Genome-wide gene expression profiling to predict resistance to anthracyclines in breast cancer patients.

Validated biomarkers predictive of response/resistance to anthracyclines in breast cancer are currently lacking. The neoadjuvant Trial of Principle (TOP) study, in which patients with estrogen receptor (ER)-negative tumors were treated with anthracycline (epirubicin) monotherapy, was specifically designed to evaluate the predictive value of topoisomerase II-alpha (TOP2A) and develop a gene expression signature to identify those patients who do not benefit from anthracyclines. Here we describe in details the contents and quality controls for the gene expression and clinical data associated with the study published by Desmedt and colleagues in the Journal of Clinical Oncology in 2011 (Desmedt et al., 2011). We also provide R code to easily access the data and perform the quality controls and basic analyses relevant to this dataset.

Evaluation of the histological size of the sentinel lymph node metastases using RT-PCR assay: a rapid tool to estimate the risk of non-sentinel lymph node invasion in patients with breast cancer.

A RT-PCR assay (GeneSearch™, Veridex, LLC), FDA approved and CE marked to detect metastases > 0.2 mm in sentinel lymph nodes (SLNs) is used intra-operatively for the management of patients with breast cancer. The assay provides qualitative results by applying cut-off values to cycle times (Ct) for mammaglobin (MG) and cytokeratin-19 (CK19) genes. Aims of this study were to evaluate the performance of the quantitative Ct values to estimate the size of nodal metastases and the risk of additional disease in non-SLNs. SLNs from 367 patients were clinically processed using both BLN assay and post-operative histology. Complementary axillary lymph node dissection (ALND) was performed concurrently in case of BLN assay positivity or tumour size > 2 cm. BLN positivity was reported in 19.6% of the patients for a sensitivity of 89%. BLN specificity (94.5%) and negative predictive value (97.5%) clearly demonstrated its reliability to guide ALND decision. All, except one, residual axillary metastases were found in BLN-positive patients. Considering the 78 patients with SLN positivity or discordant status according to both criteria, the metastases histological size was significantly correlated to the expression level of MG (ρ = 0.62) and CK19 (ρ = 0.64) genes (P < 10E-6). Moreover, ALND status positivity was significantly associated to Ct value of MG (z = 2.4; P = 0.018) and CK19 (z = 3.2; P = 0.001). The high intra-operative quality performance of the BLN assay minimizes the need for second surgeries for ALND. Results from this investigational study suggest that markers Ct value may provide, intra-operatively, valuable metastases size data and a risk prediction of additional disease in non-SLNs.

Clinical validation of a molecular assay for intra-operative detection of metastases in breast sentinel lymph nodes.

BACKGROUND: In breast cancer patients, the status of the sentinel lymph nodes (SLNs) has been shown to accurately reflect the presence of metastases in the axillary lymph nodes (ALNs). Intra-operative SLN evaluation by frozen section histology may miss positive cases, leading to a second surgery for complete ALN dissection. Permanent section histology itself has tissue sampling limitations and is partially dependent on pathologist expertise. METHODS: A prospective study (N=78) was conducted in our institution to validate a new intra-operative molecular assay, the GeneSearch breast lymph node (BLN) assay. This assay quantifies the expression of mammaglobin and cytokeratin-19 genes using quantitative RT-PCR technology to determine SLN status. Fresh SLN sections (2 mm thick) were analyzed alternatively by BLN assay or post-operative histology (haematoxylin-eosin and immunohistochemistry). The subject was considered positive when histology revealed a focus >0.2 mm. RESULTS: BLN assay results corroborated with histologic results in 75 out of 78 patients for an overall agreement of 96%, a sensitivity of 92%, and a specificity of 97%. The positive and negative predictive values of the BLN assay were of 86% (12/14) and 98% (63/64), respectively. Interestingly, a statistically significant correlation was observed between the metastases' histologic size and both assay markers' expression levels as represented by cycle time to positivity (rho > or = 0.71, all p<0.0001). CONCLUSIONS: The performance of the BLN assay in identifying nodal metastases >0.2 mm was similar to that of permanent section histology, with the added advantages of an objective and rapid output that could be used for intra-operative decision to remove additional ALN.

A significant proportion of elderly patients develop hormone-dependant "luminal-B" tumours associated with aggressive characteristics.

INTRODUCTION: To investigate the influence of ageing on the incidence of breast cancer (BC) molecular subtypes, patient age at diagnosis was correlated with bio-pathological data collected retrospectively from 2723 consecutive patients diagnosed/treated at our Institute between 2000 and 2003. METHODS: According to their bio-characteristics, 61% of the samples could be assigned to a molecular subtype: the "HER-2+", the "ER & HER2 negative" or one of the two "luminal-like" subtypes divided according to their histological grade ("A" [HER-2-/ER+/grade 1-2] and "B" [HER-2-/ER+/grade 3]). RESULTS AND CONCLUSION: Age is highly influencing the incidence of BC molecular subtypes. Patients younger than 40 develop a statistically higher rate of high grade proliferating "HER-2" (27%) and "ER & HER2 negative" (31%) BC whereas patients older than 50 develop mostly less aggressive hormone-dependant "luminal-A" BC (>67%). Nevertheless, a significant proportion of patients older than 70 develop "luminal-B" (19%) tumours associated with high proliferation, high grade, large size and nodal invasion.

HER-2 overexpression/amplification and its interaction with taxane-based therapy in breast cancer.

Breast cancer (BC) is the most common cancer in women and it is incurable when metastases are diagnosed. Taxanes, namely docetaxel and paclitaxel, are effective chemotherapeutic agents in the metastatic, neoadjuvant and adjuvant settings. HER-2 overexpression/amplification is detected in 25-30% of BCs and confers aggressive tumor behavior as well as resistance to some systemic treatments; nevertheless, its association with response to taxane-based chemotherapy is still unclear, with conflicting results in both in vitro and in vivo preclinical studies. This review will address the impact of HER-2 overexpression/amplification in BC patients treated with taxanes. Prospective, randomized trials incorporating important biological hypotheses are either ongoing or just closed, and their results will hopefully help to shed more light on this issue.

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.

p-53 gene mutations as a predictive marker in a population of advanced breast cancer patients randomly treated with doxorubicin or docetaxel in the context of a phase III clinical trial.

BACKGROUND: Preclinical data indicate that p-53 gene mutations predict resistance to doxorubicin (A) but not to docetaxel (Taxotere) (T). In the TAX 303 trial, A and T have been compared with advanced breast cancer patients. PATIENTS AND METHODS: Primary tumor samples from patients participating in the TAX 303 trial were collected. p-53 gene mutations were evaluated by denaturing high-performance liquid chromatography (DHPLC) and confirmed by sequencing. Topoisomerase II alpha (topo II alpha) protein levels were evaluated by immunohistochemistry. Clinical and biological data were correlated. RESULTS: Tumor samples for DHPLC analysis were available for 108 of 326 patients from the clinical trial. p-53 gene mutations were observed in 20% of patients. In patients with a mutated p-53 gene, a trend for a lower percentage of responders was observed in the A arm (17%) compared with the T arm (50%). In the wild-type p-53 cohort, response rates to A and T were 27% and 36%, respectively. Of the 16 patients carrying wild-type p-53- and topo II protein-positive tumors, seven (44%) responded to anthracyclines, while response rate to the same drug was 13% in the remaining cohorts [odds ratio 5.06 (95% confidence interval 1.19-21.41), P = 0.03]. The combination of the two markers had no predictive value in patients treated with docetaxel. CONCLUSIONS: (i) p-53 gene analysis indicates that gene mutations may compromise the efficacy of A while they do not interfere with the antitumor activity of T; and (ii) the evaluation of multiple molecular markers including p-53 and proliferation markers as topo II protein levels looks more promising in predicting response to anthracyclines.

HER-2/neu as a predictive marker in a population of advanced breast cancer patients randomly treated either with single-agent doxorubicin or single-agent docetaxel.

PURPOSE: To evaluate the predictive value of HER-2 in a population of advanced breast cancer patients randomly treated either with single-agent doxorubicin (A) or with single-agent docetaxel (T). EXPERIMENTAL DESIGN: Patients from this study participated in a phase III clinical trial in which doxorubicin or docetaxel was administered for advanced disease. HER-2 was evaluated by IHC. In all positive cases, FISH was used to confirm the HER-2 positive status. The different cohorts of patients identified by HER-2 were examined to assess a possible relationship between HER-2 status and treatment effect. RESULTS: Tumor samples were available for 176 of the 326 patients entered in the clinical trial (54%). HER-2 positivity was observed in 20% of the study population. A statistically significant interaction was found between response rates to the study drugs and HER-2 status, with HER-2 positive patients deriving the highest benefit from the use of T (odds ratio for HER-2 positive patients treated with T = 3.12 (95% CI 1.11-8.80), p = 0.03). The interaction between HER-2 and response rates to A and T was also confirmed by a multivariate analysis. No statistically significant interaction was found between HER-2 and drugs efficacy evaluated in terms of time to progression and overall survival, although in the HER-2 negative cohort A was at least as effective as T in term of overall survival. CONCLUSIONS: These results suggest that in HER-2 positive breast cancer patients docetaxel might be more active than doxorubicin, while in HER-2 negative patients doxorubicin might be at least as effective as docetaxel. Although the present results cannot have an impact on current practice, they allow us to formulate the hypothesis that HER-2 positive breast cancer is a heterogeneous disease with regard to sensitivity to anthracyclines and taxanes, and that this might be dependent upon other molecular markers including the p-53 and topoisomerase II alpha genes. This hypothesis is currently being tested prospectively in two different 'bench to bed-side' clinical trials.

Correlation between topoisomerase-IIalpha gene amplification and protein expression in HER-2 amplified breast cancer.

Topoisomerase-IIalpha (topo-II) is a molecular target for topo-II inhibitors, which makes it a potential predictive marker of responsiveness to these agents. We aim to correlate topo-II gene and protein status on 103 HER-2 amplified breast cancer samples. Paraffin-embedded blocks were screened by FISH for topo-II gene amplification (topo-II: CEP17 ratio >/=1.5; triple probe by Vysis inc.) and analyzed by IHC for topo-II protein expression (continuous variable; clone KiS1) and Ki-67 (positive if >25% of stained cells; clone MIB-1). Topo-II gene amplification was observed in 36.9% (38/103) of the HER-2 amplified study population. HER-2 amplification level (e.g. copy number) was not shown to be predictive for topo-II amplification. The median percentage of topo-II positively stained cells by IHC for topo-II non-amplified and amplified cases were 5% and 10%, respectively. A weak but significant correlation was observed between topo-II gene amplification level and percentage of positively stained cells (Spearman's ranks correlation coefficient of 0.23, p=0.02), the observed correlation being higher in patients with positive staining for Ki-67. Contrary to HER-2, where gene amplification is almost always correlated with protein overexpression in breast cancer, topo-II gene amplification apparently does not always lead to protein overexpression, at least when the latter is evaluated by IHC. Other factors, specifically the tumor proliferation status, may interfere with the topo-II protein status. Although the great majority of topo-II gene aberrations occur in HER-2 positive tumors, the level of HER-2 amplification does not predict for topo-II amplification.

Exquisite antitumour response to trastuzumab in a patient with no evidence of Ras-Raf-MAPK and PI3K-Akt pathways activation.

We report on the case of a patient with a diagnosis of a HER2-overexpressing metastatic breast cancer which was refractory to a combination of a Raf kinase inhibitor and docetaxel, but highly sensitive to trastuzumab, a HER2-targeted monoclonal antibody. Interestingly, there was no evidence of Ras-Raf-MAPK or PI3K-Akt pathways activation.

Rates of topoisomerase II-alpha and HER-2 gene amplification and expression in epithelial ovarian carcinoma.

OBJECTIVES: Topoisomerase II-alpha (T2a) is being actively investigated as a potential predictive marker of response to anthracyclines in breast cancer (BC). Although the role of T2a inhibitors as upfront and salvage treatment for epithelial ovarian carcinoma (EOC) remains unclear, we speculated that a small subgroup of ovarian cancer patients could derive a selective benefit from these agents. In this study, we investigated the actual rates of T2a and HER-2 amplification and overexpression by fluorescence in situ hybridisation (FISH) and immunohistochemistry (IHC), respectively. METHODS: Seventy-three samples of chemotherapy-naive patients with EOC were selected from our archives. FIGO stage and histology were available for most patients. RESULTS: Based on arbitrary cut-offs of > or =1.5 and > or =2 (ratio copies/centromere17), amplification rates for HER-2 were 15/64 (23.4%) and 8/64 (12.5%) versus 16/64 (25%) and 5/64 (7.8%) for T2a. We found only 3/72 (4.2%) cases of HER-2 overexpression (3+) versus 15/70 (21.4%) for T2a (staining of >10% of the cells). There was a modest correlation between T2a amplification and overexpression (P=0.01) and a strong correlation between T2a and HER-2 amplification when these markers were analysed as continuous variables (P<0.001). T2a amplification significantly correlated with advanced FIGO stage (P=0.02). CONCLUSION: The assessment of HER-2 and T2a amplification and overexpression by FISH and IHC, respectively, is feasible in EOC. These tests can be used for large-scale evaluation of the potential predictive and prognostic value of these markers in the future. Further studies with a special focus on T2a are needed to determine the best cut-offs for potential clinical use in the future.

Correlation between complete response to anthracycline-based chemotherapy and topoisomerase II-alpha gene amplification and protein overexpression in locally advanced/metastatic breast cancer.

UNLABELLED: Anthracycline-based regimens are among the most active but also with greater risk of both acute and long-term side effects, namely cardiotoxicity. Predictive markers of response to anthracyclines are therefore essential. Topoisomerase-IIalpha (topo-II) is the target of anthracyclines and preliminary data suggest its promising role as a predictive marker of sensitivity to these drugs. After screening a population of about 350 patients with locally advanced or metastatic breast cancer, two subgroups were selected for the present analysis: a study group (31 patients), composed of 14 complete responders (CR-a) and 17 true non-responders (PD-a) to anthracycline-based CT, and a control group (28 patients), composed of 7 CR (CR-t) and 21 true non-responders (PD-t) to taxane-based CT. True non-responders were defined as progressive disease (PD) within the first three cycles of CT. Archival tumor samples of these patients were collected, biological markers evaluated and their status correlated with response to therapy. HER-2 and topo-II gene status were evaluated by FISH (Vysis multi-color probe-positivity cut-off: >/=2 ratio for HER-2 and >/=1.5 for topo-II), topo-II protein was evaluated by IHC (positivity cut-off >10%). All cases in which HER-2 gene was non-amplified did not show topo-II gene aberrations. No association was found between HER-2 gene amplification and response to anthracyclines (5/14 (36%) CR and 5/17 (29%) PD to anthracycline-based CT were HER-2+). The topo-II gene was amplified in 3/14 (21%) CR but only in 1/17 (6%) PD to anthracyclines. Amplification of the topo-II gene was seen in 1/7 (14%) CR and in 3/21 (14%) PD to a taxane-based CT. Topo-II protein was overexpressed in 6/11 (55%) CR and in 2/17 (12%) PD to anthracyclines, while in the control group, overexpression was seen in 5/7 (71%) CR and 8/20 (40%) PD. IN CONCLUSION: i) HER-2 gene amplification did not seem to be correlated with response to anthracyclines. ii) Both topo-II gene amplification and protein overexpression seem to correlate with response to anthracyclines, although other factors, such as p53 and cell proliferation, are most likely to be involved. iii) The role of combined evaluation of several relevant markers and of potential 'molecular signatures' are currently being evaluated in prospective randomized clinical trials.

Comparison of topoisomerase-IIalpha gene status between primary breast cancer and corresponding distant metastatic sites.

BACKGROUND: Topoisomerase-IIalpha (topo-IIalpha) is a key enzyme in DNA replication and a molecular target for anticancer drugs called topoisomerase-II inhibitors, such as anthracyclines. Its value as a predictive marker of responsiveness to these cytotoxic drugs is currently being evaluated with promising results. However, even in the metastatic setting, the choice of treatment is based on the biologic characteristics of the primary tumor. Few data are available regarding the expression of biological markers between the primary tumor and the corresponding distant metastases. METHODS: Topo-IIalpha gene status was evaluated in 29 breast cancer patients in which a primary tumor sample and a corresponding metastatic sample were both available. Fluorescent in situ hybridization (FISH) with the Vysis triple probe (Vysis multi-color topo-IIalpha spectrum orange, Her-2 spectrum green and CEP17 spectrum aqua probe) was used, which allowed the concomitant evaluation of HER-2 gene status. RESULTS: As previously reported, topo-IIalpha gene aberrations are always associated with HER-2 gene amplification; indeed no topo-IIalpha gene aberrations have been observed in the HER-2 negative tumors. Conversely, 38.5% (five patients) of the HER-2 positive primary breast tumors (13 patients) were topo-IIalpha amplified, while 61.5% (eight patients) had a normal topo-IIalpha gene. No topo-IIalpha gene deletion was found in our series. Topo-IIalpha gene amplification in the primary tumor was always associated with amplification in the corresponding metastases, and no metastases with topo-IIalpha gene amplification were seen without amplification in the primary tumor. Furthermore, the amplification level of topo-IIalpha (i.e., ratio topo-IIalpha:CEP17) remained unchanged in primary and metastatic sites. CONCLUSION: Despite the low number of patients, our results seem to indicate that topo-IIalpha gene status evaluation in the primary breast tumor accurately reflects its status in the corresponding distant metastases.

Crystal structure of isopentenyl diphosphate:dimethylallyl diphosphate isomerase.

Isopentenyl diphosphate:dimethylallyl diphosphate (IPP:DMAPP) isomerase catalyses a crucial activation step in the isoprenoid biosynthesis pathway. This enzyme is responsible for the isomerization of the carbon-carbon double bond of IPP to create the potent electrophile DMAPP. DMAPP then alkylates other molecules, including IPP, to initiate the extraordinary variety of isoprenoid compounds found in nature. The crystal structures of free and metal-bound Escherichia coli IPP isomerase reveal critical active site features underlying its catalytic mechanism. The enzyme requires one Mn(2+) or Mg(2+) ion to fold in its active conformation, forming a distorted octahedral metal coordination site composed of three histidines and two glutamates and located in the active site. Two critical residues, C67 and E116, face each other within the active site, close to the metal-binding site. The structures are compatible with a mechanism in which the cysteine initiates the reaction by protonating the carbon-carbon double bond, with the antarafacial rearrangement ultimately achieved by one of the glutamates involved in the metal coordination sphere. W161 may stabilize the highly reactive carbocation generated during the reaction through quadrupole- charge interaction.

The carbamate kinase-like carbamoyl phosphate synthetase of the hyperthermophilic archaeon Pyrococcus furiosus, a missing link in the evolution of carbamoyl phosphate biosynthesis.

Microbial carbamoyl phosphate synthetases (CPS) use glutamine as nitrogen donor and are composed of two subunits (or domains), one exhibiting glutaminase activity, the other able to synthesize carbamoyl phosphate (CP) from bicarbonate, ATP, and ammonia. The pseudodimeric organization of this synthetase suggested that it has evolved by duplication of a smaller kinase, possibly a carbamate kinase (CK). In contrast to other prokaryotes the hyperthermophilic archaeon Pyrococcus furiosus was found to synthesize CP by using ammonia and not glutamine. We have purified the cognate enzyme and found it to be a dimer of two identical subunits of Mr 32,000. Its thermostability is considerable, 50% activity being retained after 1 h at 100 degrees C or 3 h at 95 degrees C. The corresponding gene was cloned by PCR and found to present about 50% amino acid identity with known CKs. The stoichiometry of the reaction (two ATP consumed per CP synthesized) and the ability of the enzyme to catalyze at high rate a bicarbonate-dependent ATPase reaction however clearly distinguish P. furiosus CPS from ordinary CKs. Thus the CPS of P. furiosus could represent a primeval step in the evolution of CPS from CK. Our results suggest that the first event in this evolution was the emergence of a primeval synthetase composed of subunits able to synthesize both carboxyphosphate and CP; this step would have preceded the duplication assumed to have generated the two subdomains of modern CPSs. The gene coding for this CK-like CPS was called cpkA.

Cloning and identification of the Sulfolobus solfataricus lrp gene encoding an archaeal homologue of the eubacterial leucine-responsive global transcriptional regulator Lrp.

The lrp gene of the extreme thermophilic archaeon Sulfolofus solfataricus, encoding a homologue of the eubacterial global leucine-responsive regulatory protein, was identified by DNA sequencing and sequence comparisons on a 6.9-kb genomic fragment cloned into Escherichia coli. The S. solfataricus Lrp subunit is a 155-aa polypeptide that bears between 24.5 and 29% sequence identity with eubacterial regulatory proteins of the Lrp/AsnC family and 30.6% and 25.8% with the archaeal homologues of respectively Methanococcus jannaschii and Pyrococcus furiosus. Transcription initiation from the strong S. solfataricus lrp promoter was analyzed by primer extension mapping. The abundance of the S. solfataricus lrp messenger strongly suggests that this protein might function in archaea as a global transcriptional regulator and genome organizer, as proposed for E. coli Lrp, rather than as a local, specific regulatory protein. Our findings suggest the presence of a eubacterial type of regulatory mechanism in archaea, a situation that is noteworthy indeed, since the transcriptional machinery of archaea is more closely related to that of eukaryotes, whereas these latter apparently do not possess a homologue of Lrp.