Elpida Charalambous

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 detect ≤1% mutant cells, provided that samples yielded cycle thresholds (Ct) <29, but this condition was met in only 38.5% of diagnostic samples. In comparison, FFPE samples (>99%) 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.

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. (Clin Cancer Res 2009;15(23):7153–60)

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.

Background—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.

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.

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.

Abstract 3579: Routinely applicable, highly multiplexed triple-negative breast cancer (TNBC) genotyping

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA In order to address TNBC diversity, determining the individual clonal genotypes of each tumor is urged as prerequisite. However, the applicability of whole genome approaches on routinely processed paraffin tissue material (FFPE) is still limited. Herein, we used targeted massive parallel sequencing (MPS) for genotyping FFPE tumors from patients with operable TNBC treated with adjuvant chemotherapy. Methods: Centrally assessed FFPE TNBC (n=260) were evaluated for tumor cell content (TCC) and submitted for DNA extraction. A custom panel targeting genomic regions in 43 genes previously implicated in TNBC was submitted for primer design (286 amplicons covering 21216bp). Barcoded FFPE libraries were massively processed on Ion Proton™ Sequencer PI and on Ion PGM™ Sequencer 318 chips (30 samples with both platforms, at least 2 runs each). Variants were called and annotated at higher-than-default-stringency conditions (63% of all detected variants excluded). Amplicons were checked for sequence specificity, blood DNA and FFPE performance prior to accepting results (informative: 280/286). Annotated SNVs (coding and non-coding) were assessed for allelic imbalance (AI) based on allelic frequency and TCC (AI: >0.3 unstable SNVs/tumor). The rate of failed samples was significantly higher with PGM (29.5%) than with Proton (3%). Results: In total, 238 informative TNBC were analyzed. AI was noticed in 169 tumors (71%) for multiple genes, more often TP53 (124 tumors, 52.1%), TERT (39.9%), MDM2 (35.7%), MET (24.4%), BRCA1 (20.2%) and NOTCH1 (18.9%). Coding mutations were identified in 40/43 genes but their number varied extensively per tumor (0-87). SNV functional mutations were observed in 55 tumors (23.1%), more often in PIK3CA (12 tumors, 5%). Non-SNV mutations were observed in 153 tumors (64.2%), more often TP53 (128 tumors, 53.8%), CDH1 (17.2%), MAP3K1 (8%), PTEN (6.3%), and PIK3CA (4.6%). High frequency mutations (>50% tumor cells) were found in TP53, PTEN, PIK3CA. Individually, the presence of AI (unstable tumors) and mutations predicted for worse survival. Importantly though, in comparison to patients with unstable mutated tumors, patients with unstable nonmutated tumors had significantly better outcome (DFS, HR:0.41, 95%CI:0.20-0.84, Wald p=0.014; OS, HR:0.36, 95%CI:0.15-0.86, p=0.021), similar to that observed for the favorable stable tumors, where the presence of mutations did not add any effect on survival. The same survival pattern was observed when evaluating AI and high frequency mutations. Conclusions: Targeted MPS with custom panels is an attractive option for highly multiplexed FFPE tissue genotyping, although differences in the efficiency of available platforms should be considered. With respect to potential clinical implications, identifying tumors with allelic (in)stability and clonal mutations seems important for assessing TNBC patient outcome and merits further validation. Citation Format: Vassiliki Kotoula, Kyriaki Papadopoulou, Elpida Charalambous, Flora Zagouri, Sotiris Lakis, Angeliki Lymperopoulou, Eleftheria Tsolaki, George Pentheroudakis, Kostas Lilakos, Dimitrios Pectasides, George Fountzilas. Routinely applicable, highly multiplexed triple-negative breast cancer (TNBC) genotyping. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3579. doi:10.1158/1538-7445.AM2014-3579

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

BackgroundThe 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.MethodsWe 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.ResultsSixty-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).ConclusionsCIMP 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.

Abstract P4-04-10: Clinically relevant tumor mutation profiles in patients with triple negative breast cancer (TNBC)

TNBC account for ∼15% of breast cancers and are most difficult to treat. However, TNBC patient outcome is very heterogeneous. In an effort to characterize the biological characteristics of TNBC, we examined 190 routinely diagnosed tumor tissues from early high-risk breast cancer patients treated with adjuvant chemotherapy (anthracyclines and/or taxanes). Highly multiplexed PCR primer pools were designed for 43 genes previously implicated in TNBC with Ion Ampliseq optimized for FFPE samples. Upon library construction and clonal amplification, amplicons were massively sequenced on Ion Proton PI chips and base-called (Torrent Suite 3.6). Variants were called and annotated (Ion Reporter 1.6), and accepted for analysis upon stringent read quality filtering at p 4 mutations per sample, most of them at low incidence but still indicative of dynamic clonal expansion. CDH1 mutations seldom occurred alone (2% of all tumors) and were probably non-founders, while mutations in some genes, e.g., AKT1 (9/12) and ARID1B (13/19) preferentially occurred in CDH1mutant tumors (p 4 mutations had longer median DFS (91 mo) as compared to those with 1 mutation (n = 68, median DFS 46.5 mo) or with 4 mutations (n = 21, median DFS 39 mo) (HR: 0.4; 95%CI: 0.2-0.8; Wald9s p = 0.010). In comparison to patients with TP53C73(24 Suppl): Abstract nr P4-04-10.

Abstract 1918: Genomic CNV testing on chromosome 17q genes reveals clinically relevant subtypes in breast cancer.

Fluorescent In Situ Hybridization (FISH) and genomic Copy Number Variation (CNV) testing may not yield identical CN status for the same gene, due to methodological differences in calling gene copies. The aim of the present study was to evaluate the clinical relevance of genomic CNV profiles on 17q (17q11.2-q12 to 17q21-q22) in the context of high-risk operable breast cancer. Methods: The CN status of NOS2, HER2, THRA, RARA and TOP2A was tested with 5’- and 3’-end-specific, exon-intron spanning qPCR CNV assays (two assays per gene). Software obtained CN values against normal DNA were evaluated from 439 informative paraffin breast cancer tissue samples with >50% tumor cells and were assessed (a) upon logarithmic transformation as continuous variables, and (b) as nominal variables with following CN categories: loss (>0-0.5 copies); no-gain or marginal accounting for DNA replication (1-4); low-gain (5-6) and high-gain (>6). For each gene, 5’- and 3’-ends were evaluated separately for gene stability and in combination for obtaining the entire gene CN status. All patients had received adjuvant anthracyclines in the frame of two phase III trials. Results: Concordance between 5’- and 3’-end CN status was observed in 94.53% of tumors for TOP2A; 91.12% for THRA; 86.79% for HER2; 69.70% for RARA; and, 53.76% for NOS2. Strong associations were observed for classic HER2 FISH status with NOS2 (p=.0007), HER2 and THRA (both p Conclusions: Breast tumors with CN losses or complex losses/gains for the genes examined on 17q seem to carry the worst prognosis among those treated in the adjuvant setting with anthracyclines and may be worthy indentifying for different treatment selection. TOP2A FISH and CNV status are not concordant, while NOS2 and RARA seem to suffer internal CNV, which needs further validation for its biologic implications. Citation Format: Vassiliki Kotoula, Mattheos Bobos, George Kouvatseas, Christos Papadimitriou, Kyriaki Papadopoulou, Elpida Charalambous, Eleftheria Tsolaki, George Fountzilas. Genomic CNV testing on chromosome 17q genes reveals clinically relevant subtypes in breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1918. doi:10.1158/1538-7445.AM2013-1918