Claudia Rauh

Circulating micro-RNAs as potential blood-based markers for early stage breast cancer detection.

Introduction MicroRNAs (miRNAs, miRs) are a class of small, non-coding RNA molecules with relevance as regulators of gene expression thereby affecting crucial processes in cancer development. MiRNAs offer great potential as biomarkers for cancer detection due to their remarkable stability in blood and their characteristic expression in many different diseases. We investigated whether microarray-based miRNA profiling on whole blood could discriminate between early stage breast cancer patients and healthy controls. Methods We performed microarray-based miRNA profiling on whole blood of 48 early stage breast cancer patients at diagnosis along with 57 healthy individuals as controls. This was followed by a real-time semi-quantitative Polymerase Chain Reaction (RT-qPCR) validation in a separate cohort of 24 early stage breast cancer patients from a breast cancer screening unit and 24 age matched controls using two differentially expressed miRNAs (miR-202, miR-718). Results Using the significance level of p<0.05, we found that 59 miRNAs were differentially expressed in whole blood of early stage breast cancer patients compared to healthy controls. 13 significantly up-regulated miRNAs and 46 significantly down-regulated miRNAs in our microarray panel of 1100 miRNAs and miRNA star sequences could be detected. A set of 240 miRNAs that was evaluated by radial basis function kernel support vector machines and 10-fold cross validation yielded a specificity of 78.8%, and a sensitivity of 92.5%, as well as an accuracy of 85.6%. Two miRNAs were validated by RT-qPCR in an independent cohort. The relative fold changes of the RT-qPCR validation were in line with the microarray data for both miRNAs, and statistically significant differences in miRNA-expression were found for miR-202. Conclusions MiRNA profiling in whole blood has potential as a novel method for early stage breast cancer detection, but there are still challenges that need to be addressed to establish these new biomarkers in clinical use.

Ki67, chemotherapy response, and prognosis in breast cancer patients receiving neoadjuvant treatment

BackgroundThe pathological complete response (pCR) after neoadjuvant chemotherapy is a surrogate marker for a favorable prognosis in breast cancer patients. Factors capable of predicting a pCR, such as the proliferation marker Ki67, may therefore help improve our understanding of the drug response and its effect on the prognosis. This study investigated the predictive and prognostic value of Ki67 in patients with invasive breast cancer receiving neoadjuvant treatment for breast cancer.MethodsKi67 was stained routinely from core biopsies in 552 patients directly after the fixation and embedding process. HER2/neu, estrogen and progesterone receptors, and grading were also assessed before treatment. These data were used to construct univariate and multivariate models for predicting pCR and prognosis. The tumors were also classified by molecular phenotype to identify subgroups in which predicting pCR and prognosis with Ki67 might be feasible.ResultsUsing a cut-off value of > 13% positively stained cancer cells, Ki67 was found to be an independent predictor for pCR (OR 3.5; 95% CI, 1.4, 10.1) and for overall survival (HR 8.1; 95% CI, 3.3 to 20.4) and distant disease-free survival (HR 3.2; 95% CI, 1.8 to 5.9). The mean Ki67 value was 50.6 ± 23.4% in patients with pCR. Patients without a pCR had an average of 26.7 ± 22.9% positively stained cancer cells.ConclusionsKi67 has predictive and prognostic value and is a feasible marker for clinical practice. It independently improved the prediction of treatment response and prognosis in a group of breast cancer patients receiving neoadjuvant treatment. As mean Ki67 values in patients with a pCR were very high, cut-off values in a high range above which the prognosis may be better than in patients with lower Ki67 values may be hypothesized. Larger studies will be needed in order to investigate these findings further.

On a dark-field signal generated by micrometer-sized calcifications in phase-contrast mammography

We show that a distribution of micrometer-sized calcifications in the human breast which are not visible in clinical x-ray mammography at diagnostic dose levels can produce a significant dark-field signal in a grating-based x-ray phase-contrast imaging setup with a tungsten anode x-ray tube operated at 40 kVp. A breast specimen with invasive ductal carcinoma was investigated immediately after surgery by Talbot-Lau x-ray interferometry with a design energy of 25 keV. The sample contained two tumors which were visible in ultrasound and contrast-agent enhanced MRI but invisible in clinical x-ray mammography, in specimen radiography and in the attenuation images obtained with the Talbot-Lau interferometer. One of the tumors produced significant dark-field contrast with an exposure of 0.85 mGy air-kerma. Staining of histological slices revealed sparsely distributed grains of calcium phosphate with sizes varying between 1 and 40 μm in the region of this tumor. By combining the histological investigations with an x-ray wave-field simulation we demonstrate that a corresponding distribution of grains of calcium phosphate in the form of hydroxylapatite has the ability to produce a dark-field signal which would-to a substantial degree-explain the measured dark-field image. Thus we have found the appearance of new information (compared to attenuation and differential phase images) in the dark-field image. The second tumor in the same sample did not contain a significant fraction of these very fine calcification grains and was invisible in the dark-field image. We conclude that some tumors which are invisible in x-ray absorption mammography might be detected in the x-ray dark-field image at tolerable dose levels.

Association of complementary methods with quality of life and life satisfaction in patients with gynecologic and breast malignancies

Goals of workIn gynecological oncology, there is growing interest in the use of complementary and alternative medicine (CAM) methods. The lack of data regarding side effects, the lack of any survival advantages, and the costs of these methods appear to have no influence on patients’ decisions on whether to use CAM. Our interest was to evaluate the association between CAM use and the patients’ quality of life/life satisfaction (QoL/LS).Materials and methodsOne thousand thirty women with breast cancer of gynecologic malignancies were asked to participate in this study, which included a questionnaire and a personal interview on CAM. User status was compared with the patient’s own description of her QoL/LS and with the cancer type.Main resultsCAM was used by 48.7% of all women (n = 502). Breast cancer patients stated that they used CAM in 50.1% and women with gynecological cancer in 44.0%. The use of mistletoe was widespread (77.3%) and was more often seen in breast cancer patients than in gynecological cancer patients (74.4% vs 67.0%). CAM users less frequently stated an overall deterioration of their health status (35.1%) compared to nonusers (50.1%). CAM use resulted in a stated improvement in family conditions (6%) in comparison with the nonusers (2%).ConclusionsWith regard to patients’ perception of health status, CAM use is associated with a better coping with their disease. Most other categories of LS are not affected by CAM use. Patient-oriented information comparing standard therapies with CAM methods should be made widely available, and patients’ expectations of CAM use should be discussed between the physician and the patient.

The role of genetic breast cancer susceptibility variants as prognostic factors

Recent genome-wide association studies identified 11 single nucleotide polymorphisms (SNPs) associated with breast cancer (BC) risk. We investigated these and 62 other SNPs for their prognostic relevance. Confirmed BC risk SNPs rs17468277 (CASP8), rs1982073 (TGFB1), rs2981582 (FGFR2), rs13281615 (8q24), rs3817198 (LSP1), rs889312 (MAP3K1), rs3803662 (TOX3), rs13387042 (2q35), rs4973768 (SLC4A7), rs6504950 (COX11) and rs10941679 (5p12) were genotyped for 25 853 BC patients with the available follow-up; 62 other SNPs, which have been suggested as BC risk SNPs by a GWAS or as candidate SNPs from individual studies, were genotyped for replication purposes in subsets of these patients. Cox proportional hazard models were used to test the association of these SNPs with overall survival (OS) and BC-specific survival (BCS). For the confirmed loci, we performed an accessory analysis of publicly available gene expression data and the prognosis in a different patient group. One of the 11 SNPs, rs3803662 (TOX3) and none of the 62 candidate/GWAS SNPs were associated with OS and/or BCS at P<0.01. The genotypic-specific survival for rs3803662 suggested a recessive mode of action [hazard ratio (HR) of rare homozygous carriers=1.21; 95% CI: 1.09-1.35, P=0.0002 and HR=1.29; 95% CI: 1.12-1.47, P=0.0003 for OS and BCS, respectively]. This association was seen similarly in all analyzed tumor subgroups defined by nodal status, tumor size, grade and estrogen receptor. Breast tumor expression of these genes was not associated with prognosis. With the exception of rs3803662 (TOX3), there was no evidence that any of the SNPs associated with BC susceptibility were associated with the BC survival. Survival may be influenced by a distinct set of germline variants from those influencing susceptibility.

Ki-67 as a prognostic molecular marker in routine clinical use in breast cancer patients

INTRODUCTION The proliferation biomarker Ki-67 is a prognostic factor for breast cancer that has been investigated in several retrospective studies and a few prospective ones. The aims of the present study were to examine interactions between Ki-67 and other biomarkers in breast cancer patients and to assess the relationship of Ki-67 to histological grading. PATIENTS AND METHODS Patients with uniform immunohistochemical staining of Ki-67 by MIB-1 were identified from the database of the University Breast Center for Franconia. Data were available for 1232 of 2523 patients with invasive breast cancer who had been treated between 1998 and 2005. Ki-67 index was determined during routine work-up of the breast cancers by several surgical pathologists according to a standardized procedure. The Ki-67 proliferation index was correlated with hormone receptor status, HER2/neu status, age, tumor staging, and prognosis. In routine clinical practice, the grading was assessed according to Elston and Ellis, along with all other parameters. RESULTS Ki-67 proliferation index>or=20% was found to be associated with all of the prognostic factors that were tested. However, it also maintained statistical significance relative to poor overall survival in a multivariate Cox proportional hazards model (hazards ratio 1.81; 95% CI, 1.17-2.78). The hazards ratio for disease-free survival did not reach statistical significance (HR 1.41; 95% CI, 0.95-2.09; P=0.086). However, in both models the grade was not an independent prognostic factor. CONCLUSIONS For routine clinical purposes, grading appears to add only limited information about the prognosis in comparison with Ki-67 expression. Further investigation of quality assurance for grading and of Ki-67 as a prognostic and predictive factor is warranted.

Quality Assured Health Care in Certified Breast Centers and Improvement of the Prognosis of Breast Cancer Patients

Background: Increasing effort has been put in the implementation and certification of breast centers in order to establish standardized, quality assured health care for breast cancer patients. The aim of this analysis was to investigate whether patients treated in certified breast centers (CBC) have a favorable prognosis as compared to patients treated outside of certified breast treatment units. Patients and Methods: The data of 3,940 patients with invasive nonmetastatic breast cancer were analyzed with regard to differences in patient and tumor characteristics and crude overall survival according to diagnosis in or outside CBC in Middle Franconia, Germany. Patient, tumor, and follow-up data were obtained from the clinical cancer registry. Results: Patients in CBC were younger, and had lower disease stages and lower grading. Independent of the effects of these variables on overall survival, being treated at a CBC added to the prediction of overall survival. Patients treated at a CBC had a hazard ratio of 0.70 (95% confidence interval 0.52–0.93) in the adjusted Cox model. Conclusions: Independent from common prognostic factors, diagnosis and treatment of breast cancer at a CBC improves the prognosis of patients. It can be hypothesized that this effect is mediated through quality assured health care provided by the certification process.

Mammographic density as a risk factor for breast cancer in a German case-control study.

Mammographic percent density (MD) is recognized as one of the strongest risk factors associated with breast cancer. This matched case–control study investigated whether MD represents an independent risk factor. Mammograms were obtained from 1025 breast cancer patients and from 520 healthy controls. MD was measured using a quantitative computer-based threshold method (0–100%). Breast cancer patients had a higher MD than healthy controls (38 vs. 32%, P<0.01). MD was significantly higher in association with factors such as age over 60 years, body mass index (BMI) of 25–30 kg/m2, nulliparity or low parity (one to two births). Average MD was inversely associated with age, BMI, parity and positively associated with age at first full-term pregnancy. MD was higher in women with at least one first-degree relative affected, but only among patients and not in the group of healthy controls (P<0.01/P=0.61). In women with an MD of 25% or more, the risk of breast cancer was doubled compared with women with an MD of less than 10% (odds ratio: 2.1; 95% confidence interval: 1.3–3.4; P<0.01); in the postmenopausal subgroup, the risk was nearly tripled (odds ratio: 2.7; 95% confidence interval: 1.6–4.7; P<0.001). This study provides further evidence that MD is an important risk factor for breast cancer. These results indicate strong associations between MD and the risk of breast cancer in a matched case–control study in Germany.

Grating-based darkfield imaging of human breast tissue

Mastectomy specimens were investigated using a Talbot-Lau X-ray imaging set-up. Significant structures in the darkfield were observed, which revealed considerably higher contrast than those observed in digital mammography. Comparison with the histomorphometric image proofs that the darkfield signal correlates with a tumor region containing small calcification grains of 3 to 30μm size.

Pain perception and detailed visual pain mapping in breast cancer survivors

Chronic pain and neural irritation after breast surgery and radiation are still relevant sequelae of the treatment. Pain quantification and localization in patient groups are difficult to standardize. In order to quantify and localize pain in a group of breast cancer patients, a Java-based program was developed to visualize the frequency of pain in “pain maps.” A questionnaire with structured questions on the perception of pain included pictograms of a body to mark possible pain areas. A group of 343 breast cancer survivors completed the questionnaires. The image information was digitalized and processed using a Java applet. Gray-scale summation pictures with numbers from “0,” indicating black (100% pain), to “255,” indicating white (0% pain), were generated. The visualization of pain by creating pain maps revealed the location of pain in breast cancer survivors on pictograms of the body. Analyzing the total number of pixels, in which pain was stated, made it possible to compare pain areas in several subgroups, showing that patients after mastectomy versus breast-conserving therapy (3,011 vs. 2,224 pixels), and patients with lymphedema versus patients without lymphedema (3,010 vs. 2,239 pixels), have larger pain areas. This study presents a method of visualizing pain areas and assigning them to a pictogram of the body in a sample of breast cancer patients. The method is easy to use and could help generate pain maps in several types of disease.