Anne-Vibeke Laenkholm

PIK3CA Mutations May Be Discordant between Primary and Corresponding Metastatic Disease in Breast Cancer

Purpose:PIK3CA mutations are frequent in breast cancer and activate the PI3K/Akt pathway. Unexpectedly, PIK3CA mutation appears in general to be associated with better outcome. In a cohort of patients where both primary and metastatic lesions were available, the objective was to assess changes in PIK3CA mutations. We wished to discern whether selective pressures occur and the influence of PIK3CA mutation on time to recurrence. Experimental Design: Formalin-fixed paraffin-embedded tumor blocks were obtained from 104 patients with paired samples from primary tumors and corresponding asynchronous metastatic breast tumors. Samples were analyzed for PIK3CA mutations (exons 9 and 20) as well as immunohistochemical evaluation for PTEN, pAKT, Ki67, ER, and HER2. Results:PIK3CA mutation was detected in 45% of the primary tumors. Overall, there was a net gain in mutation in metastatic disease, to 53%; nonetheless, there were instances where metastases were wild type in patients with PIK3CA mutant primary tumors. Laser capture microdissection on a subset of cases revealed microheterogeneity for PIK3CA mutational status in the primary tumor. PIK3CA mutants overall showed a significantly longer time to first recurrence than wild type cases (P = 0.03). Conclusion:PIK3CA mutations occur at high frequency in primary and metastatic breast cancer; these may not necessarily confer increased aggressiveness as mutants had a longer time to recurrence. Because PIK3CA status quite frequently changes between primary and metastatic disease, it emphasizes the necessity of assessing the PIK3CA status in the metastatic lesion for selection of PIK3CA inhibitor therapy. Clin Cancer Res; 17(4); 667–77. ©2010 AACR.

Activation of ErbB3, EGFR and Erk is essential for growth of human breast cancer cell lines with acquired resistance to fulvestrant

Seven fulvestrant resistant cell lines derived from the estrogen receptor α positive MCF-7 human breast cancer cell line were used to investigate the importance of epidermal growth factor receptor (ErbB1-4) signaling. We found an increase in mRNA expression of EGFR and the ErbB3/ErbB4 ligand heregulin2 (hrg2) and a decrease of ErbB4 in all resistant cell lines. Western analyses confirmed the upregulation of EGFR and hrg2 and the downregulation of ErbB4. Elevated activation of EGFR and ErbB3 was seen in all resistant cell lines and the ErbB3 activation occurred by an autocrine mechanism. ErbB4 activation was observed only in the parental MCF-7 cells. The downstream kinases pAkt and pErk were increased in five of seven and in all seven resistant cell lines, respectively. Treatment with the EGFR inhibitor gefitinib preferentially inhibited growth and reduced the S phase fraction in the resistant cell lines concomitant with inhibition of Erk and unaltered Akt activation. In concert, inhibition of Erk with U0126 preferentially reduced growth of resistant cell lines. Treatment with ErbB3 neutralizing antibodies inhibited ErbB3 activation and resulted in a modest but statistically significant growth inhibition of two resistant cell lines. These data indicate that ligand activated ErbB3 and EGFR, and Erk signaling play important roles in fulvestrant resistant cell growth. Furthermore, the decreased level of ErbB4 in resistant cells may facilitate heterodimerization of ErbB3 with EGFR and ErbB2. Our data support that a concerted action against EGFR, ErbB2 and ErbB3 may be required to obtain complete growth suppression of fulvestrant resistant cells.

Prognostic significance of urokinase-type plasminogen activator and plasminogen activator inhibitor-1 in primary breast cancer

The uPA-mediated pathway of plasminogen activation is central to cancer metastasis. Whether uPA and PAI-1 are related to local recurrence, metastatic spread or both is not clear. We present a retrospective study of 429 primary breast cancer patients with a median follow-up of 5.1 years, in which the levels of uPA and PAI-1 in tumour extracts were analysed by means of an enzyme-linked immunosorbent assay. The median values of uPA and PAI-1, which were used as cut-off points, were 4.5 and 11.1 ng mg(-1) protein respectively. The levels of uPA and PAI-1 were correlated with tumour size, degree of anaplasia, steroid receptor status and number of positive nodes. Patients with high content of either uPA or PAI-1 had increased risk of relapse and death. We demonstrated an independent ability of PAI-1 to predict distant metastasis (relative risk 1.7, confidence limits 1.22 and 2.46) and that neither uPA nor PAI-1 provided any information regarding local recurrence.

Metastasis-related Plasma Membrane Proteins of Human Breast Cancer Cells Identified by Comparative Quantitative Mass Spectrometry

The spread of cancer cells from a primary tumor to form metastasis at distant sites is a complex multistep process. The cancer cell proteins and plasma membrane proteins in particular involved in this process are poorly defined, and a study of the very early events of the metastatic process using clinical samples or in vitro assays is not feasible. We have used a unique model system consisting of two isogenic human breast cancer cell lines that are equally tumorigenic in mice; but although one gives rise to metastasis, the other disseminates single cells that remain dormant at distant organs. Membrane purification and comparative quantitative LC-MS/MS proteomics identified 13 membrane proteins that were expressed at higher levels and three that were underexpressed in the metastatic compared with the non-metastatic cell line from a total of 1919 identified protein entries. Among the proteins were ecto-5′-nucleotidase (CD73), NDRG1, integrin β1, CD44, CD74, and major histocompatibility complex class II proteins. The altered expression levels of proteins identified by LC-MS/MS were validated using flow cytometry, Western blotting, and immunocyto- and immunohistochemistry. Analysis of clinical breast cancer biopsies demonstrated a significant correlation between high ecto-5′-nucleotidase and integrin β1 expression and poor outcome, measured as tumor spread or distant recurrence within a 10-year follow-up. Further the tissue analysis suggested that NDRG1, HLA-DRα, HLA-DRβ, and CD74 were associated with the ER−/PR− phenotype represented by the two cell lines. The study demonstrates a quantitative and comparative proteomics strategy to identify clinically relevant key molecules in the early events of metastasis, some of which may prove to be potential targets for cancer therapy.

PIK3CA mutations, PTEN, and pHER2 expression and impact on outcome in HER2-positive early-stage breast cancer patients treated with adjuvant chemotherapy and trastuzumab

BACKGROUND This study was conducted to determine the frequency of PIK3CA mutations and human epidermal growth factor receptor-2 (HER2) phosphorylation status (pHER2-Tyr1221/1222) and if PIK3CA, phosphatase and tensin homolog (PTEN), or pHER2 has an impact on outcome in HER2-positive early-stage breast cancer patients treated with adjuvant chemotherapy and trastuzumab. PATIENTS AND METHODS Two hundred and forty HER2-positive early-stage breast cancer patients receiving adjuvant treatment (cyclophosphamide 600 mg/m2, epirubicin 60 mg/m2, and fluorouracil 600 mg/m2) before administration of 1 year trastuzumab were assessable. PTEN and pHER2 expression were assessed by immunohistochemistry. PIK3CA mutations (exons 9 and 20) were determined by pyrosequencing. RESULTS Five-year overall survival (OS) and invasive disease-free survival were 87.8% and 81.0%, respectively. Twenty-six percent of patients had a PIK3CA mutation, 24% were PTEN low, 45% pHER2 high, and 47% patients had increased PI3K pathway activation (PTEN low and/or PIK3CA mutation). No significant correlations were observed between the clinicopathological variables and PIK3CA, PTEN, and pHER2 status. In both univariate and multivariate analyses, patients with PIK3CA mutations or high PI3K pathway activity had a significant worse OS [multivariate: hazard ratio (HR) 2.14, 95% confidence interval (CI) 1.01-4.51, P=0.046; and HR 2.35, 95% CI 1.10-5.04, P=0.03]. CONCLUSION Patients with PIK3CA mutations or increased PI3K pathway activity had a significantly poorer survival despite adequate treatment with adjuvant chemotherapy and trastuzumab.

Identification of genes for normalization of real-time RT-PCR data in breast carcinomas

BackgroundQuantitative real-time RT-PCR (RT-qPCR) has become a valuable molecular technique in basic and translational biomedical research, and is emerging as an equally valuable clinical tool. Correlation of inter-sample values requires data normalization, which can be accomplished by various means, the most common of which is normalization to internal, stably expressed, reference genes. Recently, such traditionally utilized reference genes as GAPDH and B2M have been found to be regulated in various circumstances in different tissues, emphasizing the need to identify genes independent of factors influencing the tissue, and that are stably expressed within the experimental milieu. In this study, we identified genes for normalization of RT-qPCR data for invasive breast cancer (IBC), with special emphasis on estrogen receptor positive (ER+) IBC, but also examined their applicability to ER- IBC, normal breast tissue and breast cancer cell lines.MethodsThe reference genes investigated by qRT-PCR were RPLP0, TBP, PUM1, ACTB, GUS-B, ABL1, GAPDH and B2M. Biopsies of 18 surgically-excised tissue specimens (11 ER+ IBCs, 4 ER- IBCs, 3 normal breast tissues) and 3 ER+ cell lines were examined and the data analyzed by descriptive statistics, geNorm and NormFinder. In addition, the expression of selected reference genes in laser capture microdissected ER+ IBC cells were compared with that of whole-tissue.ResultsA group of 3 genes, TBP, RPLP0 and PUM1, were identified for both the combined group of human tissue samples (ER+ and ER- IBC and normal breast tissue) and for the invasive cancer samples (ER+ and ER- IBC) by GeNorm, where NormFinder consistently identified PUM1 at the single best gene for all sample combinations.ConclusionThe reference genes of choice when performing RT-qPCR on normal and malignant breast specimens should be either the collected group of 3 genes (TBP, RPLP0 and PUM1) employed as an average, or PUM1 as a single gene.

Determination of HER2 phosphorylation at tyrosine 1221/1222 improves prediction of poor survival for breast cancer patients with hormone receptor-positive tumors

IntroductionHigh expression of total HER2 protein confers poor prognosis for breast cancer patients. HER2 is a member of the HER family consisting of four receptors, HER1 to HER4. HER receptor activity is regulated by a variety of mechanisms, and phosphorylation of the C-terminal part of the HER receptors is a marker for active signaling. The importance of phosphorylation and thereby activation of the HER1 to HER4 receptors, however, has not been investigated concomitantly in breast tumors. In the present study we examined the importance of active HER signaling in breast tumor biopsies and paired metastases, by evaluating the expression of phosphorylated HER1, HER2, HER3, Erk, Akt and the total level of HER4 and HER2.MethodsImmunohistochemical analysis was performed on 268 primary breast tumors and 30 paired metastatic lesions from postmenopausal women with hormone receptor-positive breast tumors, who had received adjuvant tamoxifen therapy. The observed protein expression levels were analyzed for co-expression, for correlation to clinicopathological parameters and for prognostic value in relation to disease-free survival and overall survival. Lastly, the difference between protein levels in primary tumors versus metastasis was evaluated.ResultsIn the primary tumors, 8%, 18%, 14% and 15% of cases were scored positive for total HER2, pHER1, pHER2 and pHER3 expression, respectively. HER4 was expressed with strong intensity in 68% and at moderate intensity in 29% of cases. The activated forms of Akt and Erk were quite uniformly expressed in the categories; negative, moderate or strong. In univariate analysis, expression of total HER2, pHER1, pHER2 and pHER3 was significantly associated with poor disease-free survival. Strong HER4 expression was associated with prolonged disease-free as well as with overall survival. Expression of pAkt and pErk was not correlated with survival. In multivariate analysis, pHER2 expression was clearly an independent marker for poor disease-free survival and overall survival when tested against tumor size, tumor grade, nodal status and HER2. Lastly, comparison of HER receptor expression in metastatic versus primary tumors showed a significant increase in expression of pHER1 and pHER3 in the metastases.ConclusionsIn hormone receptor-positive breast cancer, determination of pHER2 yields additional prognostic information about poor prognosis compared with the current clinical standard for measuring HER2.

Phage Display–Derived Human Monoclonal Antibodies Isolated by Binding to the Surface of Live Primary Breast Cancer Cells Recognize GRP78

Clinical trials using monoclonal antibodies (mAb) against cell-surface markers have yielded encouraging therapeutic results in several cancer types. Generally, however, anticancer antibodies are only efficient against a subpopulation of cancers, and there is a strong need for identification of novel targets and human antibodies against them. We have isolated single-chain human mAbs from a large naïve antibody phage display library by panning on a single-cell suspension of freshly isolated live cancer cells from a human breast cancer specimen, and these antibodies were shown to specifically recognize cancer-associated cell-surface proteins. One of the isolated human antibody fragments, Ab39, recognizes a cell-surface antigen expressed on a subpopulation of cancer cell lines of different origins. Immunohistochemical analysis of a large panel of cancerous and normal tissues showed that Ab39 bound strongly to several cancers, including 45% breast carcinomas, 35% lung cancers, and 86% melanomas, but showed no or weak binding to normal tissues. A yeast two-hybrid screen of a large human testis cDNA library identified the glucose-regulated protein of 78 kDa (GRP78) as the antigen recognized by Ab39. The interaction was confirmed by colocalization studies and antibody competition experiments that also mapped the epitope recognized by Ab39 to the COOH terminus of GRP78. The expression of GRP78 on the surface of cancer cells, but not normal cells, makes it an attractive target for cancer therapies including mAb-based immunotherapy. Our results suggest that the human antibody Ab39 may be a useful starting point for further genetic optimization that could render it a useful diagnostic and therapeutic reagent for a variety of cancers.

Evaluation of Nine Somatic Variant Callers for Detection of Somatic Mutations in Exome and Targeted Deep Sequencing Data.

Next generation sequencing is extensively applied to catalogue somatic mutations in cancer, in research settings and increasingly in clinical settings for molecular diagnostics, guiding therapy decisions. Somatic variant callers perform paired comparisons of sequencing data from cancer tissue and matched normal tissue in order to detect somatic mutations. The advent of many new somatic variant callers creates a need for comparison and validation of the tools, as no de facto standard for detection of somatic mutations exists and only limited comparisons have been reported. We have performed a comprehensive evaluation using exome sequencing and targeted deep sequencing data of paired tumor-normal samples from five breast cancer patients to evaluate the performance of nine publicly available somatic variant callers: EBCall, Mutect, Seurat, Shimmer, Indelocator, Somatic Sniper, Strelka, VarScan 2 and Virmid for the detection of single nucleotide mutations and small deletions and insertions. We report a large variation in the number of calls from the nine somatic variant callers on the same sequencing data and highly variable agreement. Sequencing depth had markedly diverse impact on individual callers, as for some callers, increased sequencing depth highly improved sensitivity. For SNV calling, we report EBCall, Mutect, Virmid and Strelka to be the most reliable somatic variant callers for both exome sequencing and targeted deep sequencing. For indel calling, EBCall is superior due to high sensitivity and robustness to changes in sequencing depths.

Global microRNA expression profiling of high-risk ER+ breast cancers from patients receiving adjuvant tamoxifen mono-therapy: a DBCG study.

Purpose Despite the benefits of estrogen receptor (ER)-targeted endocrine therapies in breast cancer, many tumors develop resistance. MicroRNAs (miRNAs) have been suggested as promising biomarkers and we here evaluated whether a miRNA profile could be identified, sub-grouping ER+ breast cancer patients treated with adjuvant Tamoxifen with regards to probability of recurrence. Experimental Design Global miRNA analysis was performed on 152 ER+ primary tumors from high-risk breast cancer patients with an initial discovery set of 52 patients, followed by two independent test sets (N = 60 and N = 40). All patients had received adjuvant Tamoxifen as mono-therapy (median clinical follow-up: 4.6 years) and half had developed distant recurrence (median time-to-recurrence: 3.5 years). MiRNA expression was examined by unsupervised hierarchical clustering and supervised analysis, including clinical parameters as co-variables. Results The discovery set identified 10 highly significant miRNAs that discriminated between the patient samples according to outcome. However, the subsequent two independent test sets did not confirm the predictive potential of these miRNAs. A significant correlation was identified between miR-7 and the tumor grade. Investigation of the microRNAs with the most variable expression between patients in different runs yielded a list of 31 microRNAs, eight of which are associated with stem cell characteristics. Conclusions Based on the large sample size, our data strongly suggests that there is no single miRNA profile predictive of outcome following adjuvant Tamoxifen treatment in a broad cohort of ER+ breast cancer patients. We identified a sub-group of Tamoxifen-treated breast cancer patients with miRNA-expressing tumors associated with cancer stem cell characteristics.