Interconnectivity between molecular subtypes and tumor stage in colorectal cancer.

BACKGROUND: There are profound individual differences in clinical outcomes between colorectal cancers (CRCs) presenting with identical stage of disease. Molecular stratification, in conjunction with the traditional TNM staging, is a promising way to predict patient outcomes. We investigated the interconnectivity between tumor stage and tumor biology reflected by the Consensus Molecular Subtypes (CMSs) in CRC, and explored the possible value of these insights in patients with stage II colon cancer. METHODS: We performed a retrospective analysis using clinical records and gene expression profiling in a meta-cohort of 1040 CRC patients. The interconnectivity of tumor biology and disease stage was assessed by investigating the association between CMSs and TNM classification. In order to validate the clinical applicability of our findings we employed a meta-cohort of 197 stage II colon cancers. RESULTS: CMS4 was significantly more prevalent in advanced stages of disease (stage I 9.8% versus stage IV 38.5%, p < 0.001). The observed differential gene expression between cancer stages is at least partly explained by the biological differences as reflected by CMS subtypes. Gene signatures for stage III-IV and CMS4 were highly correlated (r = 0.77, p < 0.001). CMS4 cancers showed an increased progression rate to more advanced stages (CMS4 compared to CMS2: 1.25, 95% CI: 1.08-1.46). Patients with a CMS4 cancer had worse survival in the high-risk stage II tumors compared to the total stage II cohort (5-year DFS 41.7% versus 100.0%, p = 0.008). CONCLUSIONS: Considerable interconnectivity between tumor biology and tumor stage in CRC exists. This implies that the TNM stage, in addition to the stage of progression, might also reflect distinct biological disease entities. These insights can potentially be utilized to optimize identification of high-risk stage II colon cancers.

Association of microRNA-7 and its binding partner CDR1-AS with the prognosis and prediction of 1st-line tamoxifen therapy in breast cancer.

The large number of non-coding RNAs (ncRNAs) and their breadth of functionalities has fuelled many studies on their roles in cancer. We previously linked four microRNAs to breast cancer prognosis. One of these microRNAs, hsa-miR-7, was found to be regulated by another type of ncRNA, the circular non-coding RNA (circRNA) CDR1-AS, which contains multiple hsa-miR-7 binding sites. Based on this finding, we studied the potential clinical value of this circRNA on breast cancer prognosis in a cohort based on a cohort that was previously analysed for hsa-miR-7 and in an adjuvant hormone-naïve cohort for 1st-line tamoxifen treatment outcomes, in which we also analysed hsa-miR-7. A negative correlation was observed between hsa-miR-7 and CDR1-AS in both cohorts. Despite associations with various clinical metrics (e.g., tumour grade, tumour size, and relapse location), CDR1-AS was neither prognostic nor predictive of relevant outcomes in our cohorts. However, we did observe stromal CDR1-AS expression, suggesting a possible cell-type specific interaction. Next to the known association of hsa-miR-7 expression with poor prognosis in primary breast cancer, we found that high hsa-miR-7 expression was predictive of an adverse response to tamoxifen therapy and poor progression-free and post-relapse overall survival in patients with recurrent disease.

Tumor-Specific Mitochondrial DNA Variants Are Rarely Detected in Cell-Free DNA.

The use of blood-circulating cell-free DNA (cfDNA) as a "liquid biopsy" in oncology is being explored for its potential as a cancer biomarker. Mitochondria contain their own circular genomic entity (mitochondrial DNA, mtDNA), up to even thousands of copies per cell. The mutation rate of mtDNA is several orders of magnitude higher than that of the nuclear DNA. Tumor-specific variants have been identified in tumors along the entire mtDNA, and their number varies among and within tumors. The high mtDNA copy number per cell and the high mtDNA mutation rate make it worthwhile to explore the potential of tumor-specific cf-mtDNA variants as cancer marker in the blood of cancer patients. We used single-molecule real-time (SMRT) sequencing to profile the entire mtDNA of 19 tissue specimens (primary tumor and/or metastatic sites, and tumor-adjacent normal tissue) and 9 cfDNA samples, originating from 8 cancer patients (5 breast, 3 colon). For each patient, tumor-specific mtDNA variants were detected and traced in cfDNA by SMRT sequencing and/or digital PCR to explore their feasibility as cancer biomarker. As a reference, we measured other blood-circulating biomarkers for these patients, including driver mutations in nuclear-encoded cfDNA and cancer-antigen levels or circulating tumor cells. Four of the 24 (17%) tumor-specific mtDNA variants were detected in cfDNA, however at much lower allele frequencies compared to mutations in nuclear-encoded driver genes in the same samples. Also, extensive heterogeneity was observed among the heteroplasmic mtDNA variants present in an individual. We conclude that there is limited value in tracing tumor-specific mtDNA variants in blood-circulating cfDNA with the current methods available.

Using second harmonic generation to predict patient outcome in solid tumors.

BACKGROUND: Over-treatment of estrogen receptor positive (ER+), lymph node-negative (LNN) breast cancer patients with chemotherapy is a pressing clinical problem that can be addressed by improving techniques to predict tumor metastatic potential. Here we demonstrate that analysis of second harmonic generation (SHG) emission direction in primary tumor biopsies can provide prognostic information about the metastatic outcome of ER+, LNN breast cancer, as well as stage 1 colorectal adenocarcinoma. METHODS: SHG is an optical signal produced by fibrillar collagen. The ratio of the forward-to-backward emitted SHG signals (F/B) is sensitive to changes in structure of individual collagen fibers. F/B from excised primary tumor tissue was measured in a retrospective study of LNN breast cancer patients who had received no adjuvant systemic therapy and related to metastasis-free survival (MFS) and overall survival (OS) rates. In addition, F/B was studied for its association with the length of progression-free survival (PFS) in a subgroup of ER+ patients who received tamoxifen as first-line treatment for recurrent disease, and for its relation with OS in stage I colorectal and stage 1 lung adenocarcinoma patients. RESULTS: In 125 ER+, but not in 96 ER-negative (ER-), LNN breast cancer patients an increased F/B was significantly associated with a favorable MFS and OS (log rank trend for MFS: p = 0.004 and for OS: p = 0.03). On the other hand, an increased F/B was associated with shorter PFS in 60 ER+ recurrent breast cancer patients treated with tamoxifen (log rank trend p = 0.02). In stage I colorectal adenocarcinoma, an increased F/B was significantly related to poor OS (log rank trend p = 0.03), however this relationship was not statistically significant in stage I lung adenocarcinoma. CONCLUSION: Within ER+, LNN breast cancer specimens the F/B can stratify patients based upon their potential for tumor aggressiveness. This offers a "matrix-focused" method to predict metastatic outcome that is complementary to genomic "cell-focused" methods. In combination, this and other methods may contribute to improved metastatic prediction, and hence may help to reduce patient over-treatment.

Improved metastasis-free survival in nonadjuvantly treated postmenopausal breast cancer patients with chemokine receptor 5 del32 frameshift mutations.

The CC-chemokine receptor CCR5 has been associated with cancer progression and metastasis. CCR5 blockers such as Maraviroc are tested in metastatic cancer patients. A mutant allele of CCR5, CCR5-delta32 (CCR5del32), which encodes for a protein with a trans-dominant negative effect on the wildtype protein, is frequently found in populations of northern European origin. We set out to determine if the CCR5del32 genotype is associated with progression of breast cancer. Here, we genotyped 414 breast cancer patients and investigated whether the CCR5 genotype had an association with the likelihood to metastasize within specific subgroups of this cohort. The findings were subsequently confirmed in an independent cohort of 1,017 breast cancer patients. Specifically within the postmenopausal subgroup of the initial cohort (n = 325) individuals carrying the CCR5del32 genotype exhibited a significantly longer metastasis-free survival (MFS, p = 0.038). In an independent cohort, CCR5del32 genotype was confirmed to be associated with prolonged MFS only in postmenopausal patients (n = 579, hazard ratio [HR] = 0.61, 95% confidence interval [95% CI] = 0.38-0.99, p = 0.044), and not in premenopausal patients (n = 438, HR = 1.01, 95% CI = 0.70-1.48, p = 0.94). Our results indicate that CCR5del32 genotype is associated with good prognosis in postmenopausal breast cancer patients. Considering this result, postmenopausal breast cancer patients who are wildtype for CCR5 genotype might benefit from CCR5 blockers, such as Maraviroc.

Gene expression profiles in circulating tumor cells to predict prognosis in metastatic breast cancer patients.

BACKGROUND: A circulating tumor cell (CTC) count is an established prognostic factor in metastatic breast cancer (MBC). Besides enumeration, CTC characterization promises to improve outcome prediction and treatment guidance. Having shown the feasibility of quantifying clinically relevant mRNA transcripts in CTCs, we determined the prognostic value of CTC gene expression in MBC. PATIENTS AND METHODS: CTCs were isolated and enumerated from blood of 197 MBC patients who were about to start first-line systemic therapy. Of these, 180 were assessable for quantification of mRNA expression by RT-qPCR in relation to time-to-treatment failure (TTF). A prognostic CTC gene profile was generated by leave-one-out cross validation in a 103 patient discovery set and validated in 77 patients. Additionally, all 180 patients were randomly divided into two equal sets to discover and validate a second prognostic profile. RESULTS: CTC count predicted for TTF at baseline {≥5 versus <5 CTCs/7.5 ml blood, hazard ratio (HR) 2.92 [95% confidence interval (CI) 1.71-4.95] P < 0.0001}. A 16-gene CTC profile was generated in the first discovery set, which identified patients with death or TTF <9 months versus those with a better outcome. In multivariate analysis, the 16-gene profile was the only factor associated with TTF [HR 3.15 (95% CI 1.35-7.33) P 0.008]. Validation of this profile in the independent patient set pointed into the same direction, but was not statistically significant. A newly generated 8-gene profile showed similarly favorable test characteristics as the 16-gene profile, but did not significantly pass validation either. CONCLUSION: A 16-gene CTC profile was identified, which provided prognostic value on top of CTC count in MBC patients. However, validation of this profile in an independent cohort, nor of a second profile, reached statistical significance, underscoring the need to further fine-tune the still promising approach of CTC characterization.

High protein expression of EZH2 is related to unfavorable outcome to tamoxifen in metastatic breast cancer.

BACKGROUND: Metastatic breast cancer (MBC) is a highly heterogeneous disease with great differences in outcome to both chemo- and endocrine therapy. Better insight into the mechanisms underlying resistance is essential to better predict outcome to therapy and to obtain a more tailored treatment approach. We have previously described that increased mRNA expression levels of Enhancer of Zeste homolog (EZH2) are associated with worse outcome to tamoxifen therapy in MBC. Here, we explored whether this is also the case for EZH2 protein expression. PATIENTS AND METHODS: A tissue microarray (TMA) was created using formalin-fixed, paraffin-embedded estrogen receptor (ER)-positive primary breast tumor tissues of 250 MBC patients treated with first-line tamoxifen. Quantity and intensity of EZH2 expression were determined by immunohistochemistry (IHC) and both were used to generate and group scores according to a previously described method for scoring EZH2. RESULTS: In total, 116 tumors (46%) were considered to be EZH2 positive. The presence of EZH2 protein expression was significantly associated with progression-free survival (PFS) in both univariate [hazard ratio (HR) 1.51, 95% confidence interval (CI) 1.17-1.97, P = 0.002] and multivariate analysis including traditional factors associated with tamoxifen outcome (HR 1.41, 95% CI 1.06-1.88, P = 0.017). Considering quantity irrespective of intensity, tumors with >50% EZH2-positive cells had the worst PFS (HR 2.15, 95% CI 1.42-3.27, P < 0.001), whereas intensity alone did not show a significant association with PFS. Application of other methods of scoring EZH2 positivity resulted in a similar significant association between the amount of EZH2 positive cells and PFS. CONCLUSION: In addition to EZH2 mRNA levels, these results suggest that protein expression of EZH2 can be used as a marker to predict outcome to tamoxifen therapy. This provides new rationale to explore EZH2 inhibition in the clinical setting and increases the possibilities for a more personalized treatment approach in MBC patients.

Towards a personalized breast cancer treatment approach guided by circulating tumor cell (CTC) characteristics.

Circulating tumor cells (CTCs) can be found in the peripheral blood of patients with different solid tumors, including breast cancer. A CTC count is a strong established prognostic factor in various stages in several tumor types. Besides that, characterization of CTCs is expected to become an invaluable tool to predict treatment response and personalize cancer treatments. Likely, CTCs are shed by different tumor lesions and may therefore provide a comprehensive view of tumor characteristics at a certain time-point, including inter- and intratumoral heterogeneity. Obtained through a simple venipuncture, CTCs could this way serve as a "liquid biopsy". However, isolation and subsequent characterization of CTCs is technically extremely challenging, mainly due to the small number of cells amidst a large majority of leukocytes. A wide range of assays has been developed, but only the CellSearch System(®) (Veridex, Raritan, NJ, USA) has obtained FDA approval for CTC enumeration so far. For characterization purposes, no assay has been validated at all. Nevertheless, the first studies investigating the clinical value of CTC characteristics have been performed. Here, we review these clinical studies. The various techniques used to interrogate CTCs are briefly described and an overview of the clinical relevance of CTC characterization in breast cancer is given.

BCAR4 induces antioestrogen resistance but sensitises breast cancer to lapatinib.

BACKGROUND: High BCAR4 and ERBB2 mRNA levels in primary breast cancer associate with tamoxifen resistance and poor patient outcome. We determined whether BCAR4 expression sensitises breast cancer cells to lapatinib, and identifies a subgroup of patients who possibly may benefit from ERBB2-targeted therapies despite having tumours with low ERBB2 expression. METHODS: Proliferation assays were applied to determine the effect of BCAR4 expression on lapatinib treatment. Changes in cell signalling were quantified with reverse-phase protein microarrays. Quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) of ERBB2 and BCAR4 was performed in 1418 primary breast cancers. Combined BCAR4 and ERBB2 mRNA levels were evaluated for association with progression-free survival (PFS) in 293 oestrogen receptor-α (ER)-positive patients receiving tamoxifen as first-line monotherapy for recurrent disease. RESULTS: BCAR4 expression strongly sensitised ZR-75-1 and MCF7 breast cancer cells to the combination of lapatinib and antioestrogens. Lapatinib interfered with phosphorylation of ERBB2 and its downstream mediators AKT, FAK, SHC, STAT5, and STAT6. Reverse transcriptase-PCR analysis showed that 27.6% of the breast cancers were positive for BCAR4 and 22% expressed also low levels of ERBB2. The clinical significance of combining BCAR4 and ERBB2 mRNA status was underscored by the finding that the group of patients having BCAR4-positive/ERBB2-low-expressing cancers had a shorter PFS on tamoxifen treatment than the BCAR4-negative group. CONCLUSION: This study shows that BCAR4 expression identifies a subgroup of ER-positive breast cancer patients without overexpression of ERBB2 who have a poor outcome and might benefit from combined ERBB2-targeted and antioestrogen therapy.

A new approach for rapid and reliable enumeration of circulating endothelial cells in patients.

BACKGROUND: Mature circulating endothelial cells (CECs) are surrogate markers of endothelial damage/dysfunction. A lack of standardized assays and consensus on CEC phenotype has resulted in a wide variation of reported CEC numbers (4-1300 per mL). OBJECTIVES: Given the need for a quick, reliable, robust and validated CEC assay at an affordable price, we present a novel approach to enumerate CECs using a multi-parameter flow cytometric (FCM) method without immunological pre-enrichment. METHODS: CECs were defined as CD34+, CD45neg, CD146+ and DNA+ events based on the immunophenotype of endothelial cells from vein-wall dissections. As CECs express high levels of CD34, we based our assay on absolute CD34 counts after analyzing all CD34 positive events in a total blood volume of 4 mL needed for a precise enumeration of CECs at a frequency of < 1 cell µL(-1). RESULTS: The endothelial origin of CECs was confirmed by morphology, immunohistochemistry and gene expression. The new FCM assay was tested in parallel with a validated assay (i.e. CellSearch). CEC levels ranged from 4 to 79 CEC mL(-1) in healthy individuals and were significantly higher in patients with advanced solid malignancies (P = 0.0008) and in patients with hematological malignancies (P < 0.0001). CONCLUSIONS: This flow cytometric method should be useful as a fast and economical assay to enumerate and characterize CECs.

High miR-26a and low CDC2 levels associate with decreased EZH2 expression and with favorable outcome on tamoxifen in metastatic breast cancer.

For patients with metastatic breast cancer, we previously described that increased EZH2 expression levels were associated with an adverse outcome to tamoxifen therapy. Main objective of the present study is to investigate miR-26a and miR-101 levels, which both target EZH2, for their association with molecular pathways and with efficacy of tamoxifen as first-line monotherapy for metastatic breast cancer. Expression levels were measured using quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) in primary breast cancer specimens of 235 estrogen receptor-α (ER)-positive patients. Pathway analysis was performed on microarray data available for 65 of these tumors. Logistic regression and Cox uni- and multivariate analysis were performed to relate expression levels with clinical benefit and time to progression (TTP). Increasing levels of miR-26a were significantly (P < 0.005) associated with both clinical benefit and prolonged TTP, whereas miR-101 was not. Cell cycle regulation and CCNE1 and CDC2 were the only significant overlapping pathway and genes differentially expressed between tumors with high and low levels of miR-26a and EZH2, respectively. In addition, increasing mRNA levels of CCNE1 (P < 0.05) and CDC2 (P < 0.001) were related to poor outcome. Multivariate analysis revealed miR-26a and CDC2 as an optimal set of markers associated with outcome on tamoxifen therapy, independently of traditional predictive factors. To summarize, only miR-26a levels are related with treatment outcome. Cell cycle regulation is the only overlapping pathway linked to miR-26a and EZH2 levels. Low mRNA levels of EZH2, CCNE1, and CDC2, and high levels of miR-26a are associated with favorable outcome on tamoxifen.

Decreased expression of EZH2 is associated with upregulation of ER and favorable outcome to tamoxifen in advanced breast cancer.

The purpose of this study is to investigate EZH2 in a large series of breast cancer patients for its prognostic and predictive value, and to evaluate its functional role in treatment response in vitro. EZH2 levels were measured using quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) in primary breast cancer specimens and related to clinicopathologic factors and disease outcome. EZH2 expression was downregulated with siRNAs in MCF7, to assess expression alterations of putative EZH2 downstream genes and to determine cell numbers after treatment with the anti-estrogen ICI 164384. In 688 lymph node-negative patients who did not receive adjuvant systemic therapy, EZH2 was not significantly correlated with metastasis-free survival (MFS). In 278 patients with advanced disease treated with first-line tamoxifen monotherapy, the tertile with highest EZH2 levels was associated with the lowest clinical benefit (OR = 0.48; P = 0.02) and with a shorter progression-free survival (PFS) in both univariate (HR = 1.80; P < 0.001) and multivariate analysis, including traditional factors (HR = 1.61; P = 0.004). In vitro, EZH2 silencing in MCF7 caused a 38% decrease in cell numbers (P < 0.001) whereas ICI 164384 treatment resulted in a 25% decrease (P < 0.001) compared to controls. Combining EZH2 silencing with ICI treatment reduced cell numbers with 67% (P < 0.001) compared to control conditions. EZH2 downregulation was associated with an almost two-fold upregulation of the estrogen receptor alpha (ER) (P = 0.001). In conclusion, EZH2 has no prognostic value in breast cancer. High levels of EZH2 are associated with poor outcome to tamoxifen therapy in advanced breast cancer. Downregulated EZH2 leads to upregulation of the ER and better response to anti-estrogens.

Relevance of BCAR4 in tamoxifen resistance and tumour aggressiveness of human breast cancer.

BACKGROUND: Breast cancer anti-oestrogen resistance 4 (BCAR4) was identified in a search for genes involved in anti-oestrogen resistance in breast cancer. We explored whether BCAR4 is predictive for tamoxifen resistance and prognostic for tumour aggressiveness, and studied its function. METHODS: BCAR4 mRNA levels were measured in primary breast tumours, and evaluated for association with progression-free survival (PFS) and clinical benefit in patients with oestrogen receptor (ERα)-positive tumours receiving tamoxifen as first-line monotherapy for advanced disease. In a separate cohort of patients with lymph node-negative, ERα-positive cancer, and not receiving systemic adjuvant therapy, BCAR4 levels were evaluated for association with distant metastasis-free survival (MFS). The function of BCAR4 was studied with immunoblotting and RNA interference in a cell model. RESULTS: Multivariate analyses established high BCAR4 mRNA levels as an independent predictive factor for poor PFS after start of tamoxifen therapy for recurrent disease. High BCAR4 mRNA levels were associated with poor MFS and overall survival, reflecting tumour aggressiveness. In BCAR4-expressing cells, phosphorylation of v-erb-b2 erythroblastic leukaemia viral oncogene homolog (ERBB)2, ERBB3, and their downstream mediators extracellular signal-regulated kinase 1/2 and v-akt murine thymoma viral oncogene homolog (AKT) 1/2, was increased. Selective knockdown of ERBB2 or ERBB3 inhibited proliferation, confirming their role in BCAR4-induced tamoxifen resistance. CONCLUSION: BCAR4 may have clinical relevance for tumour aggressiveness and tamoxifen resistance. Our cell model suggests that BCAR4-positive breast tumours are driven by ERBB2/ERBB3 signalling. Patients with such tumours may benefit from ERBB-targeted therapy.

CITED2 and NCOR2 in anti-oestrogen resistance and progression of breast cancer.

BACKGROUND: Endocrine therapies of breast cancer are effective but ultimately fail because of the development of treatment resistance. We have previously revealed several genes leading to tamoxifen resistance in vitro by retroviral insertion mutagenesis. To understand the manner in which these genes yield tamoxifen resistance, their effects on global gene expression were studied and those genes resulting in a distinct gene expression profile were further investigated for their clinical relevance. METHODS: Gene expression profiles of 69 human breast cancer cell lines that were made tamoxifen resistant through retroviral insertion mutagenesis were obtained using oligonucleotide arrays and analysed with bioinformatic tools. mRNA levels of NCOR2 and CITED2 in oestrogen receptor-positive breast tumours were determined by quantitative RT-PCR. mRNA levels were evaluated for association with metastasis-free survival (MFS) in 620 patients with lymph node-negative primary breast cancer who did not receive systemic adjuvant therapy, and with clinical benefit in 296 patients receiving tamoxifen therapy for recurrent breast cancer. RESULTS: mRNA expression profiles of most tamoxifen-resistant cell lines were strikingly similar, except for the subgroups of cell lines in which NCOR2 or CITED2 were targeted by the retrovirus. Both NCOR2 and CITED2 mRNA levels were associated with MFS, that is, tumour aggressiveness, independently of traditional prognostic factors. In addition, high CITED2 mRNA levels were predictive for a clinical benefit from first-line tamoxifen treatment in patients with advanced disease. CONCLUSIONS: Most retrovirally targeted genes yielding tamoxifen resistance in our cell lines do not impose a distinctive expression profile, suggesting that their causative role in cell growth may be accomplished by post-transcriptional processes. The associations of NCOR2 and CITED2 with outcome in oestrogen receptor-positive breast cancer patients underscore the clinical relevance of functional genetic screens to better understand disease progression, which may ultimately lead to the development of improved treatment options.

Methylated genes as new cancer biomarkers.

Aberrant hypermethylation of promoter regions in specific genes is a key event in the formation and progression of cancer. In at least some situations, these aberrant alterations occur early in the formation of malignancy and appear to be tumour specific. Multiple reports have suggested that measurement of the methylation status of the promoter regions of specific genes can aid early detection of cancer, determine prognosis and predict therapy responses. Promising DNA methylation biomarkers include the use of methylated GSTP1 for aiding the early diagnosis of prostate cancer, methylated PITX2 for predicting outcome in lymph node-negative breast cancer patients and methylated MGMT in predicting benefit from alkylating agents in patients with glioblastomas. However, prior to clinical utilisation, these findings require validation in prospective clinical studies. Furthermore, assays for measuring gene methylation need to be standardised, simplified and evaluated in external quality assurance programmes. It is concluded that methylated genes have the potential to provide a new generation of cancer biomarkers.

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.

Identification of alternatively spliced TIMP-1 mRNA in cancer cell lines and colon cancer tissue.

TIMP-1 is a promising new candidate as a prognostic marker in colorectal and breast cancer. We now describe the discovery of two alternatively spliced variants of TIMP-1 mRNA. The two variants lacking exon 2 (del-2) and 5 (del-5), respectively, were identified in human cancer cell lines by RT-PCR. The del-2 variant was, furthermore, detected in extracts from 12 colorectal cancer tissue samples. By western blotting additional bands of lower molecular mass than full-length TIMP-1 were identified in tumor tissue, but not in plasma samples obtained from cancer patients. The two splice variants of TIMP-1 may hold important clinical information, and either alone or in combination with measurement of full-length TIMP-1 they may improve the prognostic and/or predictive value of TIMP-1 analyses.

Laser microdissection and microarray analysis of breast tumors reveal ER-alpha related genes and pathways.

About 70-80% of breast cancers express estrogen receptor alpha (ER-alpha), and estrogens play important roles in the development and growth of hormone-dependent tumors. Together with lymph node metastasis, tumor size, and histological grade, ER status is considered as one of the prognostic factors in breast cancer, and an indicator for hormonal treatment. To investigate genes and pathways that are associated with ER status and epithelial cells in breast tumor, we applied laser capture microdissection (LCM) technology to capture epithelial tumor cells from 28 lymph node-negative breast tumor samples, in which 17 patients had ER-alpha+ tumors, and 11 patients have ER-alpha- tumors. Gene expression profiles were analysed on Affymetrix Hu133A GeneChip. Meanwhile, gene profiles using total RNA isolated from bulk tumors of the same 28 patients were also generated. In total, 146 genes and 112 genes with significant P-value and having significant differential expression between ER-alpha+ and ER-alpha- tumors were identified from the LCM data set and bulk tissue data set, respectively. A total of 61 genes were found to be common in both data sets, while 85 genes were unique to the LCM data set and 51 genes were present only in the bulk tumor data set. Pathway analysis with the 85 genes using Gene Ontology suggested that genes involved in endocytosis, ceramide generation, Ras/ERK/Ark cascade, and JAT-STAT pathways may play roles related to ER. The gene profiling with LCM-captured tumor cells provides a unique approach to study epithelial tumor cells and to gain an insight into signaling pathways associated with ER.

Elevated expression of polymorphonuclear leukocyte elastase in breast cancer tissue is associated with tamoxifen failure in patients with advanced disease.

Besides a variety of other proteases, polymorphonuclear leukocyte elastase (PMN-E) is also suggested to play a role in the processes of tumour cell invasion and metastasis. Yet, there is only limited data available on the relation between the tumour level of PMN-E and prognosis in patients with primary breast cancer, and no published information exists on its relation with the efficacy of response to systemic therapy in patients with advanced breast cancer. In the present study, we have measured with enzyme-linked immunosorbent assay the levels of total PMN-E in cytosolic extracts of 463 primary breast tumours, and have correlated their levels with the rate and duration of response on first-line tamoxifen therapy (387 patients) or chemotherapy (76 patients) in patients with locally advanced and/or distant metastatic breast cancer. Furthermore, the probabilities of progression-free survival and postrelapse survival were studied in relation to the tumour levels of PMN-E. Our results show that in logistic regression analysis for response to tamoxifen treatment in patients with advanced disease, high PMN-E tumour levels were associated with a poor rate of response compared with those with low PMN-E levels (odds ratio: OR, 0.40; 95% CI, 0.22-0.73; P=0.003). After correction for the contribution of the traditional predictive factors in multivariate analysis, the tumour PMN-E status was an independent predictor of response (P=0.01). Furthermore, a high tumour PMN-E level was related with a poor progression-free survival (P<0.001) and postrelapse survival (P=0.002) in a time-dependent analysis. In contrast, the tumour level of PMN-E was not significantly related with the efficacy of response to first-line chemotherapy in patients with advanced breast cancer. Our present results suggest that PMN-E is an independent predictive marker for the efficacy of tamoxifen treatment in patients with advanced breast cancer.

The prognostic value of vascular endothelial growth factor in 574 node-negative breast cancer patients who did not receive adjuvant systemic therapy.

The growth and metastasising capacity of solid tumours are dependent on angiogenesis. Vascular endothelial growth factor is a mediator of angiogenesis. In this study we investigated whether vascular endothelial growth factor is associated with the natural course of the disease in primary invasive breast cancer. In 574 tumours of patients with node-negative invasive breast cancer the cytosolic levels of vascular endothelial growth factor were measured using a quantitative enzyme-linked immunosorbent assay. These patients did not receive adjuvant systemic therapy and were followed for a median follow-up time of 61 months (range 2-155 months) after the primary diagnosis. Correlations with well-known prognostic factors, and univariate and multivariate survival analyses were performed. Vascular endothelial growth factor level was positively associated with age and tumour size (P=0.042 and P=0.029, respectively). In addition, vascular endothelial growth factor level was inversely, but weakly correlated with progesterone receptor levels (PgR) (r(s)=-0.090, P=0.035). A high vascular endothelial growth factor level (equal or above the median level of 0.53 ng mg(-1) protein) predicted a reduced relapse-free survival and overall survival in the univariate survival rate analysis (for both P=0.005). In the multivariate analysis as well, vascular endothelial growth factor showed to be an independent predictor of poor relapse-free survival and overall survival (P=0.045 and P=0.029, respectively), in addition to age, tumour size and PgR. The results show that cytosolic levels of vascular endothelial growth factor in tumour tissue samples are independently indicative of prognosis for patients with node-negative breast cancer who were not treated with adjuvant systemic therapy. This implies that vascular endothelial growth factor is related with the natural course of breast cancer progression.