Proteomic profiling of mammary carcinomas identifies C7orf24, a gamma-glutamyl cyclotransferase, as a potential cancer biomarker.

Breast cancer is the leading cause of cancer deaths in women today and is the most common cancer (excluding skin cancers) among women in the Western world. Although cancers detected by screening mammography are significantly smaller than nonscreening ones, noninvasive biomarkers for detection of breast cancer as early as possible are an urgent need as the risk of recurrence and subsequent death is closely related to the stage of the disease at the time of primary surgery. A set of 123 primary breast tumors and matched normal tissue was analyzed by two-dimensional (2D) gel electrophoresis, and a novel protein, C7orf24, was identified as being upregulated in cancer cells. Protein expression levels of C7orf24 were evaluated by immunohistochemical assays to qualify deregulation of this protein. Analysis of C7orf24 expression showed up-regulation in 36.4 and 23.4% of cases present in the discovery sample set (123 samples) and in an independent large TMA validation data set (2197 samples) of clinically annotated breast cancer specimens, respectively. Survival analysis showed that C7orf24 overexpression defines a subgroup of breast tumors with poor clinical outcome. Up-regulation of C7orf24 was also found in other cancer types. Four of these were investigated in greater detail, and we found that a proportion of tumors (58% in cervical, 38% in lung, 72% in colon, and 46% in breast cancer) expressed C7orf24 at levels exceeding those seen in normal samples. The observed overexpression of this protein in different types of cancer suggests deregulation of C7orf24 to be a general event in epithelial carcinogenesis, indicating that this protein may play an important role in cancer cell biology and thus constitute a novel therapeutic target. Furthermore, as C7orf24 is externalized to the tissue extracellular fluid and can be detected in serum, this protein also represents a potential serological marker.

Plasma and serum levels of tissue inhibitor of metalloproteinases-1 are associated with prognosis in node-negative breast cancer: a prospective study.

The tumor level of TIMP-1 has been suggested as a new prognostic marker in breast cancer. The purpose of this study was to investigate whether TIMP-1 also carries prognostic information when measured in blood as this is a much more preferable material compared with tumor extracts. Using ELISA, TIMP-1 was measured in prospectively collected preoperative plasma and serum samples from 519 patients with primary breast cancer, and the measurements were related to patient outcome. The median age of the patients was 58 years (range, 38-80 years), and the median follow-up time was 1043 days (range, 300-1630 days). Plasma and serum TIMP-1 measurements correlated significantly with each other with a Pearson correlation coefficient of 0.75 (p < 0.0001). For univariate survival analysis, patients were divided into quartiles according to increasing TIMP-1 levels (Q1-Q4). Analysis of all patients showed that high TIMP-1 plasma levels were significantly associated with a shorter disease-free survival. Subgroup analysis showed that plasma TIMP-1 significantly predicted the prognosis of node-negative patients but not of node-positive patients. Importantly plasma TIMP-1 was able to further stratify low risk node-negative patients. High serum TIMP-1 levels were associated with a shorter disease-free survival; however, the association was not statistically significant. In contrast, serum TIMP-1 significantly predicted the prognosis of node-negative and low risk patients. In multivariate survival analysis of node-negative patients including all the classical prognostic parameters, plasma TIMP-1 remained significantly associated with prognosis when comparing Q1 with Q2 and Q4. Serum TIMP-1 remained significant when comparing Q1 with Q4. Taken together, this study is to our knowledge the first large prospective study suggesting that TIMP-1 carries independent prognostic information when measured in blood, especially plasma. This was especially true in the node-negative group of patients and in patients already defined as low risk patients using the currently available prognostic parameters.

15-prostaglandin dehydrogenase expression alone or in combination with ACSM1 defines a subgroup of the apocrine molecular subtype of breast carcinoma.

Established histopathological criteria divide invasive breast carcinomas into defined groups. Ductal of no specific type and lobular are the two major subtypes accounting for around 75 and 15% of all cases, respectively. The remaining 10% include rarer types such as tubular, cribriform, mucinous, papillary, medullary, metaplastic, and apocrine breast carcinomas. Molecular profiling technologies, on the other hand, subdivide breast tumors into five subtypes, basal-like, luminal A, luminal B, normal breast tissue-like, and ERBB2-positive, that have different prognostic characteristics. An additional subclass termed "molecular apocrine" has recently been described, but these lesions did not exhibit all the histopathological features of classical invasive apocrine carcinomas (IACs). IACs make up 0.5-3% of the invasive ductal carcinomas, and despite the fact that they are morphologically distinct from other breast lesions, there are presently no standard molecular criteria available for their diagnosis and as a result no precise information as to their prognosis. Toward this goal our laboratories have embarked in a systematic proteomics endeavor aimed at identifying biomarkers that may characterize and subtype these lesions as well as targets that may lead to the development of novel targeted therapies and chemoprevention strategies. By comparing the protein expression profiles of apocrine macrocysts and non-malignant breast epithelial tissue we have previously reported the identification of a few proteins that are specifically expressed by benign apocrine lesions as well as by the few IACs that were available to us at the time. Here we reiterate our strategy to reveal apocrine cell markers and present novel data, based on the analysis of a considerably larger number of samples, establishing that IACs correspond to a distinct molecular subtype of breast carcinomas characterized by the expression of 15-prostaglandin dehydrogenase alone or in combination with a novel form of acyl-CoA synthetase medium-chain family member 1 (ACSM1). Moreover we show that 15-prostaglandin dehydrogenase is not expressed by other breast cancer types as determined by gel-based proteomics and immunohistochemistry analysis and that antibodies against this protein can identify IACs in an unbiased manner in a large breast cancer tissue microarray making them potentially useful as a diagnostic aid.

Arm morbidity following sentinel lymph node biopsy or axillary lymph node dissection: a study from the Danish Breast Cancer Cooperative Group.

BACKGROUND: Sentinel lymph node biopsy was implemented in the treatment of early breast cancer with the aim of reducing shoulder and arm morbidity. Relatively few prospective studies have been published where the morbidity was assessed by clinical examination. Very few studies have examined the impact on shoulder mobility of node positive patients having a secondary axillary dissection because of the findings of metastases postoperatively. AIM: We aimed to investigate the objective and subjective arm morbidity in node negative and node positive patients. METHODS AND MATERIALS: In a prospective study, 395 patients with tumors less than 4 cm, were included. Patients were recruited from seven Danish breast cancer clinics. Both subjective and objective arm and shoulder morbidity were measured before, 6 and 18 months after the operation. RESULTS: Comparing node negative patients having a sentinel lymph node biopsy with node negative patients having a lymph node dissection of levels I and II of the axilla, we found significant increase in arm volume among the patients who had an axillary dissection. Only minor, but significant, differences in shoulder mobility were observed comparing the two groups of node negative patients. Highly significant difference was found comparing sensibility. Comparing the morbidity in node positive patients who had a one-step axillary dissection with patients having a two-step procedure (sentinel lymph node biopsy followed by delayed axillary dissection) revealed no difference in objective or subjective arm morbidity. CONCLUSION: Node negative patients operated with sentinel lymph node biopsy have less arm morbidity compared with node negative patients operated with axillary lymph node dissection. Node positive patients who had a secondary axillary lymph node dissection after sentinel lymph node biopsy had no difference in either objective or subjective morbidity compared with node positive patients having a one-step axillary dissection.

Organized nation-wide implementation of sentinel lymph node biopsy in Denmark.

Prior to the initiation of a nationwide study of the sentinel node staging technique the Danish Breast Cancer Cooperative Group (DBCG) defined a set of minimum requirements to be met by surgical departments before they could include patients in the study. The requirements specified a minimum patient load in the individual surgical unit, a minimum surgical training in the sentinel node biopsy technique and a minimum quality outcome in a validating learning series of SNLB procedures. A working group assisted departments in meeting these terms and later audited and certified departments before they could include patients into the study. As a result of this strategy the sentinel lymph node staging was fully implemented in all Danish surgical breast cancer centres within three years and all sentinel node biopsies in the period were recorded in the DBCG data centre. Furthermore, the strategy accelerated the ongoing process of centralizing breast surgery in specialized departments.

Sentinel node biopsy in breast cancer: five years experience from Denmark.

INTRODUCTION: Danish experience from the first five years with sentinel lymph node biopsy (SLNB) as a routine staging procedure in early breast cancer is reported. METHODS: During the period January 1, 2002 to December 31, 2006, 14 923 patients were diagnosed at Danish breast surgical centers certified for the sentinel node method. SLNB was performed in 8 338 patients (55.9%). The fraction increased steadily from 43% in 2002 to 67% in 2006. The median follow-up was 1.7 year (range 0-5.2 years). RESULTS: Patients staged with SLNB were younger, had more often BCS, had smaller tumor size, were more often hormone receptor positive, and had lower grade, than patients staged with lymph node dissection (ALND). Blue dye and radio colloid were used in combination in 82%. Lymphoscintigraphy was performed in 61%, and frozen section was performed in 87%. Originally, peritumoral injection of tracer was most often used, but the recommendations have changed, and in 2006 90% of cases had sub-or periareolar injection of radioactive tracer. In the sentinel nodes 25% had macrometastases, 17% micrometastases only, and 3.2% isolated tumor cells only (ITC). ALND was performed in 2 714 patients, whose lymph node classification by SN was known. In the group of 1 563 patients with macrometastases in SN, 45% had non-sentinel node metastases, and in the group of 942 patients with micrometastases only, 23% had more positive nodes. Regional lymph node metastases were found in 15% with ITC in sentinel nodes. Lymph node recurrence among node negative patients was observed more often after staging by SLNB (0.5%) than after ALND (0.2%, p =0.04). CONCLUSION: Two thirds of breast cancer patients can be safely staged with the sentinel node technique, half of these will need no further axillary surgery. The loco-regional control in node negative patients classified by SLNB is high, but seems not quite comparable to what is seen after ALND.

Molecular pathology of breast apocrine carcinomas: a protein expression signature specific for benign apocrine metaplasia.

Breast cancer is a heterogeneous disease that encompasses a wide range of histopathological types including: invasive ductal carcinoma, lobular carcinoma, medullary carcinoma, mucinous carcinoma, tubular carcinoma, and apocrine carcinoma among others. Pure apocrine carcinomas represent about 0.5% of all invasive breast cancers according to the Danish Breast Cancer Cooperative Group Registry, and despite the fact that they are morphologically distinct from other breast lesions, there are at present no standard molecular criteria available for their diagnosis. In addition, the relationship between benign apocrine changes and breast carcinoma is unclear and has been a matter of discussion for many years. Recent proteome expression profiling studies of breast apocrine macrocysts, normal breast tissue, and breast tumours have identified specific apocrine biomarkers [15-hydroxyprostaglandin dehydrogenase (15-PGDH) and hydroxymethylglutaryl coenzyme A reductase (HMG-CoA reductase)] present in early and advanced apocrine lesions. These biomarkers in combination with proteins found to be characteristically upregulated in pure apocrine carcinomas (psoriasin, S100A9, and p53) provide a protein expression signature distinctive for benign apocrine metaplasias and apocrine cystic lesions. These studies have also presented compelling evidence for a direct link, through the expression of the prostaglandin degrading enzyme 15-PGDH, between early apocrine lesions and pure apocrine carcinomas. Moreover, specific antibodies against the components of the expression signature have identified precursor lesions in the linear histological progression to apocrine carcinoma. Finally, the identification of proteins that characterize the early stages of mammary apocrine differentiation such as 15-PGDH, HMG-CoA reductase, and cyclooxygenase 2 (COX-2) has opened a window of opportunity for pharmacological intervention, not only in a therapeutic manner but also in a chemopreventive setting. Here we review published and recent results in the context of the current state of research on breast apocrine cancer.

Towards discovery-driven translational research in breast cancer.

Discovery-driven translational research in breast cancer is moving steadily from the study of cell lines to the analysis of clinically relevant samples that, together with the ever increasing number of novel and powerful technologies available within genomics, proteomics and functional genomics, promise to have a major impact on the way breast cancer will be diagnosed, treated and monitored in the future. Here we present a brief report on long-term ongoing strategies at the Danish Centre for Translational Breast Cancer Research to search for markers for early detection and targets for therapeutic intervention, to identify signalling pathways affected in individual tumours, as well as to integrate multiplatform 'omic' data sets collected from tissue samples obtained from individual patients. The ultimate goal of this initiative is to coalesce knowledge-based complementary procedures into a systems biology approach to fight breast cancer.

Unsuccessful radioiodine treatment of a non-toxic goiter: a case report.

A middle-aged woman with a large right-sided, non-toxic goiter with low iodine uptake was admitted to the Department of Endocrinology with the purpose of volume reduction of the goiter. Thyroid pertechnetate scintigraphy showed homogenous and diffuse uptake in both lobes. Initially thyroxine treatment was given without volume-reducing effect. Radioiodine was administered twice to deliver a total radiation dose of 70 mCi iodine (I)-131. Subsequent pertechnetate scintigraphy showed that the normal-sized, normally functioning left lobe had disappeared after radioiodine, whereas the enlarged right lobe appeared unchanged. During the following years the size of the right lobe increased, and compression symptoms developed. The thyroid gland finally had to be removed by surgery. A large solitary thyroid nodule was removed, but no left lobe was identified. After surgery the patient had no thyroid tissue and had to be substituted by thyroid hormones. Despite good results of iodine treatment of non-toxic goiters, this case describes an unintended outcome leaving a patient without thyroid tissue, and a protracted course could have been avoided if the patient had undergone surgery earlier. However, this reported case should not discredit the use of radioiodine treatment of non-toxic goiters, but focus on patients with a single large solitary adenoma in whom this treatment may be inappropriate.

[Regional function in thyroid surgery. Assessment of the specialty function and surgical risk factors]. / Landsdelsfunktion i thyroideakirurgi. Evaluering af specialefunktionen og kirurgiske risikofaktorer.

INTRODUCTION: It is recommended that a subset of benign thyroid operations, defined by The Danish National Board as potentially complicated thyroid surgery, is referred to surgical units specialised in thyroid surgery. The aim of the present study was to compare indications for operation and operative complications in patients referred as high risk operations with patients referred from the primary catchment area to a surgical unit specialising in thyroid surgery. MATERIAL AND METHODS: The study includes 570 consecutive operations performed between January 1st 1994 and December 31st 1998. RESULTS: Out of 570 operations, 239 were referred as high risk operations. Complications were significantly more frequent after high risk operations. Delayed wound bleeding requiring reoperation occurred in 3.3 per cent vs. 0.3 per cent of cases (p = 0.01), whereas the risk of unilateral recurrent nerve palsy (1 per cent vs 0.5 per cent, p = 0.3) and permanent hypocalcemia in 1.7 per cent vs. 0 per cent (p = 0.06) was statistically insignificant between the two risk groups. DISCUSSION: The study confirms an elevated risk of complications in the defined high risk group and demonstrates that the referring hospitals comply with the recommendations laid down by the National Board of Health.

Axillary sentinel node identification in breast cancer patients: degree of radioactivity present at biopsy is critical.

BACKGROUND: The radioactivity present in the patient (Act(rem) ) at sentinel node (SN) biopsy will depend on injected activity amount as well as on the time interval from tracer injection to biopsy, which both show great variations in the literature. The purpose of this study was to analyse the influence of varying Act(rem) levels on the outcome of axillary SN biopsy in patients with breast cancer (BC). MATERIAL AND METHODS: Eight hundred and fifty-eight patients with BC were consecutively referred to SN biopsy, 21% for a same-day and 79% of the patients for a 2-day procedure. Four hundred and nineteen patients underwent scintigraphy and 439 did not. For same-day procedures, 50 MBq (99m) Tc-nanocolloid (Nanocoll(®) ) was injected, and for 2-day procedures 110 MBq. For the analysis of SN biopsy outcome, the patients were divided into three Act(rem) groups: <10 (56% of the patients), 10-20 (23%), and >20 MBq (21%). During surgery, SNs were located using a hand-held gamma probe supported by image information when available and blue dye injection. Pathology included haematoxylin-eosin staining followed by immunohistochemistry. RESULTS: The number of SNs removed (mean value 1·87 versus 2·14, P = 0·0003) and the probability of finding a malignant SN (P = 0·034) were lower in the <10 MBq group of patients compared with higher Act(rem) >20 MBq. Of the 25 patients with SN non-detection, 20 patients had an Act(rem) <10 MBq. Imaging had no significant influence on the number of patients with a malignant SN (P = 0·48). CONCLUSION: Act(rem) above 10 MBq for nanocolloid tracer appears important for appropriate identification of SNs in patients with BC.

[Sentinel node technique in local relapse after breast-conserving surgery]. / Sentinel node ved lokalrecidiv af mammacancer efter brystbevarende operation.

Mastectomy is the treatment of choice in local relapse after breast-conserving surgery. We present two cases where the sentinel node technique was used. The first case presents with one negative SN after axillary dissection 11 years earlier. The second case presents with a new SN with micrometastases and axillary dissection with 11 negative nodes 2 years after a negative SN procedure. We recommend the use of the sentinel node technique in surgery for relapse in order to optimize the staging.

Apocrine cysts of the breast: biomarkers, origin, enlargement, and relation with cancer phenotype.

Up to one-third of women aged 30-50 years have cysts in their breasts and are presumed to be at increased risk of developing breast cancer. Here we present an extensive proteomic and immunohistochemistry (IHC) study of breast apocrine cystic lesions aimed at generating specific biomarkers and elucidating the relationship, if existent, of apocrine cysts with cancer phenotype. To this end we compared the expression profiles of apocrine macrocysts obtained from mastectomies from high risk cancer patients with those of cancerous and non-malignant mammary tissue biopsies collected from the same patients. We identified two biomarkers, 15-hydroxyprostaglandin dehydrogenase and 3-hydroxymethylglutaryl-CoA reductase, that were expressed specifically by apocrine type I cysts as well as by apocrine metaplastic cells in type II microcysts, terminal ducts, and intraductal papillary lesions. No expression of these markers was observed in non-malignant terminal ductal lobular units, type II flat cysts, stroma cells, or fat tissue as judged by IHC analysis of matched non-malignant tissue samples collected from 93 high risk patients enrolled in our cancer program. IHC analysis of the corresponding 93 primary tumors indicated that most apocrine changes have little intrinsic malignant potential, although some may progress to invasive apocrine cancer. None of the apocrine lesions examined, however, seemed to be a precursor of invasive ductal carcinomas, which accounted for 81% of the tumors analyzed. Our studies also provided some insight into the origin, development, and enlargement of apocrine cysts in mammary tissue. The successful identification of differentially expressed proteins that characterize specific steps in the progression from early benign lesions to apocrine cancer opens a window of opportunity for designing and testing new approaches for pharmacological intervention, not only in a therapeutic setting but also for chemoprevention, to inhibit cyst development as both 15-hydroxyprostaglandin dehydrogenase and 3-hydroxymethylglutaryl-CoA reductase are currently being targeted for chemoprevention strategies in various malignancies.

Identification of extracellular and intracellular signaling components of the mammary adipose tissue and its interstitial fluid in high risk breast cancer patients: toward dissecting the molecular circuitry of epithelial-adipocyte stromal cell interactions.

It has become clear that growth and progression of breast tumor cells not only depend on their malignant potential but also on factors present in the tumor microenvironment. Of the cell types that constitute the mammary stroma, the adipocytes are perhaps the least well studied despite the fact that they represent one of the most prominent cell types surrounding the breast tumor cells. There is compelling evidence demonstrating a role for the mammary fat pad in mammary gland development, and some studies have revealed the ability of fat tissue to augment the growth and ability to metastasize of mammary carcinoma cells. Very little is known, however, about which factors adipocytes produce that may orchestrate these actions and how this may come about. In an effort to shed some light on these questions, we present here a detailed proteomic analysis, using two-dimensional gel-based technology, mass spectrometry, immunoblotting, and antibody arrays, of adipose cells and interstitial fluid of fresh fat tissue samples collected from sites topologically distant from the tumors of high risk breast cancer patients that underwent mastectomy and that were not treated prior to surgery. A total of 359 unique proteins were identified, including numerous signaling molecules, hormones, cytokines, and growth factors, involved in a variety of biological processes such as signal transduction and cell communication; energy metabolism; protein metabolism; cell growth and/or maintenance; immune response; transport; regulation of nucleobase, nucleoside, and nucleic acid metabolism; and apoptosis. Apart from providing a comprehensive overview of the mammary fat proteome and its interstitial fluid, the results offer some insight as to the role of adipocytes in the breast tumor microenvironment and provide a first glance of their molecular cellular circuitry. In addition, the results open new possibilities to the study of obesity, which has a strong association with type 2 diabetes, hypertension, and coronary heart disease.

Smoking is a risk factor for recurrence of groin hernia.

Studies of connective tissue from patients with inguinal hernia have shown that smoking may be associated with hernia formation due to a defective connective tissue metabolism. Whether smoking is a risk factor for recurrence, too, was examined in this study. From December 1990 through December 1995, 649 patients underwent hernia repair as open sutured repair (Cooper ligament or abdominal ring repair) or as open mesh repair. Five hundred forty-four eligible patients were evaluated for recurrence 2 years postoperatively. Association between recurrence and 17 patient-, disease-, and intraoperative variables were analyzed by multiple logistic regression. The results showed that smoking was significantly and independently associated with recurrence compared to nonsmoking [odds ratio (OR = 2.22; 95% confidence interval (95% CI) = 1.19-4.15)]. Open sutured repair compared to open mesh repair was the most significant predictor for recurrence (OR = 7.23; 95% CI = 3.01-17.37). Surprisingly, local anesthesia was associated with a higher risk of recurrence compared to general anesthesia (OR = 2.44; 95% CI = 1.19-5.09). Potential confounders and other risk factors for hernia recurrence such as age, alcohol consumption, previous surgery, and anatomical characteristics of the hernia were adjusted for in the analysis. In conclusion, smoking is an important risk factor for recurrence of groin hernia, presumably due to an abnormal connective tissue metabolism in smokers.

Thromboembolic prophylaxis as a risk factor for postoperative complications after breast cancer surgery.

Hematoma and bruising (sugillation) are frequent problems after operations for primary breast cancer. In the present study we evaluated the influence of various methods of perioperative thromboembolic prophylaxis on the postoperative incidence of hematoma and suggilation. From June 1994 through August 1996, a series of 425 patients consecutively operated on for primary breast cancer were included. Thromboembolic prophylaxis was low-molecular-weight heparin (LMWH) in 310 patients and thigh-long graded compression (TED) stockings in 102 patients. Postoperative complications including deep vein thrombosis, pulmonary embolism, wound hematoma, and sugillation were recorded, and 17 variables with a potential influence on complications were analyzed by logistic regression analysis. Heparin prophylaxis compared to prophylaxis with TED stockings was significantly and independently associated with postoperative hematoma [odds ratio (OR) 3, 13; 95% confidence interval (CI) 1.38-7.13] or sugillation (OR 3.34; 95% CI 1.93-5.78). No clinically overt thromboembolic complications were diagnosed. After operations for breast cancer we found that LMWH was significantly associated with postoperative hematoma and sugillation compared to TED stockings for perioperative thromboembolic prophylaxis.