lymph node status tumor histology, and menopausal status indicated that stage classification exhibited the strongest association with telomerase activity (for stage I versus stages II-IV: odds ratio = 1.0 versus 73.4; 95% confidence interval = 2.0-959.0; P = .02)., Conclusion: Telomerase activity was detected in more than 95% of advanced stage breast cancers. It was absent in 19%-32% of less advanced cancers. Since a determination of any association between telomerase activity and patient survival is not possible at the present time, it remains to be determined whether lack of telomerase activity predicts for favorable outcome.

The enzyme O(6)-alkylguanine DNA alkyltransferase (AGT) is implicated in the repair of DNA damage by chemotherapeutic chloroethylnitrosoureas (CNUs) and plays a vital role in determining the sensitivity of cancer cells to this class of compounds. We have measured the AGT activity in 41 human female breast tissues including normal, benign and malignant specimens. The patients with breast cancer in advanced stages have shown a statistically significant 2.37-fold (p = 0.007) higher level of AGT activity compared to normal. An inter-individual variation of 1.8-fold in normal, 10.8-fold in benign and 7.6-fold in malignant tissues was observed. The average number of methyl group acceptor molecules per cell were estimated to be 19,053 +/- 903, 20,227 +/- 1,806 and 45,330 +/- 5,418 (SEM) in normal, benign and malignant tissue, respectively. These results for the first time emphasize the predictive role of AGT in selecting the responder patients by AGT determination in breast tumor biopsies and further encourage therapeutic trials based on biochemical modulation of CNU resistance against malignant cells.

N-acetyl-beta-glucosaminidase (NAG) and its isoenzymes were measured in 60 patients with breast cancer and in 30 patients suffering from benign breast tumors. NAG activity was significantly increased (P < 0.001) in cancer patients. The concentration of isoenzyme A always exceeded that of isoenzyme B, but isoenzyme B concentration was significantly (p < 0.001) higher in patients than in controls. An intermediate form, I, was more evident but not significant in patients. Evaluation of the predictive value of the NAG isoenzyme B, revealed a good sensitivity, efficiency and specificity. It seems from this study that the determination of the isoenzyme NAG B activity may be useful for better diagnosis of breast cancer.

Since many of the factors involved in tumor growth and progression act through a receptor-mediated mechanism, we applied in vitro receptor imaging techniques to study the intratumoral distribution and concentration of receptor-molecules having experimental biological relevance in such processes. Here we summarize the results of a study concerning the role of urokinase receptor (uPAR) in the acquisition of an invasive phenotype by tumor cells. Independently of the system studied, we demonstrated that in vitro receptor imaging techniques can be used to define the biological characteristics of human solid tumors and can contribute to clarify the complex network of ligand/receptor interactions leading to tumor spread.

Background: The diagnosis of breast cancer based on nipple discharge, often the only clinical manifestation of early breast cancer, is currently unsatisfactory. Because M subunits of lactate dehydrogenase (LDH) have been noted to increase in cancer tissue, the author assessed the value of using LDH isozyme patterns in nipple discharge for the diagnosis of breast cancer., Methods: LDH isozyme levels in (1) nipple discharge of patients diagnosed as having breast cancer, intraductal papilloma, mastopathy, drug-induced nipple discharge, mastitis, or benign nipple discharge; (2) control samples of normal nipple discharge (milk) 6 days, 1-5 months, and 6 months to 2 years postpartum; (3) the serum of patients presenting with nipple discharge; and (4) normal and cancerous breast tissue extracts were measured using a Ciba-Corning LDH isozyme system (Ciba Corning Diagnostic Corp., Tokyo, Japan)., Results: LDH isozyme levels in the nipple discharge of patients with benign breast diseases displayed various patterns. Levels in the nipple discharge of patients with breast cancer, including noninvasive carcinoma, tended to increase in ascending order from LDH1 to LDH5. This pattern was similar to that in breast cancer tissue and was unrelated to the pattern in serum., Conclusion: LDH isozyme assay of nipple discharge may be a useful technique for providing a supporting diagnosis of breast cancer.

We measured the tissue concentration of human urokinase receptor (uPAR) in 22 breast carcinomas and 9 benign breast lesions using in vitro quantitative autoradiography. Tissue sections were incubated with increasing concentrations of 125I-pro-urokinase in the presence or absence of unlabeled competitor. Breast carcinomas were found to contain 5 times more uPAR than benign breast lesions with respect to their protein content [523 +/- 72 versus 108 +/- 20 (SE) fmol/mg (P < 0.001)]. Simultaneous quantitation of urokinase (uPA) by immunoenzymatic assay on tissue extracts from the same specimens showed that breast carcinomas also contain 19 times more uPA than benign tumors (611 +/- 134 versus 32 +/- 8 fmol/mg) (P < 0.01). The reliability of quantitative autoradiography measurements was confirmed by uPAR cross-linking assay on membrane fraction from either U937 histiocytic lymphoma cells or breast carcinomas and immunoperoxidase staining with an anti-uPAR antibody on tumor sections. Also, immunoperoxidase staining with an anti-uPA monoclonal antibody showed that uPA is indeed localized on the plasma membrane of epithelial tumor cells in confined areas of breast carcinomas whereas cells from benign breast lesions were devoid of uPA under the same experimental conditions. In conclusion, our findings support the hypothesis that uPAR plays a central role in the acquisition of an invasive phenotye and favor its potential use as a prognostic factor in patients with breast carcinoma.

Background: Recently, the authors reported that membrane-associated phospholipase A2 (M-PLA2) was one of the acute phase reactants and increased in serum of patients with various malignant tumors., Methods: M-PLA2 concentrations in tissue specimens from 78 breast cancers, 16 benign breast tumors, and 10 normal breast tissues were determined by a specific radioimmunoassay recently developed. Immunohistochemical staining was performed on all specimens by the avidin-biotin-peroxidase method., Results: Tissue levels of M-PLA2 concentration were significantly higher in breast cancer than in benign breast tumor or normal breast tissue (P < 0.01). Correlation analyses between the tissue concentration of M-PLA2 and clinicopathologic factors showed that tissue M-PLA2 levels were significantly higher in patients with skin or muscle invasion, vessel involvement, and distant metastasis than in those without. In addition, this enzyme concentration was significantly greater in scirrhous carcinoma than in papillotubular or solid-tubular carcinoma. No association was found between M-PLA2 concentration and steroid hormone receptor status. Immunohistochemically, M-PLA2 was preferentially stained in the invading zone of breast cancer tissues, especially in scirrhous carcinoma. Patients with breast cancer with low levels of M-PLA2 showed significantly longer overall survival and disease-free survival compared with those with high levels of this enzyme at the cutoff point of 50 ng/100 mg protein. The combination of estrogen receptor status with M-PLA2 concentration could be a powerful prognostic factor in predicting such survival rates., Conclusions: M-PLA2 is closely related to the malignant potential of breast cancers, and the M-PLA2 contents in breast cancer tissues could be a new valuable prognostic factor, other than the hormone receptor, in delineating the status of human breast cancer.

Particularly in postmenopausal women, peripheral aromatase appears to be the major source of oestrogens which may encourage the growth of hormone-dependent tumours. Studies have therefore been undertaken to determine factors which influence biosynthesis of oestrogens within breast tissues. Aromatase activity was measured in (i) breast cancers by incubating tumour homogenates with [7 alpha 3H]testosterone and characterizing the production of radioactively-labelled oestradiol and (ii) breast fat by incubation of sub-cellular fractions of fibroblast cell lines with [1 beta 3H]androstenedione and monitoring the formation of 3H2O. Evidence has been presented that (i) certain treatment regimes for cancer profoundly influence aromatase activity in breast tumours, (ii) aromatase activity is elevated in breast fat associated with malignancy and (iii) breast-derived fluids and extracts can markedly affect aromatase activity in cultured fibroblasts of breast fat.

Activities of arginase, alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase were determined in sera obtained in a group of healthy women, women with verified carcinoma of the breast, benign mastopathy, a group of patients with carcinoma of various organs and a group of patients with acute viral hepatitis. Preoperative values of serum arginase activity in patients with breast carcinoma were up to 4-fold those found in healthy women. Sensitivity of the test was 86%. After the surgery, the activity decreased abruptly during the first week and normalised within 15-30 days. In benign diseases of the breast, the activity of arginase was normal. Serum arginase activity is raised in both benign and malignant liver diseases, however, the quotients alanine aminotransferase/arginase, aspartate aminotransferase/arginase and alkaline phosphatase/arginase differ significantly. Thus, use of alanine aminotransferase/arginase quotient implies a high degree of confidence in differentiating between increased arginase activity in mammary carcinoma (alanine aminotransferase/arginase = 0.572 +/- 0.278) and high arginase activity in hepatitis (alanine aminotransferase/arginase = 12.226 +/- 1.822).

The concentrations of cathepsin D (Cath D), urokinase (uPA) and two plasminogen activator inhibitors (PAI-1 and PAI-2) were analysed in the cytosols of 130 human mammary tumours (43 benign tumours and 87 primary and unilateral breast carcinomas). uPA, PAI-1 and PAI-2 levels were measured by antigenic immunoassays and Cath D by immunoradiometric assay. The median levels of the four parameters were significantly higher in the malignant tumours than in the benign ones. Cath D and uPA increases were 4-fold and 5-fold respectively. PAI-1 and PAI-2 increases were much more important, 74-fold and 29-fold respectively. In malignant tumours, median levels of Cath D and uPA did not vary according to classical prognostic factors (histologic grade, presence or absence of axillary lymph nodes, steroid receptors, UICC stage, tumour size, age, and menopausal status). However, PAI-1 decreased in ER+ and PR+ tumours and PAI-2 increased in menopausal women's tumours. When Cath D, uPA, PAI-1 and PAI-2 levels in malignant tumours were compared, positive correlations were found for all combinations. The implication of plasminogen activator inhibitors in the phenomenon was surprising and merits further investigation using tools other than global antigen measurements in tumours.

Serum cholinesterase (ChE) and Lactate dehydrogenase (LDH) activities were estimated in 40 cases of carcinoma breast, 25 cases of benign tumours and compared with healthy controls (30 cases). Significant difference in enzyme activities were obtained between benign and malignant neoplasms of the breast when compared with each other as well as when compared with healthy controls. Also, there were significant enzyme changes between non-metastatic cases and those with metastasis and when Stage I and Stage II cancers were compared with those in Stage III and Stage IV. No difference in enzyme levels were recorded between pre and post-operative cases and in different types of breast cancers. While ChE was depressed in 80 per cent cases of malignancy breast, serum LDH was raised in 73.3 per cent cases.

We have shown previously that fatty acid synthetase (FAS) is specifically induced by progestins in human breast cancer cell lines. To test the potential value of FAS as a clinical marker in breast diseases, we measured FAS expression in frozen sections of 22 benign and 27 malignant mammary tumors using in situ hybridization with the [35S]UTP alpha S-labeled FAS anti-sense mRNA. The hybridized RNA was quantified with an IMSTAR computerized image analyzer. We found FAS RNA in epithelial cells, but no labeling was detected in the connective tissue. In breast cancer, we found no correlation between FAS expression and estrogen receptor and progesterone receptor concentrations or status. However, the level of FAS was significantly (P less than .02) higher in premenopausal than in post-menopausal patients and increased with the grade of tumor differentiation (P less than .005 between the poorly and well-differentiated tumors). In benign mastopathies, high levels of FAS RNA were found in some cysts (mostly with apocrine metaplasia). In lobules, the FAS RNA level increased proportionally to the degree of proliferation determined by histological examination (P less than .015) and correlated with the H4 histone level measured in an adjacent section using in situ hybridization (r = 0.85, P less than .001). In ductal structures, a lower correlation (r = 0.64, P less than .01) was found between FAS and H4 RNA levels. We conclude that FAS RNA is overexpressed in some mammary tumors and may be useful in predicting high-risk mastopathies and less aggressive breast cancers.

Progesterone and estrogens play an important role in the control of growth, differentiation and function of mammary epithelial cells. Their mechanism of action can be studied in human metastatic breast cancer cell lines (MCF7, T47D, ZR75-1...) that contain progesterone and estrogen receptors. We used this system to try to define progestin-regulated human genes which would permit to study progestin-regulation of gene expression in cell culture and to develop clinical markers of progestin-responsiveness. This paper summarizes our investigation of the progestin-regulated 250K protein, recently identified as human fatty acid synthetase (FAS).

129 female patients with breast cancer and 79 controls undergoing biopsy for benign breast conditions had debrisoquine hydroxylator phenotype established. 129 female hospital patients with known hydroxylator phenotype were used as another control group. The breast cancer cases differed significantly from the benign controls in their debrisoquine phenotype, with 10% being poor metabolisers compared with none of the controls (P less than 0.01). However, while a comparison of the distributions of metabolic ratio (an inverse measure of debrisoquine metabolism) of breast cancer patients and hospital controls showed a significant difference by rank, there was no significant difference in the proportion of poor metabolisers in these two groups. The cases with benign disease differed from the hospital controls in both metabolic ratio distribution (P less than 0.001) and frequency of poor metabolisers (P less than 0.05). Although there was a shift in metabolic ratio distribution, debrisoquine hydroxylator phenotype was not a genetic marker for breast cancer. Why no patients undergoing biopsies for benign conditions were poor metabolisers is unknown.

Erythrophagocytosis by histiocytes in the sinuses of axillary lymph nodes is a common yet little-known phenomenon. The axillary lymph node dissections of 23 patients were studied by light microscopy and graded for the amount of erythrophagocytosis. None of the patients had evidence of a systemic hemolytic process. Nineteen of them exhibited some degree of erythrophagocytosis, and this was present even in four of the six patients who never had a prior breast biopsy. Breast biopsy was associated with massive degrees of erythrophagocytosis in 8 of 17 patients, and after a postbiopsy interval of two weeks 11 of 13 patients had hemosiderin deposition in the lymph nodes, evidence of red blood cell breakdown. This study serves to substantiate statements, made by others without confirmatory data, that large degrees of erythrophagocytosis may be seen in axillary lymph nodes after breast biopsies and that small amounts of erythrophagocytosis may be identified in lymph nodes of patients with no prior trauma history.

Acid phosphatase, leucine aminopeptidase, monoamine oxidase and non specific esterase activities were histochemically demonstrated in specimens derived from 15 infiltrating ductal carcinomas of female breast. The relative areas occupied by the enzyme-positive carcinoma cells were visually estimated and, in the cases of leucine aminopeptidase, assessed morphometrically. All enzyme activities were found to be subject to major variations within a single carcinoma and between individual carcinomas, and the activity of any single enzyme was independent of that of three others. None of the enzyme activities correlated with the estrogen and progesterone receptor values, nor the histological grade of malignancy of the tumour. Thus, histochemically demonstrable enzyme activities seem to be of no use in predicting the hormone receptor content in infiltrating ductal carcinomas of the female breast.