Ellen Schlichting

Detection of Isolated Tumor Cells in Bone Marrow Is an Independent Prognostic Factor in Breast Cancer

PURPOSE This study was performed to disclose the clinical impact of isolated tumor cell (ITC) detection in bone marrow (BM) in breast cancer. PATIENTS AND METHODS BM aspirates were collected from 817 patients at primary surgery. Tumor cells in BM were detected by immunocytochemistry using anticytokeratin antibodies (AE1/AE3). Analyses of the primary tumor included histologic grading, vascular invasion, and immunohistochemical detection of c-erbB-2, cathepsin D, p53, and estrogen receptor (ER)/progesterone receptor (PgR) expression. These analyses were compared with clinical outcome. The median follow-up was 49 months. RESULTS ITC were detected in 13.2% of the patients. The detection rate rose with increasing tumor size (P =.011) and lymph node involvement (P <.001). Systemic relapse and death from breast cancer occurred in 31.7% and 26.9% of the BM-positive patients versus 13.7% and 10.9% of BM-negative patients, respectively (P <.001). Analyzing node-positive and node-negative patients separately, ITC positivity was associated with poor prognosis in the node-positive group and in node-negative patients not receiving adjuvant therapy (T1N0). In multivariate analysis, ITC in BM was an independent prognostic factor together with node, tumor, and ER/PgR status, histologic grade, and vascular invasion. In separate analysis of the T1N0 patients, histologic grade was independently associated with both distant disease-free survival (DDFS) and breast cancer-specific survival (BCSS), ITC detection was associated with BCSS, and vascular invasion was associated with DDFS. CONCLUSION ITC in BM is an independent predictor of DDFS and BCSS. An unfavorable prognosis was observed for node-positive patients and for node-negative patients not receiving systemic therapy. A combination of several independent prognostic factors can classify subgroups of patients into excellent and high-risk prognosis groups.

Effects of Exemestane Administered for 2 Years Versus Placebo on Bone Mineral Density, Bone Biomarkers, and Plasma Lipids in Patients With Surgically Resected Early Breast Cancer

PURPOSE To evaluate potential detrimental effects of exemestane on bone and lipid metabolism. PATIENTS AND METHODS Postmenopausal women with early breast cancer were randomly assigned to exemestane 25 mg daily or placebo for 2 years in a double-blind setting. Primary objective was to evaluate the effect of exemestane on bone mineral density. Secondary objectives were effects on bone biomarkers, plasma lipids, coagulation factors, and homocysteine. Planned size was 128 patients. RESULTS One hundred forty-seven patients were enrolled. All patients completed their 24-month visit except for those discontinuing treatment at an earlier stage. The mean annual rate of bone mineral density loss was 2.17% v 1.84% in the lumbar spine (P = .568) and 2.72% v 1.48% in the femoral neck (P = .024) in the exemestane and placebo arm, respectively. The mean change in T-score after 2 years was -0.21 for exemestane and -0.11 on placebo in the hip, and -0.30 and -0.21, respectively, in the lumbar spine. Exemestane significantly increased serum level and urinary excretion of bone resorption, but also bone formation markers. Except for a modest reduction in high-density lipoprotein cholesterol (P < .001) and apolipoprotein A1 (P = .004), exemestane had no major effect on lipid profile, homocysteine levels, or coagulation parameters. CONCLUSION Exemestane modestly enhanced bone loss from the femoral neck without significant influence on lumbar bone loss. Except for a 6% to 9% drop in plasma high-density lipoprotein cholesterol, no major effects on serum lipids, coagulation factors, or homocysteine were recorded. Bone mineral density should be assessed according to the US Preventive Services Task Force guidelines.

Immunocytochemical detection of isolated epithelial cells in bone marrow: non‐specific staining and contribution by plasma cells directly reactive to alkaline phosphatase

Detection of isolated tumour cells (TCs) in bone marrow (BM) from epithelial cancer patients by immunocytochemical (ICC) analysis seems to predict future relapse, but the reported percentages of positive BMs among patients with localized cancer show large variations and the number of detected TCs is low. This emphasizes the importance of thoroughly testing the methods in use. This study was performed to clarify to what extent positive staining of haematopoietic cells (HCs) interferes with the ICC detection of epithelial cells in BM. BM mononuclear cells (MNCs) from normal donors and stage I–II breast cancer patients were stained with anti‐cytokeratin (CK) and isotype control monoclonal antibodies (MAbs) followed by alkaline phosphatase (AP)‐based visualization of immunolabelled cells. In the ICC staining of normal donors by the anti‐CK MAbs AE1/AE3 or A45‐B/B3, rare immunoreactive cells were detected in 7/20 and 8/19 BMs, respectively. Morphological examination recognized all these cells as typical HCs. In the breast cancer patients (n=257), anti‐CK‐positive cells were detected in 26·6 per cent, excluding cells with HC morphology. Using the same morphological criteria, isotype control‐positive cells were detected in 5·4 per cent of patients. Some of the false‐positive events were further analysed and cells with strong reactivity against the AP enzyme alone were detected. Double ICC staining recognized the majority of these AP directly‐reactive cells as CD45‐negative and human Ig kappa/lambda‐positive, in accordance with the phenotype of mature plasma cells. Morphological evaluation and adequate controls are important to ensure the diagnostic specificity of micrometastases in BM. It is recommended that the number of BM MNCs included in negative controls should equal the number of cells in the diagnostic specimens.

Genomic architecture characterizes tumor progression paths and fate in breast cancer patients

This study demonstrates the relation among structural genomic alterations, molecular subtype, and clinical behavior and shows that an objective score of genomic complexity can provide independent prognostic information in breast cancer. Form and Malfunction Breast cancer is an iniquitous disease with a panoply of predisposing genetic and environmental causes, the details of which have yet to be fully understood. One of every four women will be diagnosed with breast cancer, hence the early and accurate identification of specific tumor features that may affect overall survival is imperative in achieving an optimal prognosis. A widely appreciated taxonomy in the breast cancer field has enabled the molecular discernment of five pathological subtypes; however, as research dives deeper into the chromosomal underpinnings of the disease, new classifiers are needed to augment what is known with key structural details to create a more vivid tumor landscape. Now, Russnes and colleagues have generated new algorithms that can estimate the specific genomic region as well as the architectural type of rearrangement—gains or losses of chromosome arms. A cohort of breast tumors was scored using this method, and all tumors with complex rearrangements had more whole chromosome arms affected than those without complex rearrangement. Moreover, there was an overlapping correlation with the molecular subtyping features of the tumors, and the score could confer prognostic power. Distinct molecular subtypes of breast carcinomas have been identified, but translation into clinical use has been limited. We have developed two platform-independent algorithms to explore genomic architectural distortion using array comparative genomic hybridization data to measure (i) whole-arm gains and losses [whole-arm aberration index (WAAI)] and (ii) complex rearrangements [complex arm aberration index (CAAI)]. By applying CAAI and WAAI to data from 595 breast cancer patients, we were able to separate the cases into eight subgroups with different distributions of genomic distortion. Within each subgroup data from expression analyses, sequencing and ploidy indicated that progression occurs along separate paths into more complex genotypes. Histological grade had prognostic impact only in the luminal-related groups, whereas the complexity identified by CAAI had an overall independent prognostic power. This study emphasizes the relation among structural genomic alterations, molecular subtype, and clinical behavior and shows that objective score of genomic complexity (CAAI) is an independent prognostic marker in breast cancer.

Early detection of breast cancer based on gene-expression patterns in peripheral blood cells

IntroductionExisting methods to detect breast cancer in asymptomatic patients have limitations, and there is a need to develop more accurate and convenient methods. In this study, we investigated whether early detection of breast cancer is possible by analyzing gene-expression patterns in peripheral blood cells.MethodsUsing macroarrays and nearest-shrunken-centroid method, we analyzed the expression pattern of 1,368 genes in peripheral blood cells of 24 women with breast cancer and 32 women with no signs of this disease. The results were validated using a standard leave-one-out cross-validation approach.ResultsWe identified a set of 37 genes that correctly predicted the diagnostic class in at least 82% of the samples. The majority of these genes had a decreased expression in samples from breast cancer patients, and predominantly encoded proteins implicated in ribosome production and translation control. In contrast, the expression of some defense-related genes was increased in samples from breast cancer patients.ConclusionThe results show that a blood-based gene-expression test can be developed to detect breast cancer early in asymptomatic patients. Additional studies with a large sample size, from women both with and without the disease, are warranted to confirm or refute this finding.

DNA methylation patterns in luminal breast cancers differ from non-luminal subtypes and can identify relapse risk independent of other clinical variables

The diversity of breast cancers reflects variations in underlying biology and affects the clinical implications for patients. Gene expression studies have identified five major subtypes– Luminal A, Luminal B, basal‐like, ErbB2+ and Normal‐Like. We set out to determine the role of DNA methylation in subtypes by performing genome‐wide scans of CpG methylation in breast cancer samples with known expression‐based subtypes. Unsupervised hierarchical clustering using a set of most varying loci clustered the tumors into a Luminal A majority (82%) cluster, Basal‐like/ErbB2+ majority (86%) cluster and a non‐specific cluster with samples that were also inconclusive in their expression‐based subtype correlations. Contributing methylation loci were both gene associated loci (30%) and non‐gene associated (70%), suggesting subtype dependant genome‐wide alterations in the methylation landscape. The methylation patterns of significant differentially methylated genes in luminal A tumors are similar to those identified in CD24 + luminal epithelial cells and the patterns in basal‐like tumors similar to CD44 + breast progenitor cells. CpG islands in the HOXA cluster and other homeobox (IRX2, DLX2, NKX2‐2) genes were significantly more methylated in Luminal A tumors. A significant number of genes (2853, p < 0.05) exhibited expression–methylation correlation, implying possible functional effects of methylation on gene expression. Furthermore, analysis of these tumors by using follow‐up survival data identified differential methylation of islands proximal to genes involved in Cell Cycle and Proliferation (Ki‐67, UBE2C, KIF2C, HDAC4), angiogenesis (VEGF, BTG1, KLF5), cell fate commitment (SPRY1, OLIG2, LHX2 and LHX5) as having prognostic value independent of subtypes and other clinical factors.

The prognostic significance of tumour cell detection in the peripheral blood versus the bone marrow in 733 early-stage breast cancer patients

IntroductionThe detection of circulating tumour cells (CTCs) in the peripheral blood and disseminated tumour cells (DTCs) in the bone marrow are promising prognostic tools for risk stratification in early breast cancer. There is, however, a need for further validation of these techniques in larger patient cohorts with adequate follow-up periods.MethodsWe assayed CTCs and DTCs at primary surgery in 733 stage I or II breast cancer patients with a median follow-up time of 7.6 years. CTCs were detected in samples of peripheral blood mononuclear cells previously stored in liquid-nitrogen using a previously-developed multi-marker quantitative PCR (QPCR)-based assay. DTCs were detected in bone marrow samples by immunocytochemical analysis using anti-cytokeratin antibodies.ResultsCTCs were detected in 7.9% of patients, while DTCs were found in 11.7%. Both CTC and DTC positivity predicted poor metastasis-free survival (MFS) and breast cancer-specific survival (BCSS); MFS hazard ratio (HR) = 2.4 (P < 0.001)/1.9 (P = 0.006), and BCSS HR = 2.5 (P < 0.001)/2.3 (P = 0.01), for CTC/DTC status, respectively). Multivariate analyses demonstrated that CTC status was an independent prognostic variable for both MFS and BCSS. CTC status also identified a subset of patients with significantly poorer outcome among low-risk node negative patients that did not receive adjuvant systemic therapy (MFS HR 2.3 (P = 0.039), BCSS HR 2.9 (P = 0.017)). Using both tests provided increased prognostic information and indicated different relevance within biologically dissimilar breast cancer subtypes.ConclusionsThese results support the use of CTC analysis in early breast cancer to generate clinically useful prognostic information.

Predictive and prognostic impact of TP53 mutations and MDM2 promoter genotype in primary breast cancer patients treated with epirubicin or paclitaxel.

Background TP53 mutations have been associated with resistance to anthracyclines but not to taxanes in breast cancer patients. The MDM2 promoter single nucleotide polymorphism (SNP) T309G increases MDM2 activity and may reduce wild-type p53 protein activity. Here, we explored the predictive and prognostic value of TP53 and CHEK2 mutation status together with MDM2 SNP309 genotype in stage III breast cancer patients receiving paclitaxel or epirubicin monotherapy. Experimental Design Each patient was randomly assigned to treatment with epirubicin 90 mg/m2 (n = 109) or paclitaxel 200 mg/m2 (n = 114) every 3rd week as monotherapy for 4–6 cycles. Patients obtaining a suboptimal response on first-line treatment requiring further chemotherapy received the opposite regimen. Time from last patient inclusion to follow-up censoring was 69 months. Each patient had snap-frozen tumor tissue specimens collected prior to commencing chemotherapy. Principal Findings While TP53 and CHEK2 mutations predicted resistance to epirubicin, MDM2 status did not. Neither TP53/CHEK2 mutations nor MDM2 status was associated with paclitaxel response. Remarkably, TP53 mutations (p = 0.007) but also MDM2 309TG/GG genotype status (p = 0.012) were associated with a poor disease-specific survival among patients having paclitaxel but not patients having epirubicin first-line. The effect of MDM2 status was observed among individuals harbouring wild-type TP53 (p = 0.039) but not among individuals with TP53 mutated tumors (p>0.5). Conclusion TP53 and CHEK2 mutations were associated with lack of response to epirubicin monotherapy. In contrast, TP53 mutations and MDM2 309G allele status conferred poor disease-specific survival among patients treated with primary paclitaxel but not epirubicin monotherapy.

Monitoring of tissue oxygenation in shock: An experimental study in pigs

OBJECTIVE To evaluate different methods and markers for assessing adequacy of tissue oxygenation in shock. DESIGN Prospective, controlled animal trial. Two groups of six pigs, subjected to either a superior mesenteric artery occlusion shock or a hemorrhagic shock. A third group of five pigs served as controls. SETTING Hospital animal research laboratory. SUBJECTS Anesthetized, ventilated, juvenile, domestic pigs. INTERVENTIONS Clamping of the superior mesenteric artery for 5 hrs, followed by reperfusion or withdrawal of blood to achieve a mean arterial pressure of 50 mm Hg for 3 hrs was performed, followed by resuscitation using the withdrawn whole blood. Invasive hemodynamic monitoring with arterial and pulmonary artery catheters was done. A tonometer was placed in the terminal ileum. MEASUREMENTS AND MAIN RESULTS Ileal intramucosal pH, systemic base excess (or deficit), lactate concentration in systemic venous and arterial blood as well as in portal blood, ascitic fluid, and thoracic duct lymph, hemodynamics, and oxygen-related variables were measured. Five hours of intestinal ischemia caused no significant changes compared with the control group with regard to base excess or any of the hemodynamic or oxygen-related variables measured. However, lactate concentrations in the ascitic fluid and intramucosal pH were significantly altered within 1 hr of regional ischemia. Lactate concentration in the thoracic duct lymph was significantly increased after 2 hrs of ischemia, while lactate concentrations in the portal, systemic, and arterial blood were significantly increased after 2 hrs of regional ischemia. Reperfusion was associated with a high mortality rate, and only one animal survived the reperfusion period. In the hemorrhagic shock group, cardiac output and mean arterial pressure were significantly (intentionally) decreased 60 mins after the hemorrhage, while the heart rate, base excess, and systemic and portal blood lactate concentrations were significantly increased after 2 hrs of general hypoperfusion compared with those values in the control group. Ileal intramucosal pH, mixed venous oxygen saturation, oxygen delivery, oxygen extraction, and lactate concentrations in the arterial blood and thoracic duct lymph were significantly different from those values in the control group 3 hrs after the onset of hemorrhagic shock. Reperfusion induced a normalization of the hemodynamic and metabolic status of the animals. CONCLUSION Many conventional markers of tissue hypoxia are useful when assessing general hypoperfusion, whereas intestinal intramucosal pH is the only reliable and clinically useful indicator of inadequate regional intestinal tissue oxygenation.

Effect of mammography screening on surgical treatment for breast cancer in Norway: comparative analysis of cancer registry data

Objective To determine the effect of mammography screening on surgical treatment for breast cancer. Design Comparative analysis of data from Norwegian cancer registry. Setting Mammography screening, Norway (screening of women aged 50-69 was introduced sequentially from 1996 to 2004). Participants 35 408 women aged 40-79 with invasive breast cancer or ductal carcinoma in situ treated surgically from 1993 to 2008. Main outcome measures Rates of breast surgery (mastectomy plus breast conserving treatment) and rates of mastectomy for three age groups of women: 40-49, 50-69, and 70-79. Changes in rates from pre-screening period (1993-5) to introduction of screening phase (1996-2004) and then to screening period (2005-8) are presented as hazard ratios in invited and non-invited women. Results The annual rate for breast surgery from the pre-screening period (1993-5) to screening period (2005-8) in Norway increased by 70% (hazard ratio 1.70, 95% confidence interval 1.62 to 1.78), from 180 to 305 per 100 000 women in the invited age group (50-69 years). In the younger, non-invited age group (40-49 years), however, the increase was only 8% (1.08, 1.00 to 1.16), from 133 to 144 per 100 000 women per year, whereas in the older, non-invited age group (70-79 years) the rate decreased by 8% (0.92, 0.86 to 1.00), from 227 to 214 per 100 000 women per year. The rates for mastectomy decreased similarly from the pre-screening period to screening period in invited and non-invited women. From the pre-screening period to the introduction phase of screening (1996-2004), however, the annual mastectomy rate in women aged 50-69 invited to screening increased by 9% (1.09, 1.03 to 1.14), from 156 to 167 per 100 000 women, and in the younger non-invited women declined by 17% (0.83, 0.78 to 0.90), from 109 to 91 per 100 000 women. In consequence, the mastectomy rate was 31% (1.31, 1.20 to 1.43) higher in the invited than in the non-invited younger age group. Conclusions Mammography screening in Norway was associated with a noticeable increase in rates for breast cancer surgery in women aged 50-69 (the age group invited to screening) and also an increase in mastectomy rates. Although over-diagnosis is likely to have caused the initial increase in mastectomy rates and the overall increase in surgery rates in the age group screened, the more recent decline in mastectomy rates has affected all age groups and is likely to have resulted from changes in surgical policy.