Detection of HER2 amplification in circulating free DNA in patients with breast cancer

Background:

Human epidermal growth factor receptor 2 (HER2) is amplified and overexpressed in 20–25% of breast cancers. This study investigated circulating free DNA (cfDNA) for detection of HER2 gene amplification in patients with breast cancer.

Methods:

Circulating free DNA was extracted from plasma of unselected patients with primary breast cancer (22 before surgery and 68 following treatment), 30 metastatic patients and 98 female controls using the QIAamp Blood DNA Mini Kit (Qiagen). The ratio of HER2 to an unamplified reference gene (contactin-associated protein 1 (CNTNAP1)) was measured in cfDNA samples by quantitative PCR (qPCR) using SK-BR-3 cell line DNA as a positive control.

Results:

We validated the qPCR assay with DNA extracted from 23 HER2 3+ and 40 HER2-negative tumour tissue samples; the results agreed for 60 of 63 (95.2%) tumours. Amplification was detected in cfDNA for 8 of 68 patients following primary breast cancer treatment and 5 of 30 metastatic patients, but was undetected in 22 patients with primary breast cancer and 98 healthy female controls. Of the patients with amplification in cfDNA, 10 had HER2 3+ tumour status by immunohistochemistry.

Conclusions:

The results demonstrate for the first time the existence of amplified HER2 in cfDNA in the follow-up of breast cancer patients who are otherwise disease free. This approach could potentially provide a marker in patients with HER2-positive breast cancer.     

Ki-67 as prognostic marker in early breast cancer: a meta-analysis of published studies involving 12,155 patients

The Ki-67 antigen is used to evaluate the proliferative activity of breast cancer (BC); however, Ki-67's role as a prognostic marker in BC is still undefined. In order to better define the prognostic value of Ki-67/MIB-1, we performed a meta-analysis of studies that evaluated the impact of Ki-67/MIB-1 on disease-free survival (DFS) and/or on overall survival (OS) in early BC. Sixty-eight studies were identified and 46 studies including 12 155 patients were evaluable for our meta-analysis; 38 studies were evaluable for the aggregation of results for DFS, and 35 studies for OS. Patients were considered to present positive tumours for the expression of Ki-67/MIB-1 according to the cut-off points defined by the authors. Ki-67/MIB-1 positivity is associated with higher probability of relapse in all patients (HR=1.93 (95% confidence interval (CI): 1.74-2.14); P<0.001), in node-negative patients (HR=2.31 (95% CI: 1.83-2.92); P<0.001) and in node-positive patients (HR=1.59 (95% CI: 1.35-1.87); P<0.001). Furthermore, Ki-67/MIB-1 positivity is associated with worse survival in all patients (HR=1.95 (95% CI: 1.70-2.24; P<0.001)), node-negative patients (HR=2.54 (95% CI: 1.65-3.91); P<0.001) and node-positive patients (HR=2.33 (95% CI: 1.83-2.95); P<0.001). Our meta-analysis suggests that Ki-67/MIB-1 positivity confers a higher risk of relapse and a worse survival in patients with early BC.     

FGFR2 amplification has prognostic significance in gastric cancer: results from a large international multicentre study

Background:

In preclinical gastric cancer (GC) models, FGFR2 amplification was associated with increased tumour cell proliferation and survival, and drugs targeting this pathway are now in clinical trials.

Methods:

FGFR2 FISH was performed on 961 GCs from the United Kingdom, China and Korea, and the relationship with clinicopathological data and overlap with HER2 amplification were analysed.

Results:

The prevalence of FGFR2 amplification was similar between the three cohorts (UK 7.4%, China 4.6% and Korea 4.2%), and intratumoral heterogeneity was observed in 24% of FGFR2 amplified cases. FGFR2 amplification was associated with lymph node metastases (P<0.0001). FGFR2 amplification and polysomy were associated with poor overall survival (OS) in the Korean (OS: 1.83 vs 6.17 years, P=0.0073) and UK (OS: 0.45 vs 1.9 years, P<0.0001) cohorts, and FGFR2 amplification was an independent marker of poor survival in the UK cohort (P=0.0002). Co-amplification of FGFR2 and HER2 was rare, and when high-level amplifications did co-occur these were detected in distinct areas of the tumour.

Conclusion:

A similar incidence of FGFR2 amplification was found in Asian and UK GCs and was associated with lymphatic invasion and poor prognosis. This study also shows that HER2 and FGFR2 amplifications are mostly exclusive.     

Somatic mutations in the p53 gene and prognosis in breast cancer: a meta-analysis.

Many studies have investigated the association between alterations in the p53 gene and clinical outcome of breast cancer, and most investigators have reported poorer overall and disease-free survival (as indicated by a relative hazard (RH) greater than one) in breast cancer cases with somatic mutations in p53. However, different studies have produced widely differing RH estimates, ranging from no risk (RH = 1) to a relative hazard of 23, and not all of these results have been statistically significant. We have therefore reviewed all the published studies that have investigated the association between somatic mutations in the p53 gene and breast cancer prognosis and used standard techniques of meta-analysis to combine the results of these studies to produce a more precise estimate of the prognostic significance of p53 mutations. Eleven studies investigated overall survival in a total of 2319 unselected cases. The RH estimates from these ranged from 1 to 23.4 with a combined RH estimate of 2.0 (confidence interval 1.7-2.5). Three studies investigated the role of p53 in node-negative patients and in these, the combined estimate of RH was 1.7 (1.2-2.3). For three studies of node-positive breast cancer the combined risk estimate was 2.6 (1.7-3.9). The inclusion of p53 mutation screening in large breast cancer clinical trials seems warranted in the light of these results. Analysis of large numbers of cases matched for stage and therapy will allow definitive clarification of the value of p53 mutational status in prognostication, and possibly choice of therapy.     

Proliferative markers as prognostic and predictive tools in early breast cancer: where are we now?

In the last few decades, proliferative markers have been broadly evaluated as prognostic and predictive factors for early stage breast cancer patients. Several papers evaluating one or more markers have been published, often with contradictory results. As a consequence, there is still uncertainty about the role of these proliferative markers. The present paper critically reviews the current knowledge about the following markers: thymidine labeling index, S phase fraction/flow cytometry, Ki 67, thymidine kinase (TK), cyclins E, cyclin D, the cyclin inhibitors p27 and p21, and topoisomerase IIalpha. For each marker, the prognostic and predictive role was separately analyzed. Only papers published in English in peer-reviewed journals before June 2004 that include at least 100 evaluable patients were selected. In addition, the prognostic and predictive role of the proliferative markers had to be assessed through multivariate analyses. One hundred and thirty-two papers fulfilled these criteria and 159 516 patients were analyzed. Unfortunately, several methodological problems in the research to date prevent us from including any one of these proliferative markers among the standard prognostic and predictive factors. Early incorporation of translational research and new technologies with clinical trials are needed to prospectively validate biological markers and allow their use in clinical practice.     

Genetic amplification of PPME1 in gastric and lung cancer and its potential as a novel therapeutic target

Protein phosphatase methylesterase 1 (PPME1) is a protein phosphatase 2A (PP2A)-specific methyl esterase that negatively regulates PP2A through demethylation at its carboxy terminal leucine 309 residue. Emerging evidence shows that the upregulation of PPME1 is associated with poor prognosis in glioblastoma patients. By performing an array comparative genomic hybridization analysis to detect copy number changes, we have been the first to identify PPME1 gene amplification in 3.8% (5/131) of Chinese gastric cancer (GC) samples and 3.1% (4/124) of Chinese lung cancer (LC) samples. This PPME1 gene amplification was confirmed by fluorescence in situ hybridization analysis and is correlated with elevated protein expression, as determined by immunohistochemistry analysis. To further investigate the role of PPME1 amplification in tumor growth, short-hairpin RNA-mediated gene silencing was employed. A knockdown of PPME1 expression resulted in a significant inhibition of cell proliferation and induction of cell apoptosis in PPME1-amplified human cancer cell lines SNU668 (GC) and Oka-C1 (LC), but not in nonamplified MKN1 (GC) and HCC95 (LC) cells. The PPME1 gene knockdown also led to a consistent decrease in PP2A demethylation at leucine 309, which was correlated with the downregulation of cellular Erk and AKT phosphorylation. Our data indicate that PPME1 could be an attractive therapeutic target for a subset of GCs and LCs.     

Evaluation of cathepsin D as a prognostic factor in breast cancer

Summary The biologic rationale for the importance of cathepsin D in breast cancer is a provocative one. Cathepsin D is both an estrogen inducible and a constitutively produced protease, which may also act directly as a peptide growth factor. Thus it may play a role in tumor invasiveness, and also in driving cell proliferation. Reported clinical studies are conflicting, with some studies showing cathepsin D levels to correlate with clinical outcome, and other studies finding prognostic significance only in selected subsets of patients, if any. Thus cathepsin D is a potentially important prognostic marker whose clinical application awaits further definition.     

Breast self-examination and death from breast cancer: a meta-analysis

Breast self-examination (BSE) is widely recommended for breast cancer prevention. Following recent controversy over the efficacy of mammography, it may be seen as an alternative. We present a meta-analysis of the effect of regular BSE on breast cancer mortality. From a search of the medical literature, 20 observational studies and three clinical trials were identified that reported on breast cancer death rates or rates of advanced breast cancer (a marker of death) according to BSE practice. A lower risk of mortality or advanced breast cancer was only found in studies of women with breast cancer who reported practising BSE before diagnosis (mortality: pooled relative risk 0.64, 95% CI 0.56-0.73; advanced cancer, pooled relative risk 0.60, 95% CI 0.46-0.80). The results are probably due to bias and confounding. There was no difference in death rate in studies on women who detected their cancer during an examination (pooled relative risk 0.90, 95% CI 0.72-1.12). None of the trials of BSE training (in which most women reported practising it regularly) showed lower mortality in the BSE group (pooled relative risk 1.01, 95% CI 0.92-1.12). They did show that BSE is associated with considerably more women seeking medical advice and having biopsies. Regular BSE is not an effective method of reducing breast cancer mortality.     

PSMB7 is associated with anthracycline resistance and is a prognostic biomarker in breast cancer

Background:

To date individual markers have failed to correctly predict resistance against anticancer agents in breast cancer. We used gene expression patterns attributable to chemotherapy-resistant cells to detect potential new biomarkers related to anthracycline resistance. One of the genes, PSMB7, was selected for further functional studies and clinical validation.

Methods:

We contrasted the expression profiles of four pairs of different human tumour cell lines and of their counterparts resistant to doxorubicin. Observed overexpression of PSMB7 in resistant cell lines was validated by immunohistochemistry. To examine its function in chemoresistance, we silenced the gene by RNA interference (RNAi) in doxorubicin-resistant MCF-7 breast cancer cells, then cell vitality was measured after doxorubicin treatment. Microarray gene expression from GEO raw microarray samples with available progression-free survival data was downloaded, and expression of PSMB7 was used for grouping samples.

Results:

After doxorubicin treatment, 79.8±13.3% of resistant cells survived. Silencing of PSMB7 in resistant cells decreased survival to 31.8±6.4% (P>0.001). A similar effect was observed after paclitaxel treatment. In 1592 microarray samples, the patients with high PSMB7 expression had a significantly shorter survival than the patients with low expression (P<0.001).

Conclusion:

Our findings suggest that high PSMB7 expression is an unfavourable prognostic marker in breast cancer.     

Diabetes and breast cancer risk: a meta-analysis

Background:

The potential of an increased risk of breast cancer in women with diabetes has been the subject of a great deal of recent research.

Methods:

A meta-analysis was undertaken using a random effects model to investigate the association between diabetes and breast cancer risk.

Results:

Thirty-nine independent risk estimates were available from observational epidemiological studies. The summary relative risk (SRR) for breast cancer in women with diabetes was 1.27 (95% confidence interval (CI), 1.16–1.39) with no evidence of publication bias. Prospective studies showed a lower risk (SRR 1.23 (95% CI, 1.12–1.35)) than retrospective studies (SRR 1.36 (95% CI, 1.13–1.63)). Type 1 diabetes, or diabetes in pre-menopausal women, were not associated with risk of breast cancer (SRR 1.00 (95% CI, 0.74–1.35) and SRR 0.86 (95% CI, 0.66–1.12), respectively). Studies adjusting for body mass index (BMI) showed lower estimates (SRR 1.16 (95% CI, 1.08–1.24)) as compared with those studies that were not adjusted for BMI (SRR 1.33 (95% CI, 1.18–1.51)).

Conclusion:

The risk of breast cancer in women with type 2 diabetes is increased by 27%, a figure that decreased to 16% after adjustment for BMI. No increased risk was seen for women at pre-menopausal ages or with type 1 diabetes.     

BRAF mutation is a powerful prognostic factor in advanced and recurrent colorectal cancer

Background:

Activating mutation of KRAS and BRAF are focused on as potential prognostic and predictive biomarkers in patients with colorectal cancer (CRC) treated with anti-EGFR therapies. This study investigated the clinicopathological features and prognostic impact of KRAS/BRAF mutation in advanced and recurrent CRC patients.

Method:

Patients with advanced and recurrent CRC treated with systemic chemotherapy (n=229) were analysed for KRAS/BRAF genotypes by cycleave PCR. Prognostic factors associated with survival were identified by univariate and multivariate analyses using the Cox proportional hazards model.

Results:

KRAS and BRAF mutations were present in 34.5% and 6.5% of patients, respectively. BRAF mutated tumours were more likely to develop on the right of the colon, and to be of the poorly differentiated adenocarcinoma or mucinous carcinoma, and peritoneal metastasis. The median overall survival (OS) for BRAF mutation-positive and KRAS 13 mutation-positive patients was 11.0 and 27.7 months, respectively, which was significantly worse than that for patients with wild-type (wt) KRAS and BRAF (40.6 months) (BRAF; HR=4.25, P<0.001, KRAS13; HR=2.03, P=0.024). After adjustment for significant features by multivariate Cox regression analysis, BRAF mutation was associated with poor OS (HR=4.23, P=0.019).

Conclusion:

Presence of mutated BRAF is one of the most powerful prognostic factors for advanced and recurrent CRC. The KRAS13 mutation showed a trend towards poor OS in patients with advanced and recurrent CRC.     

FGFR2 gene amplification and clinicopathological features in gastric cancer

Background:

Frequency of FGFR2 amplification, its clinicopathological features, and the results of high-throughput screening assays in a large cohort of gastric clinical samples remain largely unclear.

Methods:

Drug sensitivity to a fibroblast growth factor receptor (FGFR) inhibitor was evaluated in vitro. The gene amplification of the FGFRs in formalin-fixed, paraffin-embedded (FFPE) gastric cancer tissues was determined by a real-time PCR-based copy number assay and fluorescence in situ hybridisation (FISH).

Results:

FGFR2 amplification confers hypersensitivity to FGFR inhibitor in gastric cancer cell lines. The copy number assay revealed that 4.1% (11 out of 267) of the gastric cancers harboured FGFR2 amplification. No amplification of the three other family members (FGFR1, 3 and 4) was detected. A FISH analysis was performed on 7 cases among 11 FGFR2-amplified cases and showed that 6 of these 7 cases were highly amplified, while the remaining 1 had a relatively low grade of amplification. Although the difference was not significant, patients with FGFR2 amplification tended to exhibit a shorter overall survival period.

Conclusion:

FGFR2 amplification was observed in 4.1% of gastric cancers and our established PCR-based copy number assay could be a powerful tool for detecting FGFR2 amplification using FFPE samples. Our results strongly encourage the development of FGFR-targeted therapy for gastric cancers with FGFR2 amplification.     

Survival and contralateral breast cancer in CHEK2 1100delC breast cancer patients: impact of adjuvant chemotherapy

Background:

We assessed the sensitivity to adjuvant chemotherapy in cell cycle checkpoint kinase 2 (CHEK2) vs non-CHEK2 breast cancer patients by comparing the contralateral breast cancer incidence and distant disease-free and breast cancer-specific survival between both groups, stratified for adjuvant chemotherapy.

Methods:

One Dutch hereditary non-BRCA1/2 breast cancer patient cohort (n=1220) and two Dutch cohorts unselected for family history (n=1014 and n=2488, respectively) were genotyped for CHEK2 1100delC. Hazard ratios for contralateral breast cancer, distant disease-free and breast cancer-specific death for mutation carriers vs noncarriers were calculated using the Cox proportional hazard method, stratified for adjuvant chemotherapy.

Results:

The CHEK2 mutation carriers (n=193) had an increased incidence of contralateral breast cancer (multivariate hazard ratio 3.97, 95% confidence interval 2.59–6.07). Distant disease-free and breast cancer-specific survival were similar in the first 6 years in mutation carriers compared with noncarriers, but diverted as of 6 years after breast cancer diagnosis (multivariate hazard ratios and 95% confidence intervals 2.65 (1.79–3.93) and 2.05 (1.41–2.99), respectively). No significant interaction between CHEK2 and adjuvant chemotherapy was observed.

Conclusions:

The CHEK2 1100delC-associated breast cancer is associated with a higher contralateral breast cancer rate as well as worse survival measures beyond 6 years after diagnosis. No differential sensitivity to adjuvant chemotherapy was observed in CHEK2 patients.     

Cyclin D1, EMS1 and 11q13 amplification in breast cancer

Chromosome locus 11q13 is frequently amplified in a number of human cancers including carcinoma of the breast where up to 15% carry this chromosomal abnormality. Originally 11q13 amplification was thought to involve a single amplicon spanning many megabases, but more recent data have identified four core regions within 11q13 that can be amplified independently or together in different combinations. Although the region harbors several genes with known or suspected oncogenic potential, the complex structure of the amplicons and the fact that 11q13 is gene-rich have made definitive identification of specific genes that contribute to the genesis and progression of breast cancer a difficult and continuing process. To date CCND1, encoding the cell cycle regulatory gene cyclin D1, and EMS1, encoding the filamentous actin binding protein and c-Src substrate cortactin, are the favored candidates responsible for the emergence of two of the four amplification cores.     

The prognostic value of the human kallikrein gene 9 (KLK9) in breast cancer

Background. Many members of the human kallikrein gene family were found to be differentially expressed in various malignancies and some of them are useful diagnostic/prognostic markers. KLK9 is a newly discovered human kallikrein gene that is expressed in several tissues including thymus, spinal cord, testis, prostate, breast, and ovary. Like other kallikreins, the KLK9 gene was found to be regulated by steroid hormones, mainly estrogens and progestins, in cancer cell lines. Experimental design. We studied the expression of KLK9 by quantitative RT-PCR in 169 breast cancer patients of different stages, grades and histological types. We also compared the relation between KLK9 expression and other clinicopathological variables and patient survival. Results. KLK9 expression is significantly higher in patients with early stage cancers (p = 0.039) and in patients with small tumor size (<2 cm) (p = 0.028). Kaplan-Meier survival curves demonstrated that KLK9-positive patients have longer disease-free and overall survival (p = 0.015 and 0.036, respectively). Univariate and multivariate analysis also indicates that KLK9 expression is associated with increased disease-free and overall survival. When the Cox proportional hazard regression analysis was applied to subgroups of patients, KLK9 expression was found to be a significant predictor of disease-free survival in the estrogen receptor (ER) and progesterone receptor (PR) negative subgroups of patients (Hazard Ratio 'HR' = 0.28, and 0.38, respectively, and p = 0.011 and 0.028, respectively). After adjusting for other known prognostic variables, KLK9 retained its independent prognostic value in these subgroups of patients. Similar results were obtained for overall survival. Conclusions. KLK9 is a new potential independent marker of favorable prognosis in breast cancer.     

Association between c-myc amplification and pathological complete response to neoadjuvant chemotherapy in breast cancer

Background

The aim of this study was to investigate whether c-myc amplification in human breast cancer is associated with response to neoadjuvant chemotherapy comprising paclitaxel followed by 5-FU/epirubicin/cyclophosphamide (P-FEC).

Methods

Tumour tissue samples were obtained before neoadjuvant chemotherapy (P-FEC) from 100 primary breast cancer patients (stage II/III). C-myc and HER2 amplification were examined by FISH, and oestrogen receptor (ER), progesterone receptor (PR), Ki67, and topoisomerase 2α (TOP2A) expression were examined immunohistochemically. Pathological complete response (pCR) was defined by a complete loss of tumour cells in the breast without any lymph node metastasis.

Results

C-myc amplification was observed in 40% (40/100) of breast tumours, and was significantly associated with ER-negative tumours (23/40 for ER(-) versus 17/60 for ER(+), P = 0.004), high histological grade tumours (11/18 for grade 3 versus 29/82 for grades 1 + 2, P = 0.043) and TOP2A-positive tumours (28/51 for TOP2A(+) versus 12/49 for TOP2A(-), P = 0.002). pCR rate was 20% for total patients (10.0% for ER(+) and 35.0% for ER(-)). Further, breast tumours with c-myc amplification (c-myc(+)) showed a significantly (P = 0.041) higher pCR rate (12/40) than those without such amplification (c-myc(-)) (8/60). This association between pCR and c-myc amplification was observed in ER-positive tumours (4/17 for c-myc(+) versus 2/43 for c-myc(-), P = 0.048) but not in ER-negative tumours (8/23 for c-myc(+) versus 6/17 for c-myc(-), P = 0.973).

Conclusion

Our results suggest that c-myc amplification is significantly associated with a high pCR rate to P-FEC in breast tumours, especially in ER-positive tumours.     

Epidermal growth factor receptor expression in 780 breast cancer patients: A reappraisal of the prognostic value based on an eight-year median follow-up

Purpose:Because new therapeutic approaches target tumorsexpressing epidermal growth factor receptor (EGFR), the aim was toundertake a thorough analysis of the expression profile of EGFR inbreast cancer and to reassess its prognostic value. Patients and methods:Tumor EGFR levels were determined by aspecific ligand binding assay in 780 consecutive breast cancer patientsfollowed in our institute between 1980 and 1993. Mean age was 61 years(25–85 years). All patients had undergone tumor resection withaxillary lymph node dissection: 373 patients (47.8%) underwentmastectomy, 37 (5%) subcutaneous mastectomy and 370(47.2%) tumorectomy. Results:EGFR levels ranged between non-detectable up to 789fmol/mg protein. EGFR median value was 9 fmol/mg protein and only asmall proportion of patients exhibited a relatively marked EGFRexpression. There was no link between tumor size, grade, node status andEGFR tumoral levels. There was a constant and significant decrease inEGFR tumoral levels according to patient age. A significant inverserelationship was found between estradiol receptors (ER) and EGFR. Medianfollow-up was 97 months with a minimum at 4 months and a maximum at228 months. From univariate analysis it was found that histologicalgrade, tumor size, node status and ER status were all significantpredictors of survival, considering metastasis-free as well as overallsurvival. Using multivariable analysis, only histological grade, tumorsize and node status remained independent predictors of survival. Conclusion:EGFR determination is of limited value as aprognostic indicator in breast cancer.     

Higher dietary folate intake reduces the breast cancer risk: a systematic review and meta-analysis

Background:

Many epidemiological studies have investigated the association between folate intake, circulating folate level and risk of breast cancer; however, the findings were inconsistent between the studies.

Methods:

We searched the PubMed and MEDLINE databases updated to January, 2014 and performed the systematic review and meta-analysis of the published epidemiological studies to assess the associations between folate intake level, circulating folate level and the overall risk of breast cancer.

Results:

In all, 16 eligible prospective studies with a total of 744 068 participants and 26 205 breast cancer patients and 26 case–control studies with a total of 16 826 cases and 21 820 controls that have evaluated the association between folate intake and breast cancer risk were identified. Pooled analysis of the prospective studies and case–control studies suggested a potential nonlinearity relationship for dietary folate intake and breast cancer risk. Prospective studies indicated a U-shaped relationship for the dietary folate intake and breast cancer risk. Women with daily dietary folate intake between 153 and 400 μg showed a significant reduced breast cancer risk compared with those <153 μg, but not for those >400 μg. The case–control studies also suggested a significantly negative correlation between the dietary folate intake level and the breast cancer risk. Increased dietary folate intake reduced breast cancer risk for women with higher alcohol intake level, but not for those with lower alcohol intake. No significant association between circulating folate level and breast cancer risk was found when the results of 8 identified studies with 5924 participants were pooled.

Conclusions:

Our studies suggested that folate may have preventive effects against breast cancer risk, especially for those with higher alcohol consumption level; however, the dose and timing are critical and more studies are warranted to further elucidate the questions.