Human papilloma virus (HPV) integration signature in Cervical Cancer: identification of MACROD2 gene as HPV hot spot integration site.

BACKGROUND: Cervical cancer (CC) remains a leading cause of gynaecological cancer-related mortality with infection by human papilloma virus (HPV) being the most important risk factor. We analysed the association between different viral integration signatures, clinical parameters and outcome in pre-treated CCs. METHODS: Different integration signatures were identified using HPV double capture followed by next-generation sequencing (NGS) in 272 CC patients from the BioRAIDs study [NCT02428842]. Correlations between HPV integration signatures and clinical, biological and molecular features were assessed. RESULTS: Episomal HPV was much less frequent in CC as compared to anal carcinoma (p < 0.0001). We identified >300 different HPV-chromosomal junctions (inter- or intra-genic). The most frequent integration site in CC was in MACROD2 gene followed by MIPOL1/TTC6 and TP63. HPV integration signatures were not associated with histological subtype, FIGO staging, treatment or PFS. HPVs were more frequently episomal in PIK3CA mutated tumours (p = 0.023). Viral integration type was dependent on HPV genotype (p < 0.0001); HPV18 and HPV45 being always integrated. High HPV copy number was associated with longer PFS (p = 0.011). CONCLUSIONS: This is to our knowledge the first study assessing the prognostic value of HPV integration in a prospectively annotated CC cohort, which detects a hotspot of HPV integration at MACROD2; involved in impaired PARP1 activity and chromosome instability.

Variant peptide detection utilizing mass spectrometry: laying the foundations for proteogenomic identification and validation.

BACKGROUND: Proteogenomics is an emerging field at the intersection of genomics and proteomics. Many variant peptides corresponding to single nucleotide variations (SNVs) are associated with specific diseases. The aim of this study was to demonstrate the feasibility of proteogenomic-based variant peptide detection in disease models and clinical specimens. METHODS: We sought to detect p53 single amino acid variant (SAAV) peptides in breast cancer tumor samples that have been previously subjected to sequencing analysis. Initially, two cancer cell lines having a cellular tumor antigen p53 (TP53) mutation and one wild type for TP53 were analyzed by selected reaction monitoring (SRM) assays as controls. One pool of wild type and one pool of mutated for TP53 cytosolic extracts were assayed with a shotgun proteogenomic workflow. Furthermore, 18 individual samples having a mutation in TP53 were assayed by SRM. RESULTS: Two mutant p53 peptides were successfully detected in two cancer cell lines as expected from their DNA sequence. Wild type p53 peptides were detected in both cytosolic pools, however, none of the mutant p53 peptides were identified. Mutations at the protein level were detected in two cytosolic extracts and whole tumor lysates from the same patients by SRM analysis. Six thousand and six hundred and twenty eight non-redundant proteins were identified in the two cytosolic pools, thus greatly improving a previously reported cytosolic proteome. CONCLUSIONS: In the current study we show the great potential of using proteogenomics for the direct identification of cancer-associated mutations in clinical samples and we discuss current limitations and future perspectives.

An 8-gene mRNA expression profile in circulating tumor cells predicts response to aromatase inhibitors in metastatic breast cancer patients.

BACKGROUND: Molecular characterization of circulating tumor cells (CTC) is promising for personalized medicine. We aimed to identify a CTC gene expression profile predicting outcome to first-line aromatase inhibitors in metastatic breast cancer (MBC) patients. METHODS: CTCs were isolated from 78 MBC patients before treatment start. mRNA expression levels of 96 genes were measured by quantitative reverse transcriptase polymerase chain reaction. After applying predefined exclusion criteria based on lack of sufficient RNA quality and/or quantity, the data from 45 patients were used to construct a gene expression profile to predict poor responding patients, defined as disease progression or death <9 months, by a leave-one-out cross validation. RESULTS: Of the 45 patients, 19 were clinically classified as poor responders. To identify them, the 75% most variable genes were used to select genes differentially expressed between good and poor responders. An 8-gene CTC predictor was significantly associated with outcome (Hazard Ratio [HR] 4.40, 95% Confidence Interval [CI]: 2.17-8.92, P < 0.001). This predictor identified poor responding patients with a sensitivity of 63% and a positive predictive value of 75%, while good responding patients were correctly predicted in 85% of the cases. In multivariate Cox regression analysis, including CTC count at baseline, the 8-gene CTC predictor was the only factor independently associated with outcome (HR 4.59 [95% CI: 2.11-9.56], P < 0.001). This 8-gene signature was not associated with outcome in a group of 71 MBC patients treated with systemic treatments other than AI. CONCLUSIONS: An 8-gene CTC predictor was identified which discriminates good and poor outcome to first-line aromatase inhibitors in MBC patients. Although results need to be validated, this study underscores the potential of molecular characterization of CTCs.

miR-634 restores drug sensitivity in resistant ovarian cancer cells by targeting the Ras-MAPK pathway.

BACKGROUND: Drug resistance hampers the efficient treatment of malignancies, including advanced stage ovarian cancer, which has a 5-year survival rate of only 30 %. The molecular processes underlying resistance have been extensively studied, however, not much is known about the involvement of microRNAs. METHODS: Differentially expressed microRNAs between cisplatin sensitive and resistant cancer cell line pairs were determined using microarrays. Mimics were used to study the role of microRNAs in drug sensitivity of ovarian cancer cell lines and patient derived tumor cells. Luciferase reporter constructs were used to establish regulation of target genes by microRNAs. RESULTS: MiR-634 downregulation was associated with cisplatin resistance. Overexpression of miR-634 affected cell cycle progression and enhanced apoptosis in ovarian cancer cells. miR-634 resensitized resistant ovarian cancer cell lines and patient derived drug resistant tumor cells to cisplatin. Similarly, miR-634 enhanced the response to carboplatin and doxorubicin, but not to paclitaxel. The cell cycle regulator CCND1, and Ras-MAPK pathway components GRB2, ERK2 and RSK2 were directly repressed by miR-634 overexpression. Repression of the Ras-MAPK pathway using a MEK inhibitor phenocopied the miR-634 effects on viability and chemosensitivity. CONCLUSION: miR-634 levels determine chemosensitivity in ovarian cancer cells. We identify miR-634 as a therapeutic candidate to resensitize chemotherapy resistant ovarian tumors.

PIK3CA mutations, phosphatase and tensin homolog, human epidermal growth factor receptor 2, and insulin-like growth factor 1 receptor and adjuvant tamoxifen resistance in postmenopausal breast cancer patients.

INTRODUCTION: Inhibitors of the phosphatidylinositol-3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) pathway can overcome endocrine resistance in estrogen receptor (ER) α-positive breast cancer, but companion diagnostics indicating PI3K/AKT/mTOR activation and consequently endocrine resistance are lacking. PIK3CA mutations frequently occur in ERα-positive breast cancer and result in PI3K/AKT/mTOR activation in vitro. Nevertheless, the prognostic and treatment-predictive value of these mutations in ERα-positive breast cancer is contradictive. We tested the clinical validity of PIK3CA mutations and other canonic pathway drivers to predict intrinsic resistance to adjuvant tamoxifen. In addition, we tested the association between these drivers and downstream activated proteins. METHODS: Primary tumors from 563 ERα-positive postmenopausal patients, randomized between adjuvant tamoxifen (1 to 3 years) versus observation were recollected. PIK3CA hotspot mutations in exon 9 and exon 20 were assessed with Sequenom Mass Spectometry. Immunohistochemistry was performed for human epidermal growth factor receptor 2 (HER2), phosphatase and tensin homolog (PTEN), and insulin-like growth factor 1 receptor (IGF-1R). We tested the association between these molecular alterations and downstream activated proteins (like phospho-protein kinase B (p-AKT), phospho-mammalian target of rapamycin (p-mTOR), p-ERK1/2, and p-p70S6K). Recurrence-free interval improvement with tamoxifen versus control was assessed according to the presence or absence of canonic pathway drivers, by using Cox proportional hazard models, including a test for interaction. RESULTS: PIK3CA mutations (both exon 9 and exon 20) were associated with low tumor grade. An enrichment of PIK3CA exon 20 mutations was observed in progesterone receptor- positive tumors. PIK3CA exon 20 mutations were not associated with downstream-activated proteins. No significant interaction between PIK3CA mutations or any of the other canonic pathway drivers and tamoxifen-treatment benefit was found. CONCLUSION: PIK3CA mutations do not have clinical validity to predict intrinsic resistance to adjuvant tamoxifen and may therefore be unsuitable as companion diagnostic for PI3K/AKT/mTOR inhibitors in ERα- positive, postmenopausal, early breast cancer patients.

Phosphorylated p-70S6K predicts tamoxifen resistance in postmenopausal breast cancer patients randomized between adjuvant tamoxifen versus no systemic treatment.

INTRODUCTION: Activation of the phosphatidylinositol-3-kinase (PI3K) and/or mitogen-activated protein kinase (MAPK) pathways results in anti-estrogen resistance in vitro, but a biomarker with clinical validity to predict intrinsic resistance has not been identified. In metastatic breast cancer patients with previous exposure to endocrine therapy, the addition of a mammalian target of rapamycine (mTOR) inhibitor has been shown to be beneficial. Whether or not patients on adjuvant endocrine treatment might benefit from these drugs is currently unclear. A biomarker that predicts intrinsic resistance could potentially be used as companion diagnostic in this setting. We tested the clinical validity of different downstream-activated proteins in the PI3K and/or MAPK pathways to predict intrinsic tamoxifen resistance in postmenopausal primary breast cancer patients. METHODS: We recollected primary tumor tissue from patients who participated in a randomized trial of adjuvant tamoxifen (1-3 years) versus observation. After constructing a tissue micro-array, cores from 563 estrogen receptor α positive were immunostained for p-AKT(Thr308), p-AKT(Ser473), p-mTOR, p-p706SK and p-ERK1/2. Cox proportional hazard models for recurrence free interval were used to assess hazard ratios and interactions between these markers and tamoxifen treatment efficacy. RESULTS: Interactions were identified between tamoxifen and p-AKT(Thr308), p-mTOR, p-p70S6K and p-ERK1/2. Applying a conservative level of significance, p-p70S6K remained significantly associated with tamoxifen resistance. Patients with p-p70S6K negative tumors derived significant benefit from tamoxifen (HR 0.24, P < 0.0001), while patients whose tumor did express p-p70S6K did not (HR = 1.02, P =0.95), P for interaction 0.004. In systemically untreated breast cancer patients, p-p70S6K was associated with a decreased risk for recurrence. CONCLUSIONS: Patients whose tumor expresses p-p70S6K, as a marker of downstream PI3K and/or MAPK pathway activation, have a favorable prognosis, but do not benefit from adjuvant tamoxifen. A potential benefit from inhibitors of the PI3K/Akt/mTOR pathway in these patients needs to be further explored.

Hotspot mutations in PIK3CA associate with first-line treatment outcome for aromatase inhibitors but not for tamoxifen.

PIK3CA mutations occur frequently in breast cancer, predominantly in exons 9 and 20. The aim of this retrospective study is to evaluate the PIK3CA mutation status for its relationship with prognosis and first-line endocrine therapy outcome. PIK3CA exon 9 and 20 were evaluated for mutations in 1,352 primary breast cancer specimens by SnaPshot multiplex analyses. The mutation status was studied for their relationship with metastasis-free survival (MFS) in 342 untreated lymph node-negative (LNN) patients and to time to progression (TTP) in estrogen receptor (ER)-positive patients with metastatic disease treated with first-line tamoxifen (N = 447) or aromatase inhibitors (AIs; N = 84). We detected in 423 patients hotspot mutations for PIK3CA (31 %). Mutations in exon 20 were detected in 251 patients (59 %), with H1047L and H1047R mutations in 37 (15 %) and 214 (85 %) cases, respectively. Mutations in PIK3CA exon 9 were discovered in 173 patients (41 %), with E542K and E545K mutations in 57 (32 %) and 104 (60 %) cases as most prevalent ones. Evaluation of the untreated LNN patients for prognosis showed no relationship between MFS and PIK3CA mutations, neither for exon 9 [HR = 1.04 (95 % CI 0.57-1.89), P = 0.90] nor for exon 20 [HR = 0.98 (95 % CI 0.63-1.54); P = 0.94] when compared to wild-type. The PIK3CA mutation status was also not associated with treatment outcome after first-line tamoxifen. On the other hand, patients treated with first-line AIs showed a longer TTP when having a PIK3CA mutation in exon 9 [HR = 0.40 (95 % CI 0.17-0.95); P = 0.038] or exon 20 [HR = 0.50 (95 % CI 0.27-0.91); P = 0.024] compared to wild-types, both significant in uni- and multivariate analysis including traditional predictive factors. All results remained when only HER2-negative patients were evaluated for each cohort. PIK3CA mutations in ER-positive tumors were significantly associated with a favorable outcome after first-line AIs, which needs further confirmation in other datasets. Mutations were not associated with prognosis in untreated LNN patients nor predictive outcome after first-line tamoxifen therapy in advanced disease patients.

Correlation of breast cancer susceptibility loci with patient characteristics, metastasis-free survival, and mRNA expression of the nearest genes.

To understand the biology of low-risk breast cancer alleles, and to investigate whether these loci also contribute to disease progression that was once established, we examined the association of SNPs tagging the low-risk breast cancer loci in or near FGFR2, LSP1, MAP3K1, H19, TOX3, POU5F1P1, MYC, and 2q35, with clinical, pathological characteristics, prognosis, and mRNA expression of the nearest genes. Tumor DNA samples of 2,480 breast cancer patients were available. Out of this cohort, 1,290 patients with lymph-node negative disease who did not receive adjuvant systemic therapy, the SNP status was associated with metastasis-free survival (MFS). In 1,401 patients, the mRNA expression levels of FGFR2, LSP1, MAP3K1, H19, TOX3, POU5F1P1, and MYC were determined and correlated with SNP genotypes. The SNP rs2981582 in FGFR2 was significantly associated with positive ER and PgR status (P < 0.001 and P = 0.003, respectively). No other significant associations with patient or tumor characteristics were observed. Only rs2107425 near H19 was significantly associated with shorter MFS in uni- and multi-variate analysis (HR: 1.53, CI: 1.12-2.08, P = 0.006 and HR: 1.59, CI: 1.16-2.20, P = 0.004, respectively), with the more aggressive minor allele displaying a recessive trait. The minor allele of SNP rs3803662 located near the TOX3 gene was associated with lower mRNA expression of this gene. In conclusion, except for the association of rs13283662 with TOX3 gene expression indicating a tumor suppressor role of TOX3, our findings suggest that breast cancer low-risk loci generally do not affect expression of the nearest gene in breast tumor tissue. Also the prognosis of patients is largely not affected by low-risk breast cancer loci except for the SNP near H19. How, this SNP affects prognosis warrants further study as it does not operate through altering H19 mRNA expression.

Circulating HPV DNA as a Marker for Early Detection of Relapse in Patients with Cervical Cancer.

PURPOSE: Almost all cervical cancers are caused by human papillomavirus (HPV) and patients with advanced stage are at high risk for relapse. Circulating HPV DNA (HPV ctDNA) may serve as a residual tumor marker at the end of chemoradiation or to predict relapse during the follow-up period. EXPERIMENTAL DESIGN: We analyzed serum samples from 94 HPV16- or HPV18-related CCs from the BioRAIDs prospective cohort. Samples were collected before and after treatment and during an 18-month follow-up period. Using digital droplet PCR (ddPCR), we assessed the relevance of circulating HPV E7 gene as a marker for residual disease compared to HPV integration site and PIK3CA mutations. Finally, the prognostic impact of circulating HPV E7 gene was assessed with its prediction value of relapse. RESULTS: HPV E7 gene was the most sensitive tumor marker, superior to both HPV integration sites and PIK3CA mutations in serum. Circulating HPV DNA (HPV ctDNA) was detected in 63% (59/94) of patients, before treatment. HPV ctDNA detection in serum sample was associated with high FIGO stage (P = 0.02) and para-aortic lymph node involvement (P = 0.01). The level of HPV ctDNA was positively correlated with HPV copy number in the tumor (R = 0.39, P < 0.001). Complete clearance of HPV ctDNA by the end of treatment was significantly associated with a longer PFS (P < 0.0001). Patients with persistent HPV ctDNA in serum relapsed with a median time of 10 months (range, 2-15) from HPV ctDNA detection. CONCLUSIONS: HPV ctDNA detection is a useful marker to predict relapse in cervical cancer.See related commentary by Wentzensen and Clarke, p. 5733.

Pathway analysis of gene lists associated with platinum-based chemotherapy resistance in ovarian cancer: the big picture.

OBJECTIVE: Ovarian cancer is the leading cause of death from gynecological cancers in the Western world (Parkin et al., 2005). The overall 5-year survival is only 30% (Moss and Kaye, 2002), which is for a significant part due to platinum-based chemotherapy resistance. In this study, we performed a pathway analysis on nine published gene sets associated with platinum resistance in ovarian cancer, including a study by us. With this exploratory study, we aim to identify overlapping pathways associated with platinum-based chemotherapy resistance mechanisms in ovarian cancer. METHODS: Gene Ontology (GO) analysis and Ingenuity Pathway Analysis (IPA) were performed to determine which functional processes were differentially represented in the combined gene lists of nine studies (457 genes) compared to all Unigene identifiers or the Ingenuity knowledge base. RESULTS: The GO and IPA analysis resulted in the generation of 23 gene networks, and showed that 13 GO processes (>or=2 times enriched), 71 canonical pathways (p<0.05,), eight toxicity pathways (p<0.05) and 74 biological functions (p<0.005) are significantly associated with the 9-study gene set. CONCLUSION AND RECOMMENDATIONS: Several pathways identified have previously been shown to be associated with therapy resistance: these include 'oxidative stress response mediated by Nrf2,' 'TP53 signaling' and 'TGFbeta signaling.' The role of TGFbeta signaling and related miRNAs identified in the network analysis in epithelial-to-mesenchymal transition (EMT) and stemness as well as the possible relation with platin-based chemotherapy resistance are further discussed in detail. We propose that future international cooperation should aim at a uniform pooled analysis of the wealth of ovarian cancer array data already available. This will enhance the power of each separate ovarian cancer study and can lead to promising results.

TP53 Mutations in Serum Circulating Cell-Free Tumor DNA As Longitudinal Biomarker for High-Grade Serous Ovarian Cancer.

The aim of this study was to determine an optimal workflow to detect TP53 mutations in baseline and longitudinal serum cell free DNA (cfDNA) from high-grade serous ovarian carcinomas (HGSOC) patients and to define whether TP53 mutations are suitable as biomarker for disease. TP53 was investigated in tissue and archived serum from 20 HGSOC patients by a next-generation sequencing (NGS) workflow alone or combined with digital PCR (dPCR). AmpliSeq™-focused NGS panels and customized dPCR assays were used for tissue DNA and longitudinal cfDNAs, and Oncomine NGS panel with molecular barcoding was used for baseline cfDNAs. TP53 missense mutations were observed in 17 tissue specimens and in baseline cfDNA for 4/8 patients by AmpliSeq, 6/9 patients by Oncomine, and 4/6 patients by dPCR. Mutations in cfDNA were detected in 4/6 patients with residual disease and 3/4 patients with disease progression within six months, compared to 5/11 patients with no residual disease and 6/13 patients with progression after six months. Finally, mutations were detected at progression in 5/6 patients, but not during chemotherapy. NGS with molecular barcoding and dPCR were most optimal workflows to detect TP53 mutations in baseline and longitudinal serum cfDNA, respectively. TP53 mutations were undetectable in cfDNA during treatment but re-appeared at disease progression, illustrating its promise as a biomarker for disease monitoring.

CX-5461 activates the DNA damage response and demonstrates therapeutic efficacy in high-grade serous ovarian cancer.

Acquired resistance to PARP inhibitors (PARPi) is a major challenge for the clinical management of high grade serous ovarian cancer (HGSOC). Here, we demonstrate CX-5461, the first-in-class inhibitor of RNA polymerase I transcription of ribosomal RNA genes (rDNA), induces replication stress and activates the DNA damage response. CX-5461 co-operates with PARPi in exacerbating replication stress and enhances therapeutic efficacy against homologous recombination (HR) DNA repair-deficient HGSOC-patient-derived xenograft (PDX) in vivo. We demonstrate CX-5461 has a different sensitivity spectrum to PARPi involving MRE11-dependent degradation of replication forks. Importantly, CX-5461 exhibits in vivo single agent efficacy in a HGSOC-PDX with reduced sensitivity to PARPi by overcoming replication fork protection. Further, we identify CX-5461-sensitivity gene expression signatures in primary and relapsed HGSOC. We propose CX-5461 is a promising therapy in combination with PARPi in HR-deficient HGSOC and also as a single agent for the treatment of relapsed disease.

TSC22D1 and PSAP predict clinical outcome of tamoxifen treatment in patients with recurrent breast cancer.

Purpose Two genes, TSC22 domain family, member 1 (TSC22D1) and prosaposin (PSAP) were identified in an in vitro functional screen for genes having a causative role in tamoxifen resistance. These genes were also present in our previously established 81-gene signature for resistance to first-line tamoxifen therapy. The aim of this study was to investigate the predictive value of these genes for tamoxifen therapy failure in patients with recurrent breast cancer. Experimental Design The mRNA levels of TSC22D1 and PSAP were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR) in 223 estrogen receptor-positive primary breast tumors of patients with recurrent disease treated with first-line tamoxifen therapy. The main objective of this study was the length of progression-free survival (PFS). Results High mRNA levels of TSC22D1 and PSAP were significantly associated with shorter PFS and both were independent of the traditional predictive factors (HR = 1.30, 95% CI = 1.04-1.64 P = 0.023; and HR = 1.40, 95% CI = 1.03-1.88, P = 0.029, respectively). In multivariate analysis, patients with high mRNA levels of both genes associated significantly with no clinical benefit (OR = 0.19, 95% CI = 0.06-0.62, P = 0.006) and had the shortest PFS (HR = 2.05, 95% CI = 1.29-3.25, P = 0.002). Conclusion These results confirm our previous in vitro and tumor-related findings and are indicative for the failure of tamoxifen treatment in breast-cancer patients. Both TSC22D1 and PSAP are associated with clinical outcome and may have a functional role in therapy resistance.

Comparison of gene expression profiles predicting progression in breast cancer patients treated with tamoxifen.

BACKGROUND: Molecular signatures that predict outcome in tamoxifen treated breast cancer patients have been identified. For the first time, we compared these response profiles in an independent cohort of (neo)adjuvant systemic treatment naïve breast cancer patients treated with first-line tamoxifen for metastatic disease. METHODS: From a consecutive series of 246 estrogen receptor (ER) positive primary tumors, gene expression profiling was performed on available frozen tumors using 44K oligoarrays (n = 69). A 78-gene tamoxifen response profile (formerly consisting of 81 cDNA-clones), a 21-gene set (microarray-based Recurrence Score), as well as the HOXB13-IL17BR ratio (Two-Gene-Index, RT-PCR) were analyzed. Performance of signatures in relation to time to progression (TTP) was compared with standard immunohistochemical (IHC) markers: ER, progesterone receptor (PgR) and HER2. RESULTS: In univariate analyses, the 78-gene tamoxifen response profile, 21-gene set and HOXB13-IL17BR ratio were all significantly associated with TTP with hazard ratios of 2.2 (95% CI 1.3-3.7, P = 0.005), 2.3 (95% CI 1.3-4.0, P = 0.003) and 4.2 (95% CI 1.4-12.3, P = 0.009), respectively. The concordance among the three classifiers was relatively low, they classified only 45-61% of patients in the same category. In multivariate analyses, the association remained significant for the 78-gene profile and the 21-gene set after adjusting for ER and PgR. CONCLUSION: The 78-gene tamoxifen response profile, the 21-gene set and the HOXB13-IL17BR ratio were all significantly associated with TTP in an independent patient series treated with tamoxifen. The addition of multigene assays to ER (IHC) improves the prediction of outcome in tamoxifen treated patients and deserves incorporation in future clinical studies.

Downregulation of SIAH2, an ubiquitin E3 ligase, is associated with resistance to endocrine therapy in breast cancer.

PURPOSE: In our microarray analysis we observed that Seven-in-Absentia Homolog 2 (SIAH2) levels were low in estrogen receptor (ER) positive breast tumors of patients resistant to first-line tamoxifen therapy. The aim of this study was to evaluate SIAH2 for its (a) predictive/prognostic value, and (b) functional role in endocrine therapy resistance. PATIENTS AND METHODS: SIAH2 expression was measured with quantitative Real-Time-PCR (qRT-PCR) in 1205 primary breast tumor specimens and related to disease outcome. The functional role of SIAH2 was determined in human breast cancer cell lines ZR-75-1, ZR/HERc, and MCF7. Cell lines were treated with estrogen (E2), anti-estrogen ICI164.384 or epidermal growth factor (EGF). Moreover, MCF7 was treated with ICI164.384 after silencing SIAH2 expression. RESULTS: SIAH2 was not prognostic in 603 lymph node negative patients who had not received adjuvant systemic therapy. In multivariate analysis of ER-positive tumors of 235 patients with recurrent disease, SIAH2 as continuous variable, significantly predicted first-line tamoxifen treatment failure (OR = 1.48; P = 0.05) and progression-free survival (PFS) (HR = 0.79; P = 0.007). Furthermore, in primary breast cancer patients treated with adjuvant tamoxifen, SIAH2 predicted metastasis-free survival (MFS) (HR = 0.73; P = 0.005). In vitro experiments showed that SIAH2 silencing in MCF7 cells resulted in resistance to ICI164.384-treatment when compared with mock silenced cells (P = 0.008). Interestingly, in ZR cells transfected with EGFR (ZR/HERc), SIAH2 expression was induced by E2 but downregulated by EGF. CONCLUSION: In primary breast tumor specimens as well as in vitro low SIAH2 levels associated with resistance to endocrine therapy. Moreover, SIAH2 expression showed an opposite regulation by E2 and EGF.

LH receptor gene expression is essentially absent in breast tumor tissue: implications for treatment.

Worldwide, breast cancer is the most frequently occurring malignancy in women. Early age at full term pregnancy has a protective effect against breast cancer. Evidence coming from a rat breast cancer model suggests a possible role for the pregnancy hormone hCG, a ligand of the LH receptor, as a mediator for this effect. In a previous study, we found that a common polymorphism in the LH receptor associates with tumor progression in premenopausal breast cancer patients, as carriers of the variant receptor showed a shorter disease free survival compared to non-carriers. How hCG and its receptor exert their effects on breast cancer, however, is unclear. One possibility is that these effects take place through LH receptors present in the ovaries, thereby influencing steroid hormone production. Another possibility is that the effects take place through LH receptors present in breast tumor cells themselves, as some studies have detected the receptor in both normal and neoplastic breast tissues and in breast cancer cell lines. To investigate whether a direct effect of LH signaling in breast cancer is likely, we measured LH receptor mRNA expression levels in 1551 breast tumors and 42 different human breast cancer cell lines using a qRT-PCR with a wide dynamic range. In addition, associations between LH receptor expression and clinico-pathologic factors were investigated. Assay validation showed that as little as ?10 copies per reaction volume of LH receptor cDNA could still be detected by our assay. We show that LH receptors are undetectable in 62% of breast tumor samples and 41 of 42 breast cancer cell lines. For the remaining samples we found expression levels to be very low. Although low, expression of the LH receptor appears to be associated with normal breast cells, favorable tumor characteristics and low tumor percentage. Since expression of the LH receptor in breast cancer cells is very low, it almost excludes the possibility of direct signaling effects. We therefore conclude that signaling effects of the LH receptor on breast cancer most likely take place by an indirect pathway through the ovaries.

Association of an extracellular matrix gene cluster with breast cancer prognosis and endocrine therapy response.

PURPOSE: We previously discovered an extracellular matrix (ECM) gene cluster associated with resistance to first-line tamoxifen therapy of patients with metastatic breast cancer. In this study, we determined whether the six individual ECM genes [collagen 1A1 (COL1A1), fibronectin 1 (FN1), lysyl oxidase (LOX), secreted protein acidic cysteine-rich (SPARC), tissue inhibitor of metalloproteinase 3 (TIMP3), and tenascin C (TNC)] were associated with treatment response, prognosis, or both. EXPERIMENTAL DESIGN: In 1,286 primary breast tumors, mRNA expression (quantitative real-time PCR) was related to clinicopathologic factors and disease outcome in univariate and multivariate analysis including traditional factors. RESULTS: TIMP3, FN1, LOX, and SPARC expression levels (continuous variables) were significantly associated with distant metastasis-free survival (MFS) in 680 lymph node-negative untreated patients (P<0.03). Using a calculated linear prognostic score, these patients were evenly divided into five prognostic groups with a significant difference in 10-year MFS of approximately 40% between the two extreme prognostic groups. Furthermore, high TNC expression as continuous variable was associated with (a) shorter MFS in 139 estrogen receptor-positive and lymph node-positive patients who received adjuvant tamoxifen therapy (hazard ratio, 1.53; P=0.001), and (b) no clinical benefit (odds ratio, 0.81; P=0.035) and shorter progression-free survival (hazard ratio, 1.19; P=0.002) in 240 patients in whom recurrence was treated with tamoxifen as first-line monotherapy. These results were also significant in multivariate analyses. CONCLUSION: FN1, LOX, SPARC, and TIMP3 expression levels are associated with the prognosis of patients with breast cancers, whereas TNC is associated with resistance to tamoxifen therapy. Further validation and functional studies are necessary to determine the use of these ECM genes in decisions regarding treatment and whether they can serve as targets for therapy.

Patient-derived cell line models revealed therapeutic targets and molecular mechanisms underlying disease progression of high grade serous ovarian cancer.

High grade serous ovarian cancer (HGSOC) is the most frequent type of ovarian cancer. Most patients have primary response to platinum-based chemotherapy but frequently relapse, which leads to patient death. A lack of well documented and characterized patient-derived HGSOC cell lines is so far a major barrier to define tumor specific therapeutic targets and to study the molecular mechanisms underlying disease progression. We established 34 patient-derived HGSOC cell lines and characterized them at cellular and molecular level. Particularly, we demonstrated that a cancer-testis antigen PRAME and Estrogen Receptor could serve as therapeutic targets. Notably, data from the cell lines did not demonstrate acquired resistance due to tumor recurrence that matched with clinical observations. Finally, we presented that all HGSOC had no or very low CDKN1A (p21) expression due to loss of wild-type TP53, suggesting that loss of cell cycle control is the determinant for tumorigenesis and progression. In conclusion, patient-derived cell lines reveal that PRAME is a potential tumor specific therapeutic target in HGSOC and counteracting the down-regulation of p21 caused by loss of wild-type TP53 might be the key to impede disease progression.

The use of genomic tools for the molecular understanding of breast cancer and to guide personalized medicine.

The use of gene-expression microarray analysis to assess the expression levels of all the genes in the genome has tremendous potential. Important information has been obtained about many disease processes, particularly in classifying tumors in different subtypes and risk groups. Combining gene-expression data with other genomic information and the use of sophisticated bioinformatic tools enables the discovery of potential new targets for treatment, and is helpful for high-throughput drug screening and for designing new classes of drugs for targeted therapy. Here, we provide a short overview of the recent, promising developments in the field with emphasis on breast cancer.