Phosphorylated CXCR4 expression has a positive prognostic impact in colorectal cancer.

BACKGROUND: The CXCL12-CXCR4 chemokine axis plays an important role in cell trafficking as well as in tumor progression. In colorectal cancer (CRC), the chemokine receptor CXCR4 has been shown to be an unfavorable prognostic factor in some studies, however, the role of its activated (phosphorylated) form, pCXCR4, has not yet been evaluated. Here, we aimed to investigate the prognostic value of CXCR4 and pCXCR4 in a large cohort of CRC patients. PATIENTS AND METHODS: A tissue microarray (TMA) of 684 patient specimens of primary CRCs was analyzed by immunohistochemistry (IHC) for the expression of CXCR4 and pCXCR4 by tumor cells and tumor-infiltrating immune cells (TICs). RESULTS: The combined high expression of CXCR4 and pCXCR4 showed a favorable 5-year overall survival rate (68%; 95%CI = 59-76%) compared to tumors showing a high expression of CXCR4 only (48%; 95%CI = 41-54%). High expression of pCXCR4 was significantly associated with a favorable prognosis in a test and validation group (p = 0.015 and p = 0.0001). Moreover, we found that CRCs with a high density of pCXCR4+ tumor-infiltrating immune cells (TICs) also showed a favorable prognosis in a test and validation group (p = 0.054 and p = 0.004). Univariate Cox regression analysis for TICs revealed that a high density of pCXCR4+ TICs was a favorable prognostic marker for overall survival (HR = 0.97,95%CI = 0.96-1.00; p = 0.01). In multivariate Cox regression survival analyses a high expression of pCXCR4 in tumor cells lost its association with a better overall survival (HR = 0.99; 95%CI = 0.99-1.00, p = 0.098). CONCLUSION: Our results show that high densities of CXCR4 and pCXCR4 positive TICs are favorable prognostic factors in CRC.

Dual role of tumour-infiltrating T helper 17 cells in human colorectal cancer.

BACKGROUND: The immune contexture predicts prognosis in human colorectal cancer (CRC). Whereas tumour-infiltrating CD8+ T cells and myeloid CD16+ myeloperoxidase (MPO)+ cells are associated with favourable clinical outcome, interleukin (IL)-17-producing cells have been reported to correlate with severe prognosis. However, their phenotypes and functions continue to be debated. OBJECTIVE: To investigate clinical relevance, phenotypes and functional features of CRC-infiltrating, IL-17-producing cells. METHODS: IL-17 staining was performed by immunohistochemistry on a tissue microarray including 1148 CRCs. Phenotypes of IL-17-producing cells were evaluated by flow cytometry on cell suspensions obtained by enzymatic digestion of clinical specimens. Functions of CRC-isolated, IL-17-producing cells were assessed by in vitro and in vivo experiments. RESULTS: IL-17+ infiltrates were not themselves predictive of an unfavourable clinical outcome, but correlated with infiltration by CD8+ T cells and CD16+ MPO+ neutrophils. Ex vivo analysis showed that tumour-infiltrating IL-17+ cells mostly consist of CD4+ T helper 17 (Th17) cells with multifaceted properties. Indeed, owing to IL-17 secretion, CRC-derived Th17 triggered the release of protumorigenic factors by tumour and tumour-associated stroma. However, on the other hand, they favoured recruitment of beneficial neutrophils through IL-8 secretion and, most importantly, they drove highly cytotoxic CCR5+CCR6+CD8+ T cells into tumour tissue, through CCL5 and CCL20 release. Consistent with these findings, the presence of intraepithelial, but not of stromal Th17 cells, positively correlated with improved survival. CONCLUSIONS: Our study shows the dual role played by tumour-infiltrating Th17 in CRC, thus advising caution when developing new IL-17/Th17 targeted treatments.

Disease-related surgery in patients with distant metastatic breast cancer.

INTRODUCTION: This study evaluates the frequency of and indications for disease-related surgical procedures in the palliative breast cancer (BC) situation. PATIENTS & METHODS: Based on a cohort of women who were treated for newly diagnosed BC during a 20-year period (1990-2009), we analyzed 340 patients who developed distant metastatic disease (DMD) until 2011 and died (i.e. still ongoing palliative disease courses were not included). RESULTS: One hundred and twenty-seven surgical procedures were performed in 100 patients (29.4% of all patients with metastatic disease). The most common site for surgery was breast (n = 60, 47.2%). The primary tumor was removed at first diagnosis of DMD in 43 patients (33.9%); sixteen operations (12.6%) were performed for local recurrence. In 37 patients, 50 surgical procedures (39.4%) were necessary to stabilize osseous structures due to metastases. Procedures were rarely performed on other common metastatic sites: lung: n = 1 (0.8%); liver: n = 1 (0.8%), brain: n = 4 (3.1%). When excluding surgery for primary breast tumors at initial diagnosis of DMD from analysis, 34 of 84 surgeries (40.4%) were performed in the first third of survival follow-up (i.e. period of metastatic disease survival); operations in the last two-thirds each totaled 29.8% (n = 25). The median survival after surgery was 16 months (range: 0.5-89 months). CONCLUSIONS: In a cohort of BC patients who had primary or developed secondary DMD, nearly one third of the patients received disease-related surgical procedures during their palliative disease course. This high rate of operations shows that surgery has a clearly established role in the palliative therapy concept.

[Assessment of proliferation: core biopsy or resection specimen? Discrepancies in breast cancer with low and high proliferation]. / Bestimmung der Proliferation: Stanze oder Resektat?: Diskrepanzen bei niedrig und stark proliferierenden Mammakarzinomen.

BACKGROUND: The assessment of the proliferation fraction is becoming more and more important; however, there is no consensus concerning optimal validation. Depending on the institute the proliferation fraction is determined either from a core biopsy (SB) or resection specimen (OP). PATIENTS AND METHODS: The interobserver variability and the results of SB and OP were investigated whereby two pathologists independently estimated the proliferation fraction of 90 cases of invasive breast cancer. The results (Ki-67) were quantified, categorized, and compared. RESULTS: Identical (accuracy of 5% steps) results between the 2 pathologists were achieved in 43% (n=39) for SB, 47% (n=42) for OP and 60% (n=54) for SB versus OP. When categorizing the proliferation fraction (low ≤ 15%, moderate 20-30% and high ≥ 35%) the following results were achieved: 76% (n=68) for SB, 82% (n=74) for OP and 81% (n=73) for SB versus OP. CONCLUSIONS: There was a clear interobserver variability (SB: kappa=0.32, OP: kappa=0.34) but this could be dramatically improved by forming proliferation categories (SB: kappa=0.62, OP: kappa=0.72, 60 versus 81%). In SB with a low proliferation fraction a repeated analysis in OPs can be advisable as a higher proliferation fraction is observed in up to 12% of OPs.

Harmonisation of multi-centre real-time reverse-transcribed PCR results of a candidate prognostic marker in breast cancer: an EU-FP6 supported study of members of the EORTC - PathoBiology Group.

AIM: Assessment of intra- and inter-laboratory variation in multi-centre real-time reverse-transcribed PCR (qRT-PCR)-based mRNA quantification of a prognostic marker in breast cancer using external quality assurance (EQA). METHODS: A questionnaire on the methodologies used and EQA calibrators were sent to 5 participating laboratories from 4 European countries, which measured mRNA levels of PITX2 splice variants and reference genes by qRT-PCR. RESULTS: Differences in the methodology included PCR quantification methodology and equipment, RNA extraction and cDNA synthesis procedures. The intra-laboratory coefficient of variation (CV) ranged from 5 to 23%, and the inter-laboratory CV ranged from 17 to 30%. The inter-laboratory CV was reduced to 13% by using prediluted calibrators and by harmonising the data in the central QA laboratory. Additional normalisation using reference genes did not decrease the variation further. CONCLUSIONS: Both externally provided calibrators and centralised harmonisation are required to reduce the intra-laboratory variation in multi-centre qRT-PCR results to an acceptable level.

Simultaneous quantitative detection of relevant biomarkers in breast cancer by quantitative real-time PCR.

The assessment of ERa, PgR and HER2 status is routinely performed today to determine the endocrine responsiveness of breast cancer samples. Such determination is usually accomplished by means of immunohistochemistry and in case of HER2 amplification by means of fluorescent in situ hybridization (FISH). The analysis of these markers can be improved by simultaneous measurements using quantitative real-time PCR (Qrt-PCR). In this study we compared Qrt-PCR results for the assessment of mRNA levels of ERa, PgR, and the members of the human epidermal growth factor receptor family, HER1, HER2, HER3 and HER4. The results were obtained in two independent laboratories using two different methods, SYBR Green I and TaqMan probes, and different primers. By linear regression we demonstrated a good concordance for all six markers. The quantitative mRNA expression levels of ERa, PgR and HER2 also strongly correlated with the respective quantitative protein expression levels prospectively detected by EIA in both laboratories. In addition, HER2 mRNA expression levels correlated well with gene amplification detected by FISH in the same biopsies. Our results indicate that both Qrt-PCR methods were robust and sensitive tools for routine diagnostics and consistent with standard methodologies. The developed simultaneous assessment of several biomarkers is fast and labor effective and allows optimization of the clinical decision-making process in breast cancer tissue and/or core biopsies.

The NFkappaB pathway and endocrine-resistant breast cancer.

Endocrine therapy with an estrogen receptor (ER)-targeted antiestrogen, such as tamoxifen, or estrogen ablation by aromatase inhibitors is clinically indicated for the management of all forms of ER-positive breast cancer. However, 30-50% of ER-positive breast cancer cases fail to benefit clinically from endocrine therapy alone, and recent molecular evidence suggests that 'crosstalk' pathways originating from activated receptor tyrosine kinases and/or other proliferative and survival signals may be contributing to this endocrine resistance. Molecular identification and validation of candidate ER crosstalking pathways will likely lead to clinically important prognostic markers and targets for the application of novel therapeutics in combination with standard endocrine agents. This review focuses on a critical survival and proliferation pathway involving activation of nuclear factor-kappaB (NFkappaB), a family of ubiquitously expressed transcription factors that for nearly two decades have been known to be critical regulators of mammalian immune and inflammatory responses, and more recently have been associated with chemotherapy resistance. With the demonstration that activation of NFkappaB is absolutely required for normal mammary gland development, NFkappaB involvment in human breast cancers was initially explored and linked to the development of hormone-independent (ER-negative) breast cancer. Newer clinical evidence now implicates NFkappaB activation, particularly DNA-binding by the p50 subunit of NFkappaB, as a potential prognostic marker capable of identifying a high-risk subset of ER-positive, primary breast cancers destined for early relapse despite adjuvant endocrine therapy with tamoxifen. Furthermore, initial preclinical studies suggest that treatment strategies designed to prevent or interrupt activation of NFkappaB in cell-line models of these more aggressive, ER-positive breast cancers can restore their sensitivity to such standard endocrine agents as tamoxifen.

Understanding the age dependency of breast cancer biomarkers.

This review and study of nearly 4,000 primary breast cancers evaluates the hypothesis that human aging not only increases breast cancer incidence but also alters breast cancer biology. Clinically validated biomarkers were chosen as surrogate measures of genetic instability and tumor growth, invasiveness and metastatic potential. Our results support the premise that breast cancer clinical behavior and biology are significantly affected by patient age, but call into question key aspects of the current cancer-aging paradigm.

Prognostic and predictive significance of ErbB-2 breast tumor levels measured by enzyme immunoassay.

PURPOSE: A retrospective analysis to assess the prognostic and predictive clinical value of breast tumor ErbB-2 receptor expression quantified by enzyme immunoassay (EIA), to compare levels measured by EIA with ErbB-2 status determined by immunohistochemistry (IHC), and to correlate receptor content with levels of phosphorylated (Y1248-P) ErbB-2, a measure of functional tyrosine kinase activity. MATERIALS AND METHODS: EIA quantification of ErbB-2 was performed on membrane extracts from 3,208 well-characterized primary breast cancers. Overall, relapse-free, distant disease-free, and local/regional-free patient survival data were available on 1,123 of these tumors. IHC scoring for ErbB-2 status (HercepTest; DAKO, Glostrup, Denmark) was performed on adjacent sections of 151 cases, and receptor functionality was measured in 230 tumors by an antibody specific for phosphorylated (Y1248-P) ErbB-2. RESULTS: Unlike nonmalignant breast tissues, breast tumors showed increased ErbB-2 levels in a bimodal distribution, with 12% constituting a distinct set of ErbB-2-overexpressing tumors. The intermodal threshold value for ErbB-2 overexpression distinguished tumors with reduced estrogen and progesterone receptor content, high IHC score for ErbB-2, and significantly increased levels of phosphorylated (Y1248-P) ErbB-2 receptor. By multivariate analysis, EIA-determined ErbB-2 overexpression predicted significantly reduced patient survival that was unaffected by tamoxifen or cyclophosphamide, methotrexate, and fluorouracil adjuvant therapy. CONCLUSION: Determination of ErbB-2 receptor expression by EIA offers a clinically valuable alternative to semiquantitative IHC assessment of breast tumor ErbB-2 overexpression and affords the opportunity to evaluate ErbB-2 phosphorylation, which may represent an important predictive parameter of receptor functionality.

Potential prognostic value of mitogen-activated protein kinase activity for disease-free survival of primary breast cancer patients.

Signaling through pathways involving mitogen-activated protein kinases (MAP kinases) has been implicated in the pathogenesis of cancer. Thus, the activity of MAP kinase is essential in the malignant potential of human breast tumors. p42/44(MAPK) was significantly higher expressed in tumor samples than in matching normal tissues adjacent to the tumor. p42/44(MAPK) protein expression correlated with enhanced MAP kinase activity only in a subset of tumors, indicating that over-expression of MAP kinases does not reflect the activation status of these enzymes. MAP kinase activity was significantly elevated in 131 tissue samples from primary breast tumors when compared to 18 normal tissues adjacent to tumors. A trend for higher MAP kinase activity in primary tumors of node-positive patients was observed when compared with tumors from node-negative patients. Similarly, higher MAP kinase activities were observed in specimens from patients who had a relapse within the follow-up time of 40 months when compared with patients with no relapse. A survival analysis demonstrated that the MAP kinase activity in primary breast tumors is potentially prognostic for relapse-free survival of patients.

Markers of tumor angiogenesis and proteolysis independently define high- and low-risk subsets of node-negative breast cancer patients.

PURPOSE: To compare the prognostic impact of tumor angiogenesis factors (vascular endothelial growth factor [VEGF], angiogenin, and basic fibroblast growth factor [bFGF]), tumor proteolysis factors (urokinase-type plasminogen activator [uPA] and plasminogen activator inhibitor-1 [PAI-1]), and conventional tumor markers (stage, grade, and steroid receptors) in early breast cancer. PATIENTS AND METHODS: In the primary clinical study, tumor angiogenesis and other factors were detected in frozen biopsies from 305 primary breast tumors. VEGF expression was assessed by chemiluminescence immunosorbent assay (ICMA); angiogenin, bFGF, uPA, and PAI-1 by enzyme-linked immunosorbent assay (ELISA); and steroid receptors (estrogen receptor [ER] and progesterone receptor [PgR]) by enzyme immunoassay (EIA). In the validating clinical study, another set of 190 node-negative primary breast tumor samples were collected at a separate institution. RESULTS: Univariate analysis of the primary study showed that VEGF levels were positively correlated with recurrence (P < .001). Angiogenin levels were positively correlated with disease relapse (P < .005) for the overall collective group, but not within the node-negative subset. No significant correlations were found between tumor bFGF levels and patient survival. In multivariate regression analysis, the only independent predictors of relapse-free survival (RFS) were VEGF, uPA, and lymph node status. In the validation set, the distribution of VEGF and uPA values were similar to those in the primary study; low expression of both VEGF and uPA identified patients with a < or = 20% likelihood of recurrence within 7 years. CONCLUSION: Separate primary and validating clinical studies concur that tumor VEGF level is the most important prognostic parameter among several markers of tumor angiogenesis and proteolysis.

Increased expression of estrogen-receptor exon-5-deletion variant in relapse tissues of human breast cancer.

A substantial percentage (30-70%) of human breast carcinomas that initially respond to endocrine therapy acquire resistance during the treatment. Many patients with tumor progression despite treatment with anti-estrogen tamoxifen show continued expression of estrogen receptors (ER) and/or progesterone receptors (PgR) in the relapse tissue. This indicates that, in these tumors, mechanisms other than loss of ER expression are responsible for treatment failure. We have investigated the occurrence and frequency of the exon-5-deletion variant (d5) of ER in human breast-cancer biopsies and in normal tissues. In all normal and tumor tissues tested, both wild-type (wt) and d5 were detected, indicating that expression of the d5 variant is a naturally occurring polymorphism. However, the primary tumors of patients who relapse within 15 months (n = 13) express higher ratios of d5 than do those of patients with no relapse during the same period (p = 0.4, n = 19), though this difference is statistically not significant. A significant increase in the expression level of d5 was determined in relapse as compared with the respective primary tumor (p = 0.02). These data indicate that increased expression of the ER exon-5-deletion variant in relapse tissues might be due to clonal selection of cells resistant to anti-estrogen treatment.

[Molecular factors determine primary and secondary therapy of breast carcinoma]. / Molekulare Faktoren bestimmen die primäre und sekundäre Therapie des Mammakarzinoms.

The clinical oncology realizes that the classical approach with systemic adjuvant chemo- and hormonal therapies is not sufficient and will be challenged by cellular and molecular structures which reflect the targets for new therapeutic approaches. These targets are key proteins involved in the signal transduction cascade. In human tumors these proteins have either lost their biological functionality by oncogenic mutations or are constitutively activated. The molecular classification of primary breast cancer was performed by assessing the following factors: estrogen- and progesterone receptors, ERbB-2 mutated p53, uPA, PAI-I, VEGF, DNA-Index and S-Phase. These factors are of prognostic and predictive value.

BRCA1 mutations found in archived early onset breast tumours.

Inherited mutations in the BRCA1 gene are thought to account for approximately 5% of breast cancers in women under the age of 45 years. In order to determine whether mutations could be found at the expected frequency, 60% of the protein coding region of BRCA1 was screened in 75 archived early-onset breast tumours, taken from women under 45 years of age. Two of the 75 tumours (2.7%) had detectable mutations, in close agreement to that predicted. Since BRCA1 mutations found in breast tumours are invariably germline, two immediate consequences are apparent. Firstly, family members of affected patients are likely to carry mutations as well, and should be considered for BRCA1 screening; and secondly, persons harbouring a germline BRCA1 mutation should be examined frequently and indefinitely for new primary tumours in remaining breast tissue.

Purification of the N-acetylglucosaminide alpha(1-3/4)fucosyltransferase of human milk.

The N-acetylglucosaminide alpha(1-3/4)fucosyltransferase has been purified 1.8 x 10(6)-fold from human milk by ion-exchange chromatography, affinity chromatography on GDP-agarose and HPLC. The alpha(1-3/4)fucosyltransferase behaves in gel filtration-HPLC as a molecule of M(r) 98,000, and differs from the alpha(1-3)fucosyltransferase which behaves like a molecule of about M(r) 47,000. The enzyme is a glycoprotein, and the purified preparation appears in SDS polyacrylamide gel electrophoresis as a band of M(r) 44,000. The results present the first purification of human milk alpha(1-3/4)fucosyltransferase to apparent homogeneity, and suggest that the alpha(1-3/4)- and alpha(1-3)fucosyltransferase of human milk differ in their native molecular sizes, the former being a dimer of two subunits.