Mutations In Tumor Tissue Research Articles

Evaluation of clinical outcomes by analysis of mutations in tumor tissue and circulating plasma DNA using next-generation sequencing (NGS) from STEAM, a prospective, randomized, multicenter study in metastatic colorectal cancer (mCRC).

11510 Background: STEAM (NCT01765582) assessed the efficacy and safety of concurrent (c) and sequential (s) FOLFOXIRI-bevacizumab (BEV) vs FOLFOX-BEV for first-line treatment of mCRC. Methods: The AVENIO ctDNA Expanded Kit (Research Use Only) was used to assess somatic mutations in 77 cancer-related genes by NGS in tissue, and both pre- and post-induction plasma samples (n = 182, 150 and 118 respectively) from STEAM. Four mutation classes including single-nucleotide variants (SNVs), indels, copy number amplifications (CNAs) and fusions were identified. SNVs and indels were called in tissue and plasma at allele frequencies of 5% and 0.25% respectively. Results: Overall concordance of mutations in pre-induction plasma with tissue was 83%. Concordance for the seven most mutated genes ranged from 91.5%-100%. In pts with matched samples (n = 118), RAS WT pts showed significantly longer progression-free survival (PFS) in both cFOLFOXIRI-BEV (A) and sFOLFOXIRI-BEV (B) arms versus FOLFOX-BEV (C), using genotyping of either tissue or plasma. This was not seen in RAS MUT pts. In contrast, TP53 WT showed no significant treatment differences while TP53 MUT showed longer PFS for cFOLFOXIRI-BEV versus FOLFOX-BEV. A list of mutation frequencies for all samples, as well as hierarchical clustering analysis of tissue mutations will be presented. Conclusions: The AVENIO ctDNA Expanded Kit identified mutations in 77 cancer-related genes, in both plasma and tissue, with high overall concordance. Compared to FOLFOX-BEV, longer PFS was observed for c- or s- FOLFOXIRI-BEV in RAS WT pts and for cFOLFOXIRI-BEV in TP53 MUT pts, irrespective of sample type. These results are hypothesis generating and require further clinical validation. Clinical trial information: NCT01765582. [Table: see text]

EGFR Mutation Analysis for Prospective Patient Selection in Two Phase II Registration Studies of Osimertinib

IntroductionOsimertinib is an oral, central nervous system–active, EGFR tyrosine kinase inhibitor (TKI) for the treatment of EGFR T790M–positive advanced NSCLC. Here we have evaluated EGFR mutation frequencies in two phase II studies of osimertinib (AURA extension and AURA2). MethodsAfter progression while receiving their latest line of therapy, patients with EGFR mutation–positive advanced NSCLC provided tumor samples for mandatory central T790M testing for the study selection criteria. Tumor tissue mutation analysis for patient selection was performed with the Roche cobas EGFR Mutation Test (European Conformity–in vitro diagnostic, labeled investigational use only) (Roche Molecular Systems, Pleasanton, CA). Patients should not have been prescreened for T790M mutation status. The cobas test results were compared with those of the MiSeq next-generation sequencing system (Illumina, San Diego, CA), which was used as a reference method. ResultsSamples from 324 and 373 patients screened for AURA extension and AURA2, respectively, produced valid cobas test results. The T790M detection rates were similar between AURA extension and AURA2 (64% and 63%, respectively). The pooled T790M rate was 63%, with no difference by ethnicity (63% for Asian and non-Asian patients alike) or immediately prior treatment with an EGFR TKI (afatinib, 69%; erlotinib, 69%; and gefitinib, 63%). A higher proportion of patients had T790M detected against a background of exon 19 deletions versus L858R mutation (73% versus 58% [p = 0.0002]). In both trials the cobas test demonstrated high sensitivity (positive percent agreement) and specificity (negative percent agreement) for T790M detection when compared with the next-generation sequencing reference method: positive percent agreement of 91% versus 89% and negative percent agreement of 97% versus 98%. ConclusionsIn both trials, the rate of detection of T790M mutation in patients with advanced NSCLC was approximately 63% and was unaffected by immediately prior treatment with an EGFR TKI or ethnicity.

Complete genome sequencing and clinical analysis of intrahepatic hepatitis B virus cccDNA from HCC

More than half of hepatocellular carcinomas (HCCs) are etiologically attributed to hepatitis B virus (HBV) infection, but it remains unclear whether HBV mutations are virological factors that contribute to formation of HCC or instead reflect accumulation during the progression of HBV-related disease. Rolling-cycle amplification and PCR sequencing were used to characterize covalently closed circular DNA (cccDNA) mutations in tumor tissues. Paired non-tumor tissues were used as controls. High frequencies of C1653T, T1753V, and A1762T/G1764A cccDNA mutations were observed in both tumor and non-tumor tissues. T1719G, C1329A, and T3098C mutations were related to the overall survival of HCC patients. Patients with G1719 tended to be in the high Barcelona Clinic Liver Cancer stage and had lower levels of total DNA and cccDNA per cell than patients with T1719. Additionally, invitro analysis revealed that T1719G mutation reduced viral replication efficacy. Finally, significantly higher levels of preoperative alpha-fetoprotein were observed in patients harboring the G1078T, C1653T, G1727A, C1913A, T1978C, or C3116T mutations at the cccDNA level. We speculated that HBV cccDNA mutations accumulated over the course of HBV-related disease development, and that some key mutations had prognostic value for patients with HBV-related HCC.

Comparison of the PI3KCA pathway in circulating tumor cells and corresponding tumor tissue of patients with metastatic breast cancer.

The aim of the present study was to compare the phosphatidylinositol 3-kinase (PI3KCA)-AKT serine/threonine kinase (AKT) pathway in circulating tumor cells (CTCs) and corresponding cancerous tissues. Stemness-like circulating tumor cells (slCTCs) and CTCs in epithelial-mesenchymal transition (EMT) have been implicated as the active source of metastatic spread in breast cancer (BC). In this regard, the PI3KCA-AKT signaling pathway was demonstrated to be implicated in and to be frequently mutated in BC. The present study compared this pathway in slCTCs/CTCs in EMT and the corresponding tumor tissues of 90 metastatic BC patients (pts). slCTCs and CTCs in EMT were isolated using the AdnaTest EMT-1/StemCell for the detection of aldehyde dehydrogenase 1 family member A1 (ALDH1) (singleplex PCR) and PI3KCA, AKT2 and twist family bHLH transcription factor 1 (multiplex PCR). Tumor tissue was investigated for PI3KCA hotspot mutations using Sanger sequencing of genomic DNA from micro-dissected formalin-fixed paraffin-embedded tissue, and for the expression of ALDH1 and phosphorylated AKT (pAKT), and phosphatase and tensin homolog (PTEN) loss, by immunohistochemistry. slCTCs were identified in 23% of pts (21/90 pts) and CTCs in EMT in 56% (50/90 pts) of pts. pAKT and ALDH1 positivity in tumor tissue was identified in 47 and 9% of cases, respectively, and a PTEN loss was observed in 18% of pts. A significant association was detected between pAKT expression in cancerous tissue and AKT2 expression in CTCs (P=0.037). PI3KCA mutations were detected in 32% of pts, most frequently on exons 21 (55%) and 10 (45%). Pts with PI3KCA mutations in tumor tissue had a significantly longer overall survival than pts with wild-type PI3KCA expression (P=0.007). Similar results were obtained for pts with aberrant PI3KCA signaling in CTCs and/or aberrant signaling in cancerous tissue (P=0.009). Therapy-resistant CTCs, potentially derived from the primary tumor or metastatic tissue, may be eliminated with specific PI3K pathway inhibitors, alone or in combination, to improve the prognosis of metastatic BC pts.

Analysis of EGFR mutation status in tissue and plasma for predicting response to EGFR-TKIs in advanced non-small-cell lung cancer.

The detection of mutations in the epidermal growth factor receptor (EGFR) gene in tumor tissues has been established as the gold standard for predicting the efficacy of treatment with EGFR-tyrosine kinase inhibitors (EGFR-TKIs) in advanced non-small-cell lung cancer (NSCLC). The current study aimed to investigate whether the presence of co-existing EGFR mutations in tumor tissue and in cell-free tumor DNA (ctDNA) in the plasma predicts a more favorable outcome of EGFR-TKI treatment in advanced NSCLC. A total of 287 NSCLC patients who had undergone EGFR-TKI treatment were enrolled and stratified into four subgroups: Wild-type EGFR in plasma and tissue specimens (B-/T-); mutated EGFR in plasma and tissue specimens (B+/T+); mutated EGFR in only in plasma samples (B+/T-); or mutated EGFR in only tissue specimens (B-/T+). EGFR mutations were tested using denaturing high-performance liquid chromatography and confirmed by amplification-refractory mutation system analysis. Of the 287 patients, 101 had mutations in both tissue and plasma samples and 103 had mutation in either tissue (n=65) or plasma (n=38). The median progression-free survival (mPFS) times were 9.2 and 2.0 months in the B+/T+ and B-/T- groups, respectively. The mPFS times were 7.9 months in the B-/T+ group and 11.9 months in the B+/T-group (P=0.001). Among the 187 patients with available pre-EGFR-TKI plasma samples, 70 received first-line EGFR-TKI treatment, and the mPFS in the B+/T+ group was longer than in the B-/T+ or B+/T- groups (18.8 vs. 9.4 vs. 6.9 months; P=0.003). In second-line setting of EGFR-TKI therapy, the groups of patients with EGFR mutation in ctDNA, regardless of the mutation status in the tissues, exhibited longer mPFS times compared with the B-/T+ group (10.0 vs. 5.8 months; P=0.044). The results suggest that co-existence of EGFR mutations in tissue and ctDNA predict longer PFS times for NSCLC patients who receive first-line EGFR-TKI therapy. In addition, real-time detection in ctDNA is an excellent predictor for the efficacy of second- or higher line EGFR-TKI therapy.

JCES01.23 EGFR Mutation Status Analysis in Cerebrospinal Fluid and Plasma of Advanced Lung Adenocarcinoma with Brain Metastases

We aimed to investigate the feasibility of droplet digital PCR (ddPCR) for the detection of epidermal growth factor receptor (EGFR) mutations in circulating free DNA (cfDNA) from cerebrospinal fluid (CSF) and plasma of advanced Lung Adenocarcinoma (ADC) with brain metastases (BM). Fourteen advanced ADC patients with BM carrying activating EGFR mutations in tumor tissues were enrolled in this study, and their matched CSF and plasma samples were collected. EGFR mutations were detected by the Amplification Refractory Mutation System (ARMS) in tumor tissues. EGFR mutations, including 19del, L858R, and T790M were examined in cfDNA isolated from 2milliliter CSF or plasma by ddPCR assay. The clinical response was assessed according to Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1 guidelines. Overall survival (OS) and progression free survival (PFS) after the diagnosis of BM were also evaluated. Out of 14 patients, eleven were females and three males aged from 34 to 74 years old (median age of 55 years old). In all of cases, CSF cytology were negative. In ddPCR assays, EGFR mutations were detected in CSF of three patients (21.4%; one of 19del and two of L858R), and in plasma of six patients (42.9%; one of 19del, one of L858R, one of T790M, two of L858R&T790M, and one of 19del&T790M). All EGFR T790M mutations were found during or after EGFR-TKIs treatments. The three patients with activating EGFR mutations in CSF achieved partial response (PR) of BM after treated with combination of WBRT and EGFR-TKIs. The median OS and PFS after the diagnosis of BM were 18.0 months and 9.0 months, respectively.Tabled 1PatientTissue EGFRCSF EGFRPlasma EGFRSystematic TreatmentBM Treatment119delWTT790MErotinib+ChemotherapyWBRT+Gamma knife219delWT19delErotinib+ChemotherapyWBRT3L858RL858RL858RGefitinib+ChemotherapyWBRT4L858RWTWTGefitinib+ChemotherapyWBRT519delWTWTGefitinib+ChemotherapyWBRT6L858RWTL858R/T790MErotinib+ChemotherapyWBRT7L858RWTWTGefitinibWBRT819del19Del19Del/T790MGefitinibWBRT9L858RWTWTErotinib+ChemotherapyNONE1019delWTWTErotinib+ChemotherapyWBRT1119delWTWTIcotinib+ChemotherapyWBRT12L858RWTL858R/T790MChemotherapyWBRT13L858RL858RWTIcotinibWBRT1419delWTWTGefitinib+ChemotherapyWBRT Open table in a new tab It was feasible to test EGFR mutation in CSF. CSF may serve as liquid biopsy of advanced ADC with BM by enabling measurement of cfDNA within CSF to characterize EGFR mutations.

P2.03b-010 EGFR Mutation Status Analysis in Cerebrospinal Fluid and Plasma of Advanced Lung Adenocarcinoma with Brain Metastases: Topic: Brain Meta

We aimed to investigate the feasibility of droplet digital PCR (ddPCR) for the detection of epidermal growth factor receptor (EGFR) mutations in circulating free DNA (cfDNA) from cerebrospinal fluid (CSF) and plasma of advanced Lung Adenocarcinoma (ADC) with brain metastases (BM). Fourteen advanced ADC patients with BM carrying activating EGFR mutations in tumor tissues were enrolled in this study, and their matched CSF and plasma samples were collected. EGFR mutations were detected by the Amplification Refractory Mutation System (ARMS) in tumor tissues. EGFR mutations, including 19del, L858R, and T790M were examined in cfDNA isolated from 2 milliliter CSF or plasma by ddPCR assay. The clinical response was assessed according to Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1 guidelines. Overall survival (OS) and progression free survival (PFS) after the diagnosis of BM were also evaluated. Out of 14 patients, eleven were females and three males aged from 34 to 74 years old (median age of 55 years old). In all of cases, CSF cytology were negative. In ddPCR assays, EGFR mutations were detected in CSF of three patients (21.4%; one of 19del and two of L858R), and in plasma of six patients (42.9%; one of 19del, one of L858R, one of T790M, two of L858R&T790M, and one of 19del&T790M). All EGFR T790M mutations were found during or after EGFR-TKIs treatments. The three patients with activating EGFR mutations in CSF achieved partial response (PR) of BM after treated with combination of WBRT and EGFR-TKIs. The median OS and PFS after the diagnosis of BM were 18.0 months and 9.0 months, respectively.Tabled 1PatientTissue EGFRCSF EGFRPlasma EGFRSystematic TreatmentBM Treatment119delWTT790MErotinib+ChemotherapyWBRT+Gamma knife219delWT19delErotinib+ChemotherapyWBRT3L858RL858RL858RGefitinib+ChemotherapyWBRT4L858RWTWTGefitinib+ChemotherapyWBRT519delWTWTGefitinib+ChemotherapyWBRT6L858RWTL858R/T790MErotinib+ChemotherapyWBRT7L858RWTWTGefitinibWBRT819del19Del19Del/T790MGefitinibWBRT9L858RWTWTErotinib+ChemotherapyNONE1019delWTWTErotinib+ChemotherapyWBRT1119delWTWTIcotinib+ChemotherapyWBRT12L858RWTL858R/T790MChemotherapyWBRT13L858RL858RWTIcotinibWBRT1419delWTWTGefitinib+ChemotherapyWBRT Open table in a new tab It was feasible to test EGFR mutation in CSF. CSF may serve as liquid biopsy of advanced ADC with BM by enabling measurement of cfDNA within CSF to characterize EGFR mutations.

Abstract A088: Immune profiling of inflamed microsatellite stable colorectal cancer

Abstract Immunotherapy is an important therapeutic modality that is rapidly turning into standard of care for many cancers. However, early immunotherapy efforts in colorectal cancer (CRC) were ineffective, and only recently did a successful breakthrough lead to improved survival in a small subset of metastatic colorectal cancer patients which were noted to harbor MMR deficiency (microsatellite instability: MSI). Therefore, there is an urgent need for additional biomarkers to identify CRC patients who are predicted to respond to immunotherapy. For the past year we have been interrogating the immune microenvironment of microsatellite stable (MSS) CRC patients to aid our understanding of why a fair proportion of these patients have an inflamed tumor microenvironment (TME). We have banked 140 tumor/normal CRC tissue matched pairs using our recruitment network at Hopkins. Genetic MSI testing was done on 114 of these patients, with 20 (17%) and 94 (83%) being categorized as MSI and MSS, respectively. Our results are therefore representative of the general CRC population which includes 15-20% of MSI+ tumors. We are also currently working with material obtained from CRC patients treated under the Hopkins phase 2 study testing anti PD-1 therapy in patients with MSI tumors. Immunohistochemistry (IHC) and digital image analysis of primary and trial CRC patients: We previously demonstrated the critical geographic association between infiltrating CD8+ T cells and immuno-regulatory molecule expression in the TME of MSI CRC. CRC FFPE tissue sections were stained with anti-CD3 and anti-CD8 antibodies and digital image analysis utilizing the HALO platform was performed. Inflamed MSS CRC are infiltrated by PD1high T cells which are functionally suppressed: Multiparameter flow cytometry (MFC) performed on lymphocytes freshly isolated from our primary CRC tumors demonstrated that a large proportion of both CD4+ and CD8+ T cells infiltrating what we denominate inflamed MSS tumors express higher levels of PD-1 compared to conventional MSS patients. Very importantly, these PD-1high T cells are capable of producing a large amount of IFN-γ after short term PMA/ionomycin stimulation. CD8+ T cells co-localize with immune checkpoint expression in inflamed MSS CRC: We further explored the nature of the T-cell infiltrates by performing laser capture microdissection (LCM) on inflamed MSS tumors and MSS patients who stabilized disease during anti PD-1 therapy, separately dissecting the TIL and invasive front compartments and then performed qRT-PCR for selected genes encoding signature T-cell cytokines as well as core transcription factors for each of the three major Th subsets, Th1/Tc1 (type I CTL; TBX21 and IFN-γ are common to Th1 and Tc1), Th2, and Th17. We additionally analyzed genes associated with CTL and Treg and also general inflammatory cytokines. Finally, we analyzed expression of genes encoding both co-inhibitory membrane ligands and receptors (checkpoints) and metabolic enzymes that have been shown to regulate lymphocyte activity. Inflamed MSS tumors exhibited a similar CD8+ T cell infiltration with a Th1/Tc1 immune signature associated with the counter expression of immune checkpoints as observed in MSI patients. Prediction of MHC class I-restricted Mutation Associated Neoantigens (MANAs): Nonsynonymous mutations in tumor tissue are expected to generate MANAs, which are 8-11 amino-acid peptides generated from proteosomal degradation and recognized by tumor-specific CD8+ T cells in the context of HLA-I restriction. By comparing tumor to normal colon exome we are currently identifying mutations and MANAs in MSS CRC patients who showed high levels of lymphocyte infiltration and PD-1 expression. MANAs in inflamed MSS CRC will be tested for their recognition by autologous TIL. Analysis of the TCR Vβ repertoire in primary CRC and anti-PD-1-treated CRC: In addition to the IHC characterization of CRC T cell infiltration, we are using TCRVβ clonality analysis of the intra-tumor immune response as a metric of the intensity of the antitumor immune response. We postulate that the number and the frequency of unique TCRVβ sequences in the tumor tissue should reflect the density and the immunogenicity of neoepitopes, respectively. In conclusion, we have identified a subset of MSS CRCs that exhibit an “MSI-like” immunologic microenvironment. The final aim of our studies is to validate that MSS CRC patients with a preexisting anti-tumor immune response can benefit from immunotherapeutic interventions. Citation Format: Nicolas Jose Llosa, Franck Housseau, Nicholas Siegel, Kellie N. Smith, Hongni Fan, Robert M. Anders, Dung Le, Luis Diaz, Jr., Cynthia Sears, Drew M. Pardoll. Immune profiling of inflamed microsatellite stable colorectal cancer [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A088.

Molecular Testing in Breast Cancer: A Guide to Current Practices.

-Molecular diagnostics play a role in the management of many cancers, including breast cancer. -To provide an update on molecular testing in current clinical practice, targeted at practicing pathologists who are not breast cancer specialists. -This study is a narrative literature review. -In addition to routine hormone (estrogen and progesterone) receptor testing, new and recurrent tumors are tested for HER2 amplification by in situ hybridization or overexpression by immunohistochemistry. Intrinsic subtyping of tumors represents a fundamental advance in our understanding of breast cancer biology, but currently it has an indirect role in patient management. Clinical next-generation sequencing (tumor profiling) is increasingly used to identify potentially actionable mutations in tumor tissue. Multianalyte assays with algorithmic analysis, including MammaPrint, Oncotype DX, and Prosigna, play a larger role in breast cancer than in many other malignancies. Given that a proportion of breast cancers are familial, testing of nontumor tissue for cancer predisposition mutations also plays a role in breast cancer care.

Association between human papillomavirus and EGFR mutations in advanced lung adenocarcinoma.

Previous studies have demonstrated an association between human papillomavirus (HPV) and mutations in the epidermal growth factor receptor (EGFR) gene in lung cancer patients; however, few studies have investigated this association in advanced lung adenocarcinoma patients undergoing gefitinib treatment. The present study investigated the association between HPV and EGFR mutations in advanced lung adenocarcinoma patients. A total of 95 advanced lung adenocarcinoma patients were enrolled in the study. The HPV infection status and presence of EGFR mutations in tumor tissue was evaluated. Patient clinical characteristics were also determined and compared with HPV infection and EGFR mutation status to analyze their impact on progression-free survival. HPV DNA was identified in 27/95 (28.4%) lung adenocarcinoma tumors and was most common in patients with lymph node metastasis (P=0.016). A total of 44/95 (46.3%) cases exhibited EGFR mutations, which were predominantly observed in female patients and non-smokers. The presence of HPV DNA was significantly associated with EGFR mutations (P=0.012) and multivariate analysis also revealed that HPV DNA was significantly associated with EGFR mutations (odds ratio=3.971) in advanced lung adenocarcinoma. Patients with both HPV infections and EGFR mutations exhibit a marked decrease in the risk of lung cancer progression when compared with those without HPV infection or EGFR mutations (adjusted HR=0.640; 95% confidence interval: 0.488-0.840; P=0.001). HPV infection was significantly associated with EGFR mutations in advanced lung adenocarcinoma patients. Furthermore, patients with HPV infections exhibited the longest progression-free survival times, which may be due to good response to tyrosine kinase inhibitor- or platinum-based-adjuvant therapy in these patients. Patients with EGFR mutations exhibited a better prognosis when compared with those exhibiting wild-type EGFR, regardless of HPV status.

EGFR mutations in tumor tissue and blood collected from lung cancer patients by Insight Onco NGS technique.

e20017Background: Activating or resistant somatic mutations (19 Del, L858R, T790M) occurring in the specific sites of EGFR gene are the powerful predictive biomarker for EGFR tyrosine kinase inhibi...

Comparative analysis of circulating tumor DNA stability In K3EDTA, Streck, and CellSave blood collection tubes

ObjectivesOptimal conditions for blood collection for circulating tumor DNA (ctDNA) are still being developed. Although both Streck and EDTA tubes are commonly used, their ability to stabilize ctDNA as a function of time and temperature post-collection has not been thoroughly studied. Additionally, the potential utility of CellSave tubes (commonly used for circulating tumor cell) for ctDNA measurements has not been studied. Design and methodsBlood was collected into Streck, EDTA, and CellSave tubes from ten patients with metastatic breast cancer enrolled in the MI-ONCOSEQ tumor sequencing program at the University of Michigan and kept either on ice or at room temperature until plasma isolation. Plasma was processed after 2, 6, and 48h post-collection. We used droplet digital PCR (ddPCR) to quantify plasma ctDNA and wild-type DNA for six patients who had tumor tissue mutations represented in commercially available ddPCR assays. ResultsctDNA abundance was similar and stable for up to 6h in all tube types, and there was no effect of storage temperature on the yield for Streck and EDTA tubes. After 48h, however, one out of four patients with detectable ctDNA showed a ~50% decline in ctDNA in the EDTA tube, and three out of six patients showed a 2–3-fold increase in wild-type DNA in the EDTA tube. ConclusionsStreck, EDTA, and CellSave tubes showed similar performance in preserving ctDNA for up to 6h before plasma isolation. Streck and CellSave tubes more consistently stabilized ctDNA and wild-type DNA at 48h than EDTA tubes.

Circulating cell-free DNA has a high degree of specificity to detect exon 19 deletions and the single-point substitution mutation L858R in non-small cell lung cancer

Detection of an epidermal growth factor receptor (EGFR) mutation in circulating cell-free DNA (cfDNA) is a noninvasive method to collect genetic information to guide treatment of lung cancer with tyrosine-kinase inhibitors (TKIs). However, the association between cfDNA and detection of EGFR mutations in tumor tissue remains unclear. Here, a meta-analysis was performed to determine whether cfDNA could serve as a substitute for tissue specimens for the detection of EGFR mutations. The pooled sensitivity, specificity, and areas under the curve of cfDNA were 0.60, 0.94, and 0.9208 for the detection of EGFR mutations, 0.64, 0.99, and 0.9583 for detection of the exon 19 deletion, and 0.57, 0.99, and 0.9605 for the detection of the L858R mutation, respectively. Our results showed that cfDNA has a high degree of specificity to detect exon 19 deletions and L858R mutation. Due to its high specificity and noninvasive characteristics, cfDNA analysis presents a promising method to screen for mutations in NSCLC and predict patient response to EGFR-TKI treatment, dynamically assess treatment outcome, and facilitate early detection of resistance mutations.

Mutations in TP53 increase the risk of SOX2 copy number alterations and silencing of TP53 reduces SOX2 expression in non-small cell lung cancer.

BackgroundAmplifications of the transcription factor, SRY (sex determining region Y)-box 2 (SOX2), are common in non-small cell lung cancer (NSCLC). SOX2 signaling is important in maintaining the stem cell-like phenotype of cancer cells and contributes to the pathogenesis of lung cancer. TP53 is known to inhibit gene amplifications and to repress many stem cell-associated genes following DNA damage. The aim of this study was to investigate if TP53 mutational status affected SOX2 copy number variation and gene expression in early-stage NSCLC patients; moreover, to assess if TP53 regulates SOX2 expression in human lung cancer cells.Methods258 early-stage lung cancer patients were included in the study. Exons 4–9 in the TP53 gene were sequenced for mutations in tumor tissues. SOX2 copy number as well as TP53 and SOX2 gene expression were analyzed in tumor and in adjacent non-tumorous tissues by qPCR. TP53 and SOX2 were silenced using gene-specific siRNAs in human lung adenocarcinoma A427 cells, and the expression of TP53, SOX2 and subset of selected miRNAs was analyzed by qPCR. The odds ratios (ORs) for associations between copy number variation and lung cancer were estimated by conditional logistic regression, and the correlation between gene status and clinicopathological characteristics was assessed by Chi-square or Fisher’s exact test. Gene expression data was analyzed using nonparametric Mann–Whitney test.ResultsTP53 mutations were associated with an increased risk of acquiring a SOX2 copy number alteration (OR = 2.08, 95 % CI: 1.14–3.79, p = 0.017), which was more frequently occurring in tumor tissues (34 %) than in adjacent non-tumorous tissues (3 %). Moreover, SOX2 and TP53 expression levels were strongly correlated in tumor tissues. In vitro studies showed that a reduction in TP53 was associated with decreased SOX2 expression in A427 cells. Furthermore, TP53 knockdown reduced the miRNA hsa-miR-145, which has previously been shown to regulate SOX2 expression.ConclusionsTP53 signaling may be important in the regulation of SOX2 copy number and expression in NSCLC tumors, and the miRNA hsa-miR-145-5p may be one potential driver. This prompts for further studies on the mechanisms behind the TP53-induced regulation of SOX2 expression and the possible importance of hsa-miR-145 in lung cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2061-3) contains supplementary material, which is available to authorized users.

Abstract P1-04-06: Prevalence of PIK3CA mutations in tumor tissue of a consecutive cohort of breast cancer patients (n=700)

Abstract Introduction: The AKT / mTOR pathway is activated by phosphorylation of the lipid kinase phosphatidylinositol 3-kinase (PI3K) and regulates proliferation, apoptosis, survival and adhesion of tumor cells. In up to 30% of breast cancers, dysregulation of PI3K is reported, resulting from mutations in the PIK3CA gene that encodes the catalytic subunit (p110a). Hotspots of mutations were described in exon 9 (helical domain, E542K, G > A) and exon 20 (kinase domain, H1047R, A > G). Prevalence and the prognostic impact of the PIK3CA mutations as well as the predictive value with regard to endocrine therapy are controversially discussed. In this study we describe the prevalence of PIK3CA mutations in a consecutive cohort of breast cancer patients and its association to tumor characteristics and known prognostic factors. Material & Methods: Fresh frozen tumor samples were obtained from a consecutive cohort of 700 breast cancer patients (inclusion criteria: pTx pNx M0 Gx HRx HER2x) who were primarily operated in 6 centres between 2009 and 2011. Tumor DNA was extracted and analyzed by conventional and quantitative PCR (exon 9: cosmid 763 and exon 20: cosmid 775). Patient data, tumor characteristics and prognostic factors were obtained from patients charts. ER, PgR and HER2 results were based on local pathology, uPA and PAI-1 values were determined centrally. Results: All isolated DNA (n=700) were analyzable by PCR and showed a mutation rate of 22% in the entire cohort; including two tumors with both mutations. Among the tumors with a positive steroid hormone receptor (HR) status (n=606; 87%), we found a mutation rate of 24% for PIK3CA, whereas only 9 (10%) of 94 HR-negative tumors were mutated. Tumors with a PIK3CA mutation were significantly associated with HR positivity / HER2 negativity (p=0,001). 14% of the cohort was HER2 positive. In 98 cases with a HER2 positive tumor 17 DNAs were mutated (17%) and 139 DNAs of the 602 HER2 negative tumors had a positive PIK3Ca mutation status (23%). Of the mutated tumors, 94 % were HR positive, and 11% HER2 positive. Only three samples were HER2 positive and HR-negative. 6% of the mutated tumors were triple negative. No significant association was found to age, menopausal status, tumor stage, nodal status, grading and uPA/PAI-1 status. Most mutant tumors were histological grade 2 (p=0,001). A quarter of the intermediate risk group according to St. Gallen risk estimation had a somatic PIK3CA mutation. Conclusion: In our cohort, nearly all mutated tumors are hormone receptor positive and a minority of all mutated samples is HER2 positive. This data adds important information to the heterogeneous results of other previously published patient cohorts. It suggests a possible role of PI3K-dysregulation with regard to resistance against endocrine therapy and anti-HER2-treatment. Citation Format: Martina Vetter, Kristin Reinhardt, Silke Wegener, Juergen Dittmer, Tilmann Lantzsch, Christoph Uleer, Susanne Peschel, Jutta John, Volker Hanf, Joerg Buchmann, Karl-Friedrich Bürrig, Edith Weigert, Christoph Thomssen, Eva J Kantelhardt. Prevalence of PIK3CA mutations in tumor tissue of a consecutive cohort of breast cancer patients (n=700) [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P1-04-06.

A mutation-sensitive switch assay to detect five clinically significant epidermal growth factor receptor mutations.

Epidermal growth factor receptor (EGFR) mutations can affect the therapeutic efficacy of drugs used to treat nonsmall-cell lung cancer (NSCLC). We aimed to develop methods to detect five common EGFR somatic mutations in tumor tissues from NSCLC patients by using a nanoscale mutation-sensitive switch consisting of a high-fidelity polymerase and phosphorothioate-modified allele-specific primers. The five clinically significant EGFR mutations examined here are S768I, T790M, L858R, and 15- and 18-bp deletion mutations in exon 19. Our assays showed sensitivities of 100 copies and specificities of more than three log scales for matched templates relative to mismatched templates by routine polymerase chain reaction (PCR), real-time PCR, and multiplex PCR. This assay would be superior to DNA sequencing in situations where mutant DNA is not abundant.

GE-01 * IDH1-MUTATED PITUITARY ADENOMA CHALLENGES TRADITIONAL CHARACTERIZATION OF MAFFUCCI SYNDROME

Maffucci syndrome is a rare, non-inheritable condition of mesodermal dysplasia, presenting in childhood with formation of multiple enchondromas and soft-tissue hemangiomas. Approximately, 40% of enchondromas undergo malignant transformation into chondrosarcomas. The underlying genetic etiology lies in somatic mosaicism of mutations in isocitrate dehydrogenase 1 (IDH1) or IDH2, which have been detected simultaneously in both tumors and blood leukocytes of affected patients. We describe a 7-year old male who presented with extensive enchondromatosis and subcutaneous hemangiomas, clinically diagnosed as Maffucci syndrome. Years later, he developed sub-acute dysphasia, dysarthria, and loss of left visual acuity. A brain MRI demonstrated two heterogeneously enhancing lesions, one centered in the left jugular foramen and the other located in the supra-sellar region. Surgical resections of both intracranial tumors were performed over a period of ten months. Histopathological review revealed diagnoses of a grade II chondrosarcoma as well as a non-functional pituitary adenoma after subsequent neuroendocrine studies were unremarkable. Genotypic studies of the tumors were performed to identify common IDH1/2 mutations. DNA sequencing utilized standard Sanger sequencing in conjunction with a target-specific peptide nucleic acid (PNA) to detect IDH1 mutations in tumor tissues. The results demonstrated identical IDH1 mutations (c.394C > T; R132C) in both tumors. This result represents the first genetic evidence to establish causality between pituitary adenoma formation and tumor predisposition in Maffucci syndrome. As neuro-ectodermal tissue, this IDH1-mutated pituitary adenoma challenges the notion that Maffucci syndrome only involves mesodermal tissues. As such, in pediatric patients newly diagnosed with Maffucci syndrome, it may be appropriate to monitor for development of pituitary pathology and neuroendocrine dysfunction. Conversely, in the rare sub-population of pediatric neuro-oncology patients with concomitant non-nervous system tumors, further investigation is warranted to rule out Maffucci syndrome, as there may be considerable risk for malignant transformation as well as additional tumor formation.

FBXW7 mutation analysis and its correlation with clinicopathological features and prognosis in colorectal cancer patients.

This study aimed to determine the prognostic value of mutations in the tumor suppressor gene FBXW7 for clinical outcomes in colorectal cancer (CRC). Between January 2000 and December 2009, FBXW7 mutations in tumor tissues from 1,519 CRC patients at Taipei Veterans General Hospital were assessed using a MassArray system. We compared the clinicopathological variables and prognosis between the wild-type and mutant tumor tissue groups. FBXW7 mutations were present in 114/1,519 CRC patients (7.5%). In stage I/II CRC patients, mutant FBXW7 was more common than wild-type FBXW7 (62.3% vs. 50.8%). CRC patients with FBXW7 mutations did not differ significantly in their 5-year overall survival (OS). Stage I/II CRC patients with FBXW7 mutations had lower OS, but this difference was not significant (71.6% vs. 78.2%). Among FBXW7 tumors, S582L was the most frequent mutation type (19.3%), followed by R465H (16.6%), R505C (14.9%) and R479Q (14.9%). Subgroup analysis of FBXW7 mutants showed that R465H/R465C/R479Q had better 5-year OS than other mutant types (76.9% vs. 56.0%; p=0.012). There was no strong association between patient prognosis and FBXW7 mutations in our large-scale study.

Peripheral Blood for Epidermal Growth Factor Receptor Mutation Detection in Non-Small Cell Lung Cancer Patients

OBJECTIVE: It is important to analyze and track Epidermal Growth Factor Receptor (EGFR) mutation status for predicting efficacy and monitoring resistance throughout EGFR-tyrosine kinase inhibitors (TKIs) treatment in non-small cell lung cancer (NSCLC) patients. The objective of this study was to determine the feasibility and predictive utility of EGFR mutation detection in peripheral blood. METHODS: Plasma, serum and tumor tissue samples from 164 NSCLC patients were assessed for EGFR mutations using Amplification Refractory Mutation System (ARMS). RESULTS: Compared with matched tumor tissue, the concordance rate of EGFR mutation status in plasma and serum was 73.6% and 66.3%, respectively. ARMS for EGFR mutation detection in blood showed low sensitivity (plasma, 48.2%; serum, 39.6%) but high specificity (plasma, 95.4%; serum, 95.5%). Treated with EGFR-TKIs, patients with EGFR mutations in blood had significantly higher objective response rate (ORR) and insignificantly longer progression-free survival (PFS) than those without mutations (ORR: plasma, 68.4% versus 38.9%, P = 0.037; serum, 75.0% versus 39.5%, P = 0.017; PFS: plasma, 7.9 months versus 6.1 months, P = 0.953; serum, 7.9 months versus 5.7 months, P = 0.889). In patients with mutant tumors, those without EGFR mutations in blood tended to have prolonged PFS than patients with mutations (19.7 months versus 11.0 months, P = 0.102). CONCLUSIONS: EGFR mutations detected in blood may be highly predictive of identical mutations in corresponding tumor, as well as showing correlations with tumor response and survival benefit from EGFR-TKIs. Therefore, blood for EGFR mutation detection may allow NSCLC patients with unavailable or insufficient tumor tissue the opportunity to benefit from personalized treatment. However, due to the high false negative rate in blood samples, analysis for EGFR mutations in tumor tissue remains the gold standard.

Automated Objective Determination of Percentage of Malignant Nuclei for Mutation Testing

Detection of DNA mutations in tumor tissue can be a critical companion diagnostic test before prescription of a targeted therapy. Each method for detection of these mutations is associated with an analytic sensitivity that is a function of the percentage of tumor cells present in the specimen. Currently, tumor cell percentage is visually estimated resulting in an ordinal and highly variant result for a biologically continuous variable. We proposed that this aspect of DNA mutation testing could be standardized by developing a computer algorithm capable of accurately determining the percentage of malignant nuclei in an image of a hematoxylin and eosin-stained tissue. Using inForm software, we developed an algorithm, to calculate the percentage of malignant cells in histologic specimens of colon adenocarcinoma. A criterion standard was established by manually counting malignant and benign nuclei. Three pathologists also estimated the percentage of malignant nuclei in each image. Algorithm #9 had a median deviation from the criterion standard of 5.4% on the training set and 6.2% on the validation set. Compared with pathologist estimation, Algorithm #9 showed a similar ability to determine percentage of malignant nuclei. This method represents a potential future tool to assist in determining the percent of malignant nuclei present in a tissue section. Further validation of this algorithm or an improved algorithm may have value to more accurately assess percentage of malignant cells for companion diagnostic mutation testing.