The CpG island methylator phenotype is concordant between primary colorectal carcinoma and matched distant metastases.

BACKGROUND: The CpG island methylator phenotype (CIMP) in stage III colon cancer (CRC) has been associated with improved survival after treatment with adjuvant irinotecan-based chemotherapy. In this analysis, we determine whether CIMP status in the primary CRC is concordant with the CIMP status of matched metastases in order to determine if assessment of CIMP status in the primary tumor can be used to predict CIMP status of metastatic disease, which is relevant for patient management as well as for understanding the biology of CIMP CRCs. METHODS: We assessed the CIMP status of 70 pairs of primary CRC and matched metastases using a CRC-specific panel of five markers (CACNA1G, IGF2, NEUROG1, RUNX3, and SOCS1) where CIMP positive was defined as 3/5 positive markers at a percent methylated reference threshold of ≥10%. Concordance was compared using the Fisher's exact test and P < 0.05 was considered significant. RESULTS: Sixty-nine of the pairs (98.6%) showed concordant CIMP status in the primary tumor and matched metastasis; five (7.0%) of the pairs were concordantly CIMP positive. Only one pair (1.4%) had divergent CIMP status, demonstrating CIMP positivity (4/5 markers positive) in the primary tumor, while the matched metastasis was CIMP negative (0 markers positive). CONCLUSIONS: CIMP status is generally concordant between primary CRCs and matched metastases. Thus, CIMP status in the primary tumor is maintained in matched metastases and can be used to inform CIMP-based therapy options for the metastases.

Expression Patterns of Growth and Survival Genes with Prognostic Implications in Advanced Pancreatic Cancer.

AIM: The aim of this study was to evaluate the mRNA expression pattern of growth- and survival-related genes and assess their prognostic significance in patients with advanced pancreatic cancer. PATIENTS AND METHODS: In total, 98 patients were included in this retrospective translational research study and were evaluated for Kirsten rat sarcoma viral oncogene homolog (KRAS) mutational status, and v-akt murine thymoma viral oncogene homolog 1 (AKT1), AKT serine/threonine kinase 2 (AKT2), AKT serine/threonine kinase 3 (AKT3), cyclin D1 (CCND1), epidermal growth factor receptor (EGFR), mitogen-activated protein kinase 1 (MAPK1), hepatocellular growth factor receptor (MET), avian myelomatosis viral oncogene homolog (MYC), nuclear factor kappa B subunit 1 (NFKb1), phosphatase and tensin homolog (PTEN) and mechanistic target of rapamycin (FRAP1) genes mRNA expression. Among these patients, 73 received first-line gemcitabine combined with erlotinib (N=57) or gefitinib (N=16). RESULTS: KRAS mutation did not correlate with mRNA gene expression. Unsupervised hierarchical clustering according to mRNA gene expression successfully distinguished four prognostically distinct groups of tumors. Overexpression of all genes was associated with best prognosis, while suppression or heterogeneous expression patterns of the examined genes were associated with expression patterns of growth- and survival-related genes, classifying pancreatic tumors into distinct groups with possibly different outcomes.

MYC copy gain, chromosomal instability and PI3K activation as potential markers of unfavourable outcome in trastuzumab-treated patients with metastatic breast cancer.

BACKGROUND: There is an unmet need for more efficient patient stratification for receiving trastuzumab in the metastatic breast cancer (mBC) setting, since only part of such patients benefit from the addition of this agent to chemotherapy. The aim of this study was to investigate the prognostic value of biomarkers including MYC and MET in mBC patients treated with trastuzumab-based regimens. METHODS: mBC patients, locally tested as HER2-positive, treated with trastuzumab and chemotherapy between 1998 and 2010 were evaluated. Paraffin tumors (n = 229) were retrospectively centrally assessed by immunohistochemistry (IHC) for HER2, ER, PgR and Ki67; fluorescence in situ hybridization (FISH) for HER2, TOP2A and centromere (CEN) 17, MYC and CEN8, MET and CEN7; qPCR for MYC, MET copy number (CN); and, for PI3K activation (PIK3CA mutations; PTEN and phospho-mTOR protein expression). Increased CEN CN was assessed based on normal cut-offs. Time to progression (TTP) and survival were evaluated from the initiation of trastuzumab as first line treatment. RESULTS: Among all tumors, 90 were HER2-negative upon central testing (ambiguous HER2) and the rest were true HER2-positive. Further, 156 patients presented with mBC upon relapse of pre-treated disease (R-mBC) and 65 were diagnosed at stage IV (de novo mBC). Concordance between FISH and qPCR on gene CN status was fair for MYC (Kappa = 0.458) and absent for MET. The presence of MYC CN gain with qPCR and the absence of PI3K activation were infrequent events (7 and 8 % of evaluable tumors, respectively), while 41 % of tumors had increased CEN CN in one or more chromosomes, indicative of chromosomal instability. The most consistent finding in the entire cohort and in the above patient subgroups with respect to outcome was the unfavourable effect of MYC CN gain, which was retained upon multivariable analysis (e.g., survival in the entire cohort, HR 6.02; 95 % CI 2.67-13.6; p < 0.001). Further unfavourable prognosticators were increased CEN CN in one chromosome in R-mBC but not in de novo mBC (multivariable interaction p = 0.048), PI3K activation in R-mBC (multivariable p = 0.004) and increased Ki67 for patient TTP. CONCLUSIONS: MYC gene copies, centromere status and PI3K activation may adversely impact trastuzumab treated mBC patient outcome and seem worthy validating in larger series.

Significance of PIK3CA Mutations in Patients with Early Breast Cancer Treated with Adjuvant Chemotherapy: A Hellenic Cooperative Oncology Group (HeCOG) Study.

BACKGROUND: The PI3K-AKT pathway is frequently activated in breast cancer. PIK3CA mutations are most frequently found in the helical (exon 9) and kinase (exon 20) domains of this protein. The aim of the present study was to examine the role of different types of PIK3CA mutations in combination with molecular biomarkers related to PI3K-AKT signaling in patients with early breast cancer. METHODS: Tumor tissue samples from 1008 early breast cancer patients treated with adjuvant chemotherapy in two similar randomized trials of HeCOG were examined. Tumors were subtyped with immunohistochemistry (IHC) and FISH for ER, PgR, Ki67, HER2 and androgen receptor (AR). PIK3CA mutations were analyzed by Sanger sequencing (exon 20) and qPCR (exon 9) (Sanger/qPCR mutations). In 610 cases, next generation sequencing (NGS) PIK3CA mutation data were also available. PIK3CA mutations and PTEN protein expression (IHC) were analyzed in luminal tumors (ER and/or PgR positive), molecular apocrine carcinomas (MAC; ER/PgR negative / AR positive) and hormone receptor (ER/PgR/AR) negative tumors. RESULTS: PIK3CA mutations were detected in 235/1008 tumors (23%) with Sanger/qPCR and in 149/610 tumors (24%) with NGS. Concordance between the two methods was good with a Kappa coefficient of 0.76 (95% CI 0.69-0.82). Lobular histology, low tumor grade and luminal A tumors were associated with helical domain mutations (PIK3CAhel), while luminal B with kinase domain mutations (PIK3CAkin). The overall incidence of PIK3CA mutations was higher in luminal as compared to MAC and hormone receptor negative tumors (p = 0.004). Disease-free and overall survival did not significantly differ with respect to PIK3CA mutation presence and type. However, a statistically significant interaction between PIK3CA mutation status and PTEN low protein expression with regard to prognosis was identified. CONCLUSIONS: The present study did not show any prognostic significance of specific PIK3CA mutations in a large group of predominantly lymph-node positive breast cancer women treated with adjuvant chemotherapy. Further analyses in larger cohorts are warranted to investigate possible differential effect of distinct PIK3CA mutations in small subgroups of patients.

Evaluation of two highly-multiplexed custom panels for massively parallel semiconductor sequencing on paraffin DNA.

AIM: Massively parallel sequencing (MPS) holds promise for expanding cancer translational research and diagnostics. As yet, it has been applied on paraffin DNA (FFPE) with commercially available highly multiplexed gene panels (100s of DNA targets), while custom panels of low multiplexing are used for re-sequencing. Here, we evaluated the performance of two highly multiplexed custom panels on FFPE DNA. METHODS: Two custom multiplex amplification panels (B, 373 amplicons; T, 286 amplicons) were coupled with semiconductor sequencing on DNA samples from FFPE breast tumors and matched peripheral blood samples (n samples: 316; n libraries: 332). The two panels shared 37% DNA targets (common or shifted amplicons). Panel performance was evaluated in paired sample groups and quartets of libraries, where possible. RESULTS: Amplicon read ratios yielded similar patterns per gene with the same panel in FFPE and blood samples; however, performance of common amplicons differed between panels (p<0.001). FFPE genotypes were compared for 1267 coding and non-coding variant replicates, 999 out of which (78.8%) were concordant in different paired sample combinations. Variant frequency was highly reproducible (Spearman's rho 0.959). Repeatedly discordant variants were of high coverage / low frequency (p<0.001). Genotype concordance was (a) high, for intra-run duplicates with the same panel (mean±SD: 97.2±4.7, 95%CI: 94.8-99.7, p<0.001); (b) modest, when the same DNA was analyzed with different panels (mean±SD: 81.1±20.3, 95%CI: 66.1-95.1, p = 0.004); and (c) low, when different DNA samples from the same tumor were compared with the same panel (mean±SD: 59.9±24.0; 95%CI: 43.3-76.5; p = 0.282). Low coverage / low frequency variants were validated with Sanger sequencing even in samples with unfavourable DNA quality. CONCLUSIONS: Custom MPS may yield novel information on genomic alterations, provided that data evaluation is adjusted to tumor tissue FFPE DNA. To this scope, eligibility of all amplicons along with variant coverage and frequency need to be assessed.

Adjusting breast cancer patient prognosis with non-HER2-gene patterns on chromosome 17.

BACKGROUND: HER2 and TOP2A gene status are assessed for diagnostic and research purposes in breast cancer with fluorescence in situ hybridization (FISH). However, FISH probes do not target only the annotated gene, while chromosome 17 (chr17) is among the most unstable chromosomes in breast cancer. Here we asked whether the status of specifically targeted genes on chr17 might help in refining prognosis of early high-risk breast cancer patients. METHODS: Copy numbers (CN) for 14 genes on chr17, 4 of which were within and 10 outside the core HER2 amplicon (HER2- and non-HER2-genes, respectively) were assessed with qPCR in 485 paraffin-embedded tumor tissue samples from breast cancer patients treated with adjuvant chemotherapy in the frame of two randomized phase III trials. PRINCIPAL FINDINGS: HER2-genes CN strongly correlated to each other (Spearman's rho >0.6) and were concordant with FISH HER2 status (Kappa 0.6697 for ERBB2 CN). TOP2A CN were not concordant with TOP2A FISH status (Kappa 0.1154). CN hierarchical clustering revealed distinct patterns of gains, losses and complex alterations in HER2- and non-HER2-genes associated with IHC4 breast cancer subtypes. Upon multivariate analysis, non-HER2-gene gains independently predicted for shorter disease-free survival (DFS) and overall survival (OS) in patients with triple-negative cancer, as compared to luminal and HER2-positive tumors (interaction p = 0.007 for DFS and p = 0.011 for OS). Similarly, non-HER2-gene gains were associated with worse prognosis in patients who had undergone breast-conserving surgery as compared to modified radical mastectomy (p = 0.004 for both DFS and OS). Non-HER2-gene losses were unfavorable prognosticators in patients with 1-3 metastatic nodes, as compared to those with 4 or more nodes (p = 0.017 for DFS and p = 0.001 for OS). CONCLUSIONS: TOP2A FISH and qPCR may not identify the same pathology on chr17q. Non-HER2 chr17 CN patterns may further predict outcome in breast cancer patients with known favorable and unfavorable prognosis.

Association of VEGF-A splice variant mRNA expression with outcome in bevacizumab-treated patients with metastatic breast cancer.

BACKGROUND: The prognostic utility of vascular endothelial growth factor A (VEGF-A) splice variants in patients with advanced breast cancer treated with bevacizumab has not been studied. PATIENTS AND METHODS: A total of 111 patients with metastatic breast cancer treated with weekly docetaxel or ixabepilone without bevacizumab (cohort A) and 100 treated with weekly paclitaxel and bevacizumab (cohort B) were studied. Formalin-fixed tumors were macrodissected for reverse transcription quantitative polymerase chain reaction relative quantification of VEGF-A165, -189, and -206 isoforms spliced at exon 8 proximal splice site (VEGF-Axxxa) and at exon 8 distal splice site (VEGF-Axxxb). RESULTS: For high VEGF-Axxxa, the hazard ratios (HRs) for progression were 1.08 (P = .71) in non-bevacizumab-treated patients (cohort A) and 0.66 (P = .22) in bevacizumab-treated patients (cohort B), and the HRs for death were 1.45 (P = .13) and 0.50 (P = .049), respectively. The interaction of VEGF-Axxxa with bevacizumab administration was significant (P = .011) for overall survival (OS). High tissue VEGF-Axxxb was not prognostic in cohort A but was predictive for bevacizumab benefit in cohort B (HR for progression, 0.57 [P = .04]; HR for death, 0.51 [P = .02]). Exploratory analyses done only in cohort B suggested that abundance of VEGFR1 messenger RNA (mRNA) in peripheral blood and low VEGFR2 mRNA in tissue correlated with poor outcome. In multivariate analysis, high tissue mRNA of angiogenic VEGF-Axxxa in the presence of bevacizumab therapy predicted for favorable progression-free survival (HR for progression, 0.39; P = .0227) and OS (HR for death, 0.32; P = .0140). CONCLUSION: Tissue mRNA expression of angiogenic VEGF-Axxxa isoforms was retrospectively associated with adverse prognosis in the absence of bevacizumab and with favorable outcome when bevacizumab was administered in patients with advanced breast cancer.

A study of gene expression markers for predictive significance for bevacizumab benefit in patients with metastatic colon cancer: a translational research study of the Hellenic Cooperative Oncology Group (HeCOG).

BACKGROUND: Bevacizumab, an antibody neutralizing Vascular Endothelial Growth Factor (VEGF), is licensed for the management of patients with advanced colon cancer. However, tumor biomarkers identifying the molecular tumor subsets most amenable to angiogenesis modulation are lacking. METHODS: We profiled expession of 24526 genes by means of whole genome 24 K DASL (c-DNA-mediated, Annealing, Selection and Ligation) arrays, (Illumina, CA) in 16 bevacizumab-treated patients with advanced colon cancer (Test set). Genes with correlation to 8-month Progression-free status were studied by means of qPCR in two independent colon cancer cohorts: 49 patients treated with bevacizumab + chemotherapy (Bevacizumab qPCR set) and 72 patients treated with chemotherapy only (Control qPCR set). Endpoints were best tumor response before metastasectomy (ORR) and progression-free survival (PFS). RESULTS: Five genes were significantly correlated to 8-month progression-free status in the Test set: overexpression of KLF12 and downregulation of AGR2, ALDH6A1, MCM5, TFF2. In the two independent datasets, irinotecan- or oxaliplatin-based chemotherapy was administered as first-line treatment and metastasectomies were subsequently applied in 8-14% of patients. No prognostically significant gene classifier encompassing all five genes could be validated in the Bevacizumab or Control qPCR sets. The complex gene expression profile of all-low tumor (ALDH6A1 + TFF2 + MCM5) was strongly associated with ORR in the Bevacizumab qPCR set (ORR 85.7%, p = 0.007), but not in the Control set (ORR 36.4%, p = 0.747). The Odds Ratio for response for the all-low tumor (ALDH6A1 + TFF2 + MCM5) profile versus any other ALDH6A1 + TFF2 + MCM5 profile was 15 (p = 0.018) in the Bevacizumab qPCR set but only 0.72 (p = 0.63) in the Control set. The tumor expression profile of (KLF12-high + TFF2-low) was significantly associated with PFS only in the Bevacizumab qPCR set: bevacizumab-treated patients with (KLF12-high + TFF2-low) tumors had superior PFS (median 14 months, 95% CI 2-21) compared to patients with any other (KLF12 + TFF2) expression profile (median PFS 7 months, 95% CI 5-10, p = 0.021). The Hazard Ratio for disease progression for (KLF12-high + TFF2-low) versus any other KLF12 + TFF2 expression profile was 2.92 (p = 0.03) in the Validation and 1.29 (p = 0.39) in the Control set. CONCLUSIONS: Our «three-stage¼ hypothesis-generating study failed to validate the prognostic significance of a five-gene classifier in mCRC patients. Exploratory analyses suggest two gene signatures that are potentially associated with bevazicumab benefit in patients with advanced colon cancer.

The prognostic significance of WNT pathway in surgically-treated colorectal cancer: ß-catenin expression predicts for disease-free survival.

BACKGROUND: The wingless-type MMTV integration site family of proteins (WNT) pathway is highly involved in colorectal cancer development. The aim of this study was to explore the prognostic significance and clinicopatological correlations of this pathway in a cohort of surgically-treated patients with non-metastatic colorectal cancer in relation to the site of expression of pathway proteins. MATERIALS AND METHODS: Immunohistochemical expression of nuclear cyclin D1, membranous E-cadherin and P-cadherin, membranous and nuclear ß-catenin in the invasive front (IF), the tumor center (TC), as well as their mean, were assessed in 106 paraffin-embedded tissue samples. Adenomatous Polyposis Coli (APC), Axin-2 (AXIN2), cyclin-D1 (CCND1), Matrix Metalloproteinase-7 (MMP7), Secreted Frizzled Related Protein (SFRP) 1, 2 and 4 and WNT5A were evaluated by RT PCR. RESULTS: Membranous ß-catenin expression was statistically reduced in the IF. Cyclin-D1 was reduced in tumors arising closer to the rectum. Reduced nuclear expression of cyclin-D1 in the IF was associated with lymphatic, venous and perineural invasion. Loss of membranous ß-catenin in the TC was more common among N2 tumors. Higher SFRP4 mRNA was associated with advanced T stage. In univariate analysis, membranous expression of ß-catenin in TC and IF, and their mean, was associated with longer disease-free survival (DFS). In multivariate analysis, tumor stage and mean ß-catenin expression were prognostic for longer DFS (hazard ratio=0.33; p=0.01). ß-Catenin expression in the IF remained significant when the mean expression was not included in the multivariate analysis (hazard ratio=0.41; p=0.028). CONCLUSION: Mean membranous expression of ß-catenin, as well as that in the IF, is prognostic for longer DFS in patients with non metastatic colorectal cancer.

Interactions of the Hdm2/p53 and proteasome pathways may enhance the antitumor activity of bortezomib.

PURPOSE: p53 is inactivated in many human malignancies through missense mutations or overexpression of the human homologue of Mdm2 (Hdm2), an E3 ubiquitin ligase that ubiquitinates p53, thereby promoting its proteasomal degradation. The cis-imidazoline nutlin-3 can disrupt the p53-Hdm2 interaction and activate p53, inducing apoptosis in vitro in many malignancies, including multiple myeloma (MM). EXPERIMENTAL DESIGN: We hypothesized that suppression of Hdm2-mediated p53 ubiquitination may augment sequelae of p53 accumulation caused by proteasomal inhibition. We compared the response of MM cells versus several epithelial cancer models to the proteasome inhibitor bortezomib in combination with nutlin-3. RESULTS: The combination of sublethal concentrations of bortezomib plus nutlin-3 induced additive cytotoxicity against bortezomib-sensitive MM cell lines. Importantly, however, in breast, prostate, colon, and thyroid (papillary, follicular, anaplastic, and medullary) carcinoma cell lines, this combination triggered synergistic cytotoxicity, and increased expression of p53, p21, Hdm2, Bax, Noxa, PUMA, and cleavage of caspase-3 and poly ADP ribose polymerase. Coculture with bone marrow stromal cells attenuated MM cell sensitivity to nutlin-3 monotherapy and was associated with evidence of suppression of p53 activity in MM cells, whereas combined bortezomib-nutlin-3 treatment maintained cytotoxicity even in the presence of bone marrow stromal cells. CONCLUSIONS: This differential response of MM versus epithelial carcinomas to combination of nutlin-3 with bortezomib sheds new light on the role of p53 in bortezomib-induced apoptosis. Concurrent Hdm2 inhibition with bortezomib may extend the spectrum of bortezomib applications to malignancies with currently limited sensitivity to single-agent bortezomib or, in the future, to MM patients with decreased clinical responsiveness to bortezomib-based therapy.

Targeted KRAS mutation assessment on patient tumor histologic material in real time diagnostics.

BACKGROUND: Testing for tumor specific mutations on routine formalin-fixed paraffin-embedded (FFPE) tissues may predict response to treatment in Medical Oncology and has already entered diagnostics, with KRAS mutation assessment as a paradigm. The highly sensitive real time PCR (Q-PCR) methods developed for this purpose are usually standardized under optimal template conditions. In routine diagnostics, however, suboptimal templates pose the challenge. Herein, we addressed the applicability of sequencing and two Q-PCR methods on prospectively assessed diagnostic cases for KRAS mutations. METHODOLOGY/PRINCIPAL FINDINGS: Tumor FFPE-DNA from 135 diagnostic and 75 low-quality control samples was obtained upon macrodissection, tested for fragmentation and assessed for KRAS mutations with dideoxy-sequencing and with two Q-PCR methods (Taqman-minor-groove-binder [TMGB] probes and DxS-KRAS-IVD). Samples with relatively well preserved DNA could be accurately analyzed with sequencing, while Q-PCR methods yielded informative results even in cases with very fragmented DNA (p<0.0001) with 100% sensitivity and specificity vs each other. However, Q-PCR efficiency (Ct values) also depended on DNA-fragmentation (p<0.0001). Q-PCR methods were sensitive to detect99%) could accurately be analyzed at a sensitivity level of 10% (external validation of TMGB results). DNA quality and tumor cell content were the main reasons for discrepant sequencing/Q-PCR results (1.5%). CONCLUSIONS/SIGNIFICANCE: Diagnostic targeted mutation assessment on FFPE-DNA is very efficient with Q-PCR methods in comparison to dideoxy-sequencing. However, DNA fragmentation/amplification capacity and tumor DNA content must be considered for the interpretation of Q-PCR results in order to provide accurate information for clinical decision making.

A splice variant of HER2 corresponding to Herstatin is expressed in the noncancerous breast and in breast carcinomas.

Herstatin (HST) is an alternatively spliced HER2 product with growth-inhibitory properties in experimental cancer systems. The role of HST in adult human tissues and disease remains unexplored. Here, we investigated HST expression at the mRNA and protein (immunohistochemistry [IHC]) level in parallel with parameters reflecting HER activation in 187 breast carcinomas and matched noncancerous breast tissues (NCBT). Noncancerous breast tissues demonstrated the highest HST/HER2 transcript ratios corresponding to a few positive epithelial and stromal cells by IHC. Although HST/HER2 transcript ratios in tumors were inversely associated with HER2 IHC grading (P = .0048 for HER2 IHC-1+ and P = .0006 for HER2 IHC-2+ vs HER2-negative tumors), relative HST expression within the same tumor/NCBT system remained constant. HST/HER2 ratios did not predict the presence of HST protein, which was found in 46 (25%) of 187 tumors. A subgroup of HER2 IHC-3+ tumors exhibited high HST/HER2 transcript ratios, strong HST protein positivity, and cytoplasmic phospho-Akt/PKB and p21(CIP1/WAF1) localization. In conclusion, HST may act as a paracrine factor in the adult breast. Because HST is described as an endogenous pan-HER inhibitor, the presence of this protein in breast carcinomas may portent the inefficiency of exogenous efforts to block HER2 dimerization, whereas its absence may justify such interventions.

Epidermal growth factor receptor as a therapeutic target in human thyroid carcinoma: mutational and functional analysis.

CONTEXT: The epidermal growth factor receptor (EGFR), a transmembrane tyrosine kinase (TK) receptor that mediates proliferation and survival signaling, is expressed in a wide variety of normal and neoplastic tissues. EGFR inhibitors have produced objective responses in patients with non-small-cell lung carcinomas harboring activating EGFR TK domain somatic mutations. OBJECTIVE AND METHODS: Because the EGFR pathway has been reported to be important for the pathophysiology of thyroid carcinoma, we investigated the expression and mutational status of EGFR in 14 thyroid carcinoma cell lines as well as its functional role by evaluating their in vitro sensitivity to AEE788, a new dual-family EGFR/ErbB2 and vascular endothelial growth factor receptor TK inhibitor. We also evaluated the mutational status, mRNA and protein expression, as well as phosphorylation status of EGFR in a panel of thyroid carcinoma specimens. RESULTS: EGFR expression and phosphorylation in the thyroid carcinoma cell lines and tissue specimens were present but not stronger than in noncancerous thyroid tissue. EGFR TK domain mutations were detected in two of 62 histological specimens (3.2%) but not in cell lines. All thyroid carcinoma cell lines were significantly less sensitive (IC(50) at least 25-fold higher) in vitro to AEE788 than a primary culture of EGFR-mutant lung carcinoma cells. CONCLUSIONS: Thyroid carcinoma cells overall are poorly responsive to clinically relevant concentrations of AEE788 in vitro. The presence of EGFR-activating TK domain mutations may identify a small minority of thyroid cancer patients that may benefit from EGFR inhibitors, but additional preclinical evidence of efficacy is needed.

Hereditary breast and ovarian cancer in Cyprus: identification of a founder BRCA2 mutation.

The entire coding regions of the two breast cancer susceptibility genes BRCA1 and BRCA2 from breast cancer patients from 40 Cypriot families with multiple cases of breast and ovarian cancer were sequenced. A total of four protein-truncating mutations were found in six families. In BRCA1, a novel truncating mutation 5429delG was found in exon 21. In BRCA2, three truncating mutations were detected: a frameshift 8984delG in exon 22 and two nonsense mutations C1913X in exon 11 and K3326X in exon 27. It is noted that mutation 8984delG was found in three separate families, and haplotype analysis showed that this may be a founder mutation in the Cypriot population. In addition, a pair of rare variants, Q356R and S1512I, was detected in BRCA1 in patients belonging to two Cypriot families. The simultaneous presence of this pair of missense mutations may be associated with the breast cancer phenotype in the Cypriot population. We conclude that the BRCA2 gene appears to play a more important role in familial breast cancer in the Cypriot population than BRCA1.

BRCA2 germline mutations in Cypriot patients with familial breast/ovarian cancer.

Germline mutations in the BRCA2 gene have been shown to be associated with familial female and male breast cancer. Mutations occur throughout the entire coding region of the gene, and there is considerable ethnic and geographical diversity in the deleterious mutations detected in different populations. No data exist on the role of the BRCA2 gene in the Cypriot population. In this study we present the results of characterizing mutations in the BRCA2 gene, in 26 Cypriot families with multiple cases of breast/ovarian cancer. The entire coding region, including splice sites, of BRCA2 were sequenced using cycle sequencing. In total 29 BRCA2 variants were detected which include 3 truncating mutations, 8 missense mutations, 6 polymorphisms and 12 intronic variants. The 3 truncating mutations are frameshift mutation 8984delG (exon 22), and two nonsense mutations, namely C1913X (exon 11) which is a novel mutation, and K3326X (exon 27). It is of interest that frameshift mutation 8984delG was the most frequent, since it was detected in 5 patients from three different families. Among the 6 polymorphisms detected, polymorphism T77T is novel and similarly 4 of the 12 intronic variants were also novel, namely IVS1+8G>A, IVS1-96insA, IVS4+36A>G and IVS11-51G>T. These results show that deleterious BRCA2 mutations, occur at the same frequency, about 20%, in Cypriot families, as that recorded in other European populations. We conclude that the BRCA2 gene plays a significant role in the familial breast cancer phenotype in the Cypriot population.