Recurrent Tumor Samples Research Articles

The TNF receptor family member Fn14 is highly expressed in recurrent glioblastoma and in GBM patient-derived xenografts with acquired temozolomide resistance.

Glioblastoma (GBM) is a difficult to treat brain cancer that nearly uniformly recurs, and recurrent tumors are largely therapy resistant. Our prior work has demonstrated an important role for the tumor necrosis factor-like weak inducer of apoptosis (TWEAK) receptor fibroblast growth factor-inducible 14 (Fn14) in GBM pathobiology. In this study, we investigated Fn14 expression in recurrent GBM and in the setting of temozolomide (TMZ) resistance. Fn14 mRNA expression levels in nonneoplastic brain, primary (newly diagnosed) GBM, and recurrent GBM (post-chemotherapy and radiation) specimens were obtained from The Cancer Genome Atlas data portal. Immunohistochemistry was performed using nonneoplastic brain, patient-matched primary and recurrent GBM, and gliosarcoma (GSM) specimens to examine Fn14 protein levels. Western blot analysis was used to compare Fn14 expression in parental TMZ-sensitive or matched TMZ-resistant patient-derived xenografts (PDXs) established from primary or recurrent tumor samples. The migratory capacity of control and Fn14-depleted TMZ-resistant GBM cells was assessed using the transwell migration assay. We found that Fn14 is more highly expressed in recurrent GBM tumors than their matched primary GBM counterparts. Fn14 expression is also significantly elevated in GSM tumors. GBM PDX cells with acquired TMZ resistance have higher Fn14 levels and greater migratory capacity than their corresponding parental TMZ-sensitive cells, and the migratory difference is due, at least in part, to Fn14 expression in the TMZ-resistant cells. This study demonstrates that the Fn14 gene is highly expressed in recurrent GBM, GSM, and TMZ-resistant GBM PDX tumors. These findings suggest that Fn14 may be a valuable therapeutic target or drug delivery portal for treatment of recurrent GBM and GSM patients.

EGFR copy number alterations in primary tumors, metastatic lymph nodes, and recurrent and multiple primary tumors in oral cavity squamous cell carcinoma

BackgroundThe EGFR and downstream signaling pathways play an important role in tumorigenesis in oral squamous cell carcinoma (OSCC). Gene copy number alteration is one mechanism for overexpressing the EGFR protein and was also demonstrated to be related to lymph node metastasis, tumor invasiveness and perineural invasion. Therefore, we hypothesized that EGFR gene copy number alteration in the primary tumor could predict amplification in recurrent tumors, lymph node metastatic foci or secondary primary tumors.MethodsWe recruited a group of newly diagnosed OSCC patients (n = 170) between Mar 1997 and Jul 2004. Metastatic lymph nodes were identified from neck dissection specimens (n = 57). During follow-up, recurrent lesions (n = 41) and secondary primary tumors (SPTs, n = 17) were identified and biopsied. The EGFR gene amplifications were evaluated by fluorescence in situ hybridization (FISH) assay in primary tumors, metastatic lymph nodes, recurrences and SPTs.ResultsOf the 170 primary OSCCs, FISH showed low EGFR amplification/polysomy in 19 (11.4%) patients and amplification in 33 (19.8%) patients. EGFR gene amplification was related to lymph node metastasis (χ2 trend test: p = 0.018). Of 57 metastatic lymph nodes, nine (15.8%) had EGFR polysomy and 14 (24.6%) had EGFR gene amplification. The concordance rate of EGFR gene copy number in primary tumors and lymph node metastasis was 68.4% (McNemar test: p = 0.389). Of 41 recurrent tumors, five (12.2%) had EGFR polysomy and five (12.2%) had gene amplification. The concordance rate of EGFR gene copy number between primary tumors and recurring tumors was 65.9% (McNemar test: p = 0.510). The concordance rate between primary tumors and SPTs was 70.6%. EGFR amplification in either primary tumors, metastatic lymph nodes or recurrent tumors had no influence on patient survival.ConclusionWe can predict two-thirds of the EGFR gene copy number alterations in lymph node metastasis or recurrent tumors from the analysis of primary tumors. For OSCC patients who are unable to provide lymph node or recurrent tumor samples for EGFR gene copy number analysis, examining primary tumors could provide EGFR clonal information in metastatic, recurrent or SPT lesions.

Clinical and genetic analysis of recurrent adult-type granulosa cell tumor of the ovary: Persistent preservation of heterozygous c.402C>G FOXL2 mutation.

BackgroundAdult-type granulosa cell tumors of the ovary (aGCTs) are rare tumors that represent 2–5% of ovarian malignancies. The prognosis of this tumor is favorable, and it is characterized by slow progression. 10–30% of these tumors recur after 4–7 years of the primary surgery and the 5-year survival rate from the first recurrence is 55%, for the incompletely resected patients. At this time, complete resection is the only prognostic factor for better outcome, and establishing a novel strategy for identification and/or treatment of recurrent tumors is crucial. After the discovery of heterozygous c.402C>G FOXL2 mutations in 97% of cases of aGCT, much effort has been made to find the role of the mutation on the pathogenesis of aGCT, however, little is known about the role of the mutation in disease progression.MethodsWe analyzed the clinical data of 56 aGCT patients to find a marker of recurrence. In particular, we compared the FOXL2 status in 5 matched primary and recurrent samples by immunohistochemistry, and TaqMan allelic discrimination assay to address the role of FOXL2 in potential mechanisms of recurrence.ResultsThe clinical data analysis was consistent with complete resection as an indicator of disease eradication, though the sample size was limited. The genetic analysis showed all the samples, including recurrent tumor samples up to 14 years after the primary surgery, expressed heterozygous c.402C>G FOXL2 mutation and the FOXL2 protein expression.ConclusionThis report describes the preservation of heterozygous c.402C>G FOXL2 mutation in recurrent aGCTs. This finding adds further credence to the concept that the c.402C>G FOXL2 mutation is oncogenic and integral to this disease.

HGG-16. A COMPARISON OF PRIMARY AND RECURRENT PEDIATRIC HIGH-GRADE GLIOMAS VIA RNA SEQUENCING AND DRUG SCREENING

Abstract BACKGROUND: High-grade gliomas (HGG) comprise 6–10% of all brain tumors in children but account for the majority of deaths, with a median survival rate of 15 months. Recurrent HGG is incurable, and median survival is five months from recurrence. METHODS: To study the changes that take place between primary diagnosis and recurrence, we performed RNA sequencing on matched initial and recurrent tumor samples from eight pediatric patients diagnosed with HGG. We then used six patient-derived pediatric HGG cell lines, three from biopsy and three from autopsy, as an in vitro model of primary and recurrent tumors. We screened these lines for survival with a panel of all FDA-approved chemotherapy drugs. RESULTS: The median age of subjects was 9.5 years at initial diagnosis. 7 of 8 subjects received radiation, and 3 of 8 tumor pairs had BRAF V600E mutations. Gene set enrichment analysis of the samples revealed that as compared with the initial tumors, recurrences are strongly enriched for gene expression characteristic of neural progenitor cells on three separate analyses, suggesting regression of the recurrent tumors to a less differentiated, more anaplastic state (Normalized Enrichment Scores 7.25–8.71, False Discovery Rate <0.001 for all). On drug screening, the autopsy-derived lines were significantly more sensitive to several agents, including trametinib, vinblastine, and etoposide. DISCUSSION: Pediatric HGG appears to take on a more stem-like state at recurrence compared to primary diagnosis. Cell lines from recurrent tumors show increased sensitivity to several agents already in use for pediatric brain tumors, raising the potential for improved treatments. Future work includes analysis of mutations that develop between primary diagnosis and recurrence, as well as development of cell line and animal models of matched primary and recurrent pairs to further investigate changes in tumor biology and potential therapies.

GENE-06. CO-OCCURRENCE OF DOUBLE MUTATION H3F3A/BRAF IN PEDIATRIC GANGLIOGLIOMAS

AbstractGrade I or III ganglioglioma (GG) is characterized by alterations in the MAPK pathway, including BRAF mutation. Diffuse midline glioma H3 K27M mutated was added to the WHO 2016 classification as a new grade IV entity. A co-occurrence of H3F3A/BRAF mutations has been reported in midline tumors. Firstly, we searched for BRAF V600E and H3 K27M mutations in a series of 54 pediatric midline grade I GG to determine the frequency of double mutations in grade I GG. We then studied the clinico-radiological and histo-molecular features of 12 grade I to III double-mutated tumors to explore the therapeutic and prognostic relevance. The frequency of the co-occurrence of H3F3A/BRAF mutations in grade I midline GG at diagnosis was 9.3%. Double-immunostaining revealed that BRAF V600E and H3 K27M mutant proteins colocalized in both the glial and neuronal components, The series of double-mutated GG included 8 grade I, and 4 grade III at diagnosis. The median survival was 8 years. Three grade I tumors showed a malignant transformation at 0.5, 3.5 and 7 years and two of them showed a loss of the BRAF mutation in recurrent tumor samples. Five patients with grade I were alive with stable disease without adjuvant therapy at the last follow-up. One grade III showed a paradoxical response under BRAF inhibitor. In this work we stress that H3F3A mutation can occur, in association with the BRAF mutation, in grade I GG, and that these have a better spontaneous outcome than classic diffuse midline H3 K27M-mutant gliomas. These data highlight the importance of an integrated histomolecular diagnosis especially in the context of targeted therapy.

High-intermediate risk endometrial cancer: Can gene expression predict recurrence?

5591 Background: Studies have shown that adjuvant therapy increases progression free survival, but does not affect overall survival in patients with high-intermediate risk (H-IR) endometrial cancer (EMCA). Our objective was to develop a gene expression signature that may help identify H-IR EMCA patients with the highest risk of recurrence to help guide treatment strategies. Methods: Data was collected on all patients that met H-IR EMCA criteria diagnosed between 2000-2010 at UAB (n = 292). Of the patients that did not receive adjuvant treatment, 13 patients that recurred were matched to 13 patients that did not recur and original tumor was compared. Of those that recurred, 5 patients had original and recurrent tumor available for analysis. Gene expression data was collected using the Nanostring nCounter PanCancer Pathway 770 gene panel. Data was analyzed using nSolver Advanced Analysis Software. A fold change (FC) of ≥ ± 2 (p &lt; 0.05) was used to identify genes with a significant expression difference. Results: Comparing the 13 patients that recurred to the 13 that did not, there were 5 genes with FC ≥ +2: BAIAP3, PLCB1, IL1R1, NOS3 and RAD50. There were 29 genes with FC ≥ -2; the top 3 genes with decreased expression (FC ≥ -10) were: BMP7, FGF18, WNT7A. Genes in the Cell Cycle (CC) pathway were significantly different in the patients that recurred (p = 0.02). There were 61 genes with FC ≥ +2 when comparing the original tumor to recurrent tumor; the top 3 genes with increased expression (FC ≥ 10) were: FGF18, CCND1, HIST1H3H (p &lt; 0.05). There were 50 genes with FC ≥ -2; the top 3 genes with decreased expression (FC ≥ -1000) were: HOXA11, LEFTY2 and SFRP4. Wnt, Hedgehog, Chromatin Modification, DNA repair, TGF-β, MAPK, and CC pathways were significantly different in the recurrent samples compared to the original tumor (p &lt; 0.05). Conclusions: Our data suggests that gene expression panels could better identify patients that warrant adjuvant treatment. The CC pathway, which is significantly different in the original tumor from those that recurred and those that did not, was further altered in the recurrent tumor samples. Additional studies are on-going to validate these findings and to further investigate DNA mutation differences in larger cohort of patients.

Altered status of programmed death-ligand 1 after recurrence in resected lung adenocarcinoma patients.

PurposeProgrammed death-ligand 1 (PD-L1) is found to be overexpressed in non-small cell lung cancer. The present study intended to evaluate the status of PD-L1 expression in patients with resection and recurrent lung adenocarcinoma.Patients and methodsMatched resection and recurrent tumor samples were harvested from 65 lung adenocarcinoma patients. Immunohistochemistry was used to evaluate the status of PD-L1 expression. Kaplan–Meier method was used for survival analysis.ResultsA total of 65 patients of lung adenocarcinoma were enrolled. They underwent complete resection and had recurrence after adjuvant treatment. PD-L1 expression was identified in 43.1% (28/65) of resection samples vs 55.4% (36/65) of recurrent samples. Ten patients shifted from negative to positive, whereas another two samples showed the opposite. Patients with PD-L1 expression showed worse disease-free survival than the PD-L1-negative counterparts. The expression of PD-L1 in recurrent samples was a significant favorable factor for epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) (11.2 vs 8.2 months, P=0.030).ConclusionThe status of PD-L1 expression may alter between resection and recurrent samples. Also, the status of PD-L1 expression after recurrence is a better prognostic factor for EGFR-TKIs.

Detection of ESR1 mutations in plasma and tumors from metastatic breast cancer patients using next-generation sequencing.

Liquid biopsy using digital PCR (dPCR) has been widely used for the screening of ESR1 mutations, since they are frequently identified in the hotspot. However, dPCR is limited to the known mutations. Therefore, we aimed to analyze the utility of next-generation sequencing (NGS) to discover novel ESR1 mutations. Whole exon sequencing of the ESR1 gene using NGS was performed in 16 primary and 47 recurrent tumor samples and 38 plasma samples from hormone receptor-positive metastatic breast cancer patients. Functional analyses were then performed for the novel mutations we detected. We identified no mutations in primary tumors and six mutations in five recurrent tumors, including three types of known mutations (Y537C, Y537N, and D538G) and two novel mutations (E279V and G557R). We also identified seven mutations in five plasma samples, including three types of known mutations (S463P, Y537S, and D538G) and one mutation not reported in COSMIC database (L536H). All nine patients with ESR1 mutations were treated with aromatase inhibitors (AIs) prior to sampling, and the mutations were frequently detected in patients who received AI treatments in the metastatic setting. Among the three novel mutations (E279V, L536H, and G557R), L536H, but not E279V and G557R, showed ligand-independent activity. All three mutant proteins showed nuclear localization and had no relation with non-genomic ER pathways. Although the molecular mechanisms of the E279V and G557R mutations remain unclear, our data suggest the utility of NGS as a liquid biopsy for metastatic breast cancer patients and the potential to identify novel ESR1 mutations.

MiR-1, a Potential Predictive Biomarker for Recurrence in Prostate Cancer After Radical Prostatectomy

BackgroundIncreasing evidence suggests that aberrant microRNAs expressions are significantly associated with cancer progression. Previous studies have reported that the relative expression of miR-1 is significantly downregulated in recurrent prostate cancer (PCa) samples when compared with nonrecurrent PCa tissues. However, whether miR-1 can serve as a novel predictive biomarker for PCa recurrence still remains unclear. Materials and MethodsThe patients with clinically localized PCa who underwent radical prostatectomy by the same medical team at the Department of Urology, Ningbo No.2 Hospital were enrolled in this study. We examined the miR-1 expression levels in recurrent and nonrecurrent tumor samples by quantitative reverse transcription polymerase chain reaction. Univariate and multivariate Cox proportional hazards analyses were used for the evaluation of potential predictors of PCa recurrence. ResultsDuring the study period, 78 patients (including 27 in the recurrent group and 51 in the nonrecurrent group) who were diagnosed with PCa and who underwent radical prostatectomy were included in the final analysis. MiR-1 was significantly downregulated in recurrent PCa tissues when compared with nonrecurrent tumor samples (P < 0.001). The univariate and multivariate Cox proportional hazards analyses indicated that miR-1 was the only independent prognostic factor for PCa recurrence (hazard ratio = 1.86; 95% CI: 1.21-2.94; P = 0.011). The area under the curve value of miR-1 for PCa recurrence was 0.885 (P < 0.001) with the sensitivity of 0.863 and specificity of 0.889 based on receiver operating characteristic curve analysis. ConclusionsThis study identifies that miR-1 in PCa tissues can function as an important independent predictive factor for PCa recurrence.

Primary and recurrent ovarian high-grade serous carcinomas display similar microRNA expression patterns relative to those of normal ovarian tissue.

Most patients with epithelial ovarian cancer eventually die due to recurrence. However, little is known about the microRNA (miRNA) expression pattern and its involvement in recurrent ovarian cancer. We analyzed miRNA expression profiles related to the recurrence of advanced ovarian high-grade serous carcinoma (HGSC) using miRNA microarrays. Between May 2006 and December 2012, 37 ovarian HGSC patients underwent secondary cyto-debulking surgery at recurrence. Among them, only 8 pairs of primary and recurrent tumor samples were deemed to be adequate for analysis. The expression profiles of primary ovarian HGSC compared with normal ovarian tissue were significantly consistent with those of recurrent ovarian HGSC compared with normal ovarian tissue (correlation coefficient = 0.81, P = 0.0078). Among 31 miRNAs that increased by more than 4-fold in primary tumors compared with normal ovarian tissue, 27 were also significantly increased in recurrent tumor samples. Likewise, among 35 miRNAs that decreased by more than 4-fold in primary tumors compared with normal ovarian tissue, 34 were also significantly decreased in recurrent tumor samples. We identified 60 miRNAs that were significantly increased in recurrent serous ovarian carcinoma compared with primary tumor tissue, including miR-630, miR-370, and miR-575. Additionally, 52 miRNAs were significantly decreased in recurrent samples, including miR-509-3p, miR-514a-3p, and miR-506-3p. Our results demonstrate that primary and recurrent ovarian HGSC displayed similar miRNA expression patterns. Nevertheless, altered miRNA expression could be implicated in the recurrence of ovarian HGSC, and further study is needed to validate these data in independent cases using a homogeneous methodology.

Abstract 2461: Rhabdomyosarcoma tumors recur by a PAX3-FOXO1-independent mechanism in a human myoblast xenograft model

Abstract The PAX3-FOXO1 fusion gene is generated by a 2;13 chromosomal translocation, and is a characteristic feature of a subset of rhabdomyosarcoma (RMS) with aggressive behavior and poor prognosis. This study utilizes an inducible expression system in human myoblasts to dissect the molecular mechanism of PAX3-FOXO1 action during RMS tumorigenesis and progression.Constitutive and doxycycline-inducible expression constructs were used to generate a human myoblast cell line that constitutively expresses MYCN and reversibly expresses PAX3-FOXO1, respectively. PAX3-FOXO1 expression was assessed by Western blotting, qRT-PCR and immunohistochemistry. Oncogenicity in these engineered myoblasts was studied in vitro by focus formation and in vivo by intramuscular injection of NOD-SCID mice. Cell proliferation, apoptosis and myogenic differentiation were assessed by western blot or immunohistochemical assays.In focus formation assays, doxycycline-treated myoblasts expressing constitutive MYCN and doxycycline inducible PAX3-FOXO1 showed a high level of oncogenic transformation. When doxycycline was removed during the course of this assay, smaller foci formed with prominent myogenic differentiation and cell death. Intramuscular injection of these engineered myoblasts resulted in RMS tumor formation when fusion protein expression was induced by feeding mice a doxycycline-supplemented diet. After small palpable tumors formed, doxycycline withdrawal resulted in decreased PAX3-FOXO1 expression and tumor regression. Microscopic examination of regressing tumors revealed widespread myogenic differentiation and cell death. In most cases, the tumors recurred several weeks later despite the absence of inducing agent. Analysis of recurrent tumor samples revealed that a subset emerged in the absence of PAX3-FOXO1 expression. A cell line generated from a PAX3-FOXO1-independent recurrence demonstrated transformation in vitro in the absence of doxycycline. Though cell lines derived from primary tumors were dependent on PAX3-FOXO1 and differentiated when doxycycline was removed, the recurrent tumor-derived cells did not differentiate under these conditions and instead proliferated continuously. Furthermore, reinjection of these recurrent tumor-derived cells resulted in tumor formation in mice in the absence of doxycycline, and even more rapid tumor formation in the presence of doxycycline.Our study provides evidence that the PAX3-FOXO1 fusion protein is necessary to develop primary tumors but recurrent tumors can develop by a PAX3-FOXO1-independent mechanism. The recurrent tumors are postulated to have acquired secondary oncogenic events that were activated or selected by initial PAX3-FOXO1 expression. These secondary events have an additive oncogenic effect with PAX3-FOXO1 expression, and then contribute to tumor recurrence in the absence of PAX3-FOXO1 expression. Citation Format: Puspa R. Pandey, Bishwanath Chatterjee, Javed Khan, Stephen M. Hewitt, Markku Martti Miettinen, Frederic G. Barr. Rhabdomyosarcoma tumors recur by a PAX3-FOXO1-independent mechanism in a human myoblast xenograft model. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2461.

Genomic characterization of recurrent high-grade astroblastoma

Astroblastomas are rare primary brain tumors, diagnosed based on histologic features. Not currently assigned a WHO grade, they typically display indolent behavior, with occasional variants taking a more aggressive course. We characterized the immunohistochemical characteristics, copy number (high-resolution array comparative genomic hybridization, OncoCopy) and mutational profile (targeted next-generation exome sequencing, OncoPanel) of a cohort of seven biopsies from four patients to identify recurrent genomic events that may help distinguish astroblastomas from other more common high-grade gliomas. We found that tumor histology was variable across patients and between primary and recurrent tumor samples. No common molecular features were identified among the four tumors. Mutations commonly observed in astrocytic tumors (IDH1/2, TP53, ATRX, and PTEN) or ependymoma were not identified. However one case with rapid clinical progression displayed mutations more commonly associated with GBM (NF1(N1054H/K63)*, PIK3CA(R38H) and ERG(A403T)). Conversely, another case, originally classified as glioblastoma with nine-year survival before recurrence, lacked a GBM mutational profile. Other mutations frequently seen in lower grade gliomas (BCOR, BCORL1, ERBB3, MYB, ATM) were also present in several tumors. Copy number changes were variable across tumors. Our findings indicate that astroblastomas have variable growth patterns and morphologic features, posing significant challenges to accurate classification in the absence of diagnostically specific copy number alterations and molecular features. Their histopathologic overlap with glioblastoma will likely confound the observation of long-term GBM "survivors". Further genomic profiling is needed to determine whether these tumors represent a distinct entity and to guide management strategies.

Abstract IA12: Determinants of resistance and response in high-grade serous ovarian cancer.

Abstract In contrast to other solid cancers, overall survival for patients with high-grade serous ovarian cancer (HGSC) is little changed over the last 30 years and it has been difficult to advance beyond platinum-based combination chemotherapy. As HGSC are driven by genomic copy number change rather than recurrent point mutation, we undertook whole genome sequence (WGS) analysis of a large HGSC cohort in order to survey structural variation at high resolution. Utilizing 114 tumors from 92 patients, we performed transcriptome, methylation, and miRNA expression analyses to support the WGS data, and compared resistant, refractory, and sensitive primary disease. We evaluated tumor and normal DNA samples from patients with primary refractory, primary resistant or primary sensitive disease to better understand the drivers of these clinical phenotypes. We also characterized paired pre-treatment chemosensitive and post-resistance samples, and end-stage HGSC obtained from patients who underwent a research autopsy. We found that gene breakage involving tumor suppressors such as NF1 and RB1 is a common form of inactivation in HGSC and in other circumstances contributes to acquired chemotherapy resistance in some patients. We found evidence of a chemotherapy footprint in the genome of recurrent tumor samples. We observed a striking degree of diversity of other mechanisms of acquired resistance, including multiple independent germline BRCA1 or BRCA2 reversion events in individual patients, loss of BRCA1 promoter methylation, a shift in molecular subtype to a desmoplastic phenotype, and a recurrent promoter fusion associated with overexpression of a driver of drug resistance, validated in an independent dataset. Collectively these findings underscore the plasticity of the HGSC genome and indicate that pre-empting resistance by modifying current first line therapy or countering it when it emerges will require a diversity of approaches. Citation Format: David D. L. Bowtell. Determinants of resistance and response in high-grade serous ovarian cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr IA12.

Identification of variants in primary and recurrent glioblastoma using a cancer-specific gene panel and whole exome sequencing.

Glioblastoma (GBM) is an aggressive, malignant brain tumor typically resulting in death of the patient within one year following diagnosis; and those who survive beyond this point usually present with tumor recurrence within two years (5-year survival is 5%). The genetic heterogeneity of GBM has made the molecular characterization of these tumors an area of great interest and has led to identification of molecular subtypes in GBM. The availability of sequencing platforms that are both fast and economical can further the adoption of tumor sequencing in the clinical environment, potentially leading to identification of clinically actionable genetic targets. In this pilot study, comprised of triplet samples of normal blood, primary tumor, and recurrent tumor samples from three patients; we compared the ability of Illumina whole exome sequencing (ExomeSeq) and the Ion AmpliSeq Comprehensive Cancer Panel (CCP) to identify somatic variants in patient-paired primary and recurrent tumor samples. Thirteen genes were found to harbor variants, the majority of which were exclusive to the ExomeSeq data. Surprisingly, only two variants were identified by both platforms and they were located within the PTCH1 and NF1 genes. Although preliminary in nature, this work highlights major differences in variant identification in data generated from the two platforms. Additional studies with larger samples sizes are needed to further explore the differences between these technologies and to enhance our understanding of the clinical utility of panel based platforms in genomic profiling of brain tumors.

ZEB1 as an indicator of tumor recurrence for areca quid chewing-associated oral squamous cell carcinomas.

Oral squamous cell carcinoma (OSCC) is the sixth most prevalent malignancy worldwide and the third most common cancer in developing nation. Most OSCC patients relapse within months after receiving treatment. Therefore, searching the biomarkers of recurrence is urgently required to improve OSCC patient survival. We set out to explore whether expression of ZEB1 could be triggered in oral epithelial cells (SG and FaDu) by arecoline in vitro. Control and ZEB1-knockdown arecoline-stimulated SG and FaDu were subjected to migration/invasiveness/anchorage-independent growth assay. Primary and recurrent OSCC tissues from areca quid chewers were analyzed using real-time RT-PCR analysis for ZEB1 expression. Arecoline led to dose-dependent elevation of ZEB1 expression in SG and FaDu cells. Downregulation of ZEB1 by lentiviral infection significantly reversed arecoline-induced oncogenicity including migration ability, cell invasiveness, and anchorage-independent growth in SG and FaDu cells. Clinically, the level of ZEB1 expression was higher in recurrent OSCC tumor samples but lower in primary lesions. Targeting ZEB1 might offer a new strategy for the treatment of OSCC patients. ZEB1 can serve as a progression and relapse marker in OSCC patients.

BI-29 * VARIANT ANALYSIS OF PRIMARY AND RECURRENT GLIOBLASTOMA USING ION AMPLISEQTM COMPREHENSIVE CANCER PANEL AND WHOLE EXOME SEQUENCING

BACKGROUND: Glioblastoma is the most deadly and frequently occurring adult primary brain tumor. The characterization of genetic variants and molecular signatures in glioblastoma is heavily reliant upon genomic sequencing. The availability of rapid and economical sequencing platforms is necessary for the widespread adoption of high-throughput sequencing in the clinical environment. METHODS: Utilizing patient matched triplet samples consisting of normal blood and snap-frozen primary and recurrent glioblastoma tumor samples from the Ohio Brain Tumor Study, we compared whole exome sequencing data from TCGA to sequencing data obtained from Ion AmpliSeqTM Comprehensive Cancer Panel (CCP). RESULTS: As we anticipated, the number of variants identified from the exome sequencing data (n = 619) was greater than those identified from the Ion AmpliSeqTM CCP data (n = 22). Surprisingly, there were only six variants common across both data sets. In addition, none of the variants from the Ion AmpliSeqTM CCP data were shared across patient samples. CONCLUSIONS: Our pilot results suggest disparities in both the number and category of mutations identified from analysis of data generated from the Ion AmpliSeqTM CCP and whole exome sequencing. Future studies are needed to elucidate the nature of these differences and to determine the clinical relevance of variants that may be associated with glioblastoma recurrence and response to treatment. High-throughput sequencing based cancer panels may be improved by the development of brain tumor specific panels.

Abstract PD3-1: Serial next generation sequencing (NGS) of poor prognosis luminal tumors across treatment history reveals both de novo and acquired alterations potentially associated with endocrine resistance

Abstract Cancer therapies exert selective pressure on genomically heterogeneous breast cancers and may select for alterations in genes associated with drug resistance or metastasis. We evaluated this hypothesis by comparing sequential primary, post-neoadjuvant chemotherapy, and recurrent metastatic tumor samples from 5 patients with aggressive hormone receptor positive (HR+) breast cancer. Triplet samples (primary untreated, primary post-neoadjuvant chemotherapy, metastatic recurrence) were assessed in 5 patients with HR+ breast cancer. All patients were treated with cyclophosphamide and doxorubicin followed by paclitaxel or docetaxel, and adjuvant radiation and hormonal therapy. They all recurred during adjuvant antiestrogen treatment (12-43 months post-surgery). Metastatic sites included liver (2), ovary, brain, and pleural fluid. In these 5 tumor sets, we queried genomic alterations across 3,769 exons from 236 cancer genes using the FoundationOne™ NGS assay. Gene amplifications were frequently discordant among the triplet biopsies; all patients showed copy number alterations and 3 of 5 patients had two or more additional gene amplifications confirmed only in the biopsy of the metastasis. ESR1 (ERa) mutations were identified in the metastatic lesions of 2/5 patients but not in the diagnostic biopsy or the post-neoadjuvant therapy mastectomy specimen; both also harbored de novo and concurrent GATA3 truncations. One of the ESR1 mutations, Y537S, is known to result in an estrogen-independent, transcriptionally active receptor. The same patient showed amplification of 11q13 genes CCND1, EMSY, and FGF19, an amplicon known to be associated with endocrine resistance. Concordant FGFR1 and ZNF703 amplifications, both at 8p12, were observed de novo in 2 other patients; these alterations have beenassociated with the luminal B subtype and resistance to antiestrogens. FGFR1/ZNF703 alterations were mutually exclusive with ESR1 mutations. The 5th tumor contained a PIK3CA ‘hot spot’ mutation in all 3 sequential biopsies. This tumor also exhibited IGF1R, IRS2, MYC and MCL1 gene amplification only in the metastasis. Overall, IGF1R amplifications were observed in 3/5 patients. In addition, amplification of ZNF217 at 20q13.2, an amplicon with known association with poor prognosis, was observed de novo in 2 patients that exhibited rapid recurrence on adjuvant therapy (12 and 17 months). Acquired somatic alterations that deregulate ERα signaling may allow for escape from estrogen deprivation leading to treatment resistance. We speculate ESR1 mutations, rarely observed in primary breast tumors, are selected for during adjuvant hormonal treatment in aggressive HR+ breast cancer. Testing for de novo and acquired alterations throughout the clinical course of patients with ER+ cancers may identify targetable alterations, such as FGFR1 and IGF1R gene amplifications and others, against which targeted drugs are available. Studies are underway to validate these findings in additional patients with sequential samples and to investigate the function and therapeutic implications of ESR1 mutations. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr PD3-1.

Fibroblast growth factor receptor 1 amplification is a common event in squamous cell carcinoma of the head and neck

Recently, we characterized fibroblast growth factor receptor 1 amplification as a target for a rational therapy in lung squamous cell carcinoma. Patients harboring this genetic event are currently eligible for treatment with antifibroblast growth factor receptor small-molecule inhibitors in phase I clinical trials. This has the potential to significantly improve standard therapy for lung squamous cell carcinoma patients. The aim of this study was to elucidate whether fibroblast growth factor receptor 1 amplification is also a common genetic event in head and neck squamous cell carcinoma. For this purpose, we assembled a cohort of 555 patients, including 264 with metastatic disease and 147 with recurrent disease. Formalin-fixed, paraffin-embedded material of primary tumors, metastases and recurrences were assessed for fibroblast growth factor receptor 1 copy number status using fluorescence in situ hybridization. Human papilloma virus status was detected by p16 immunohistochemistry staining and PCR-ELISA. Molecular parameters were correlated with each other and with clinicopathological data. We found 15% of primary head and neck squamous cell carcinoma to display a fibroblast growth factor receptor 1 amplification. In nearly all cases, metastatic and recurrent tumor samples shared the same amplification status as the corresponding primary tumor. Fibroblast growth factor receptor 1 amplification was associated with nicotine and alcohol consumption, but was mutually exclusive with human papilloma virus infection. Amplification of the gene was associated with parameters of worse outcome. Our data identify fibroblast growth factor receptor 1 amplification as a frequent event in primary and metastatic head and neck squamous cell carcinoma and represents a potential biomarker for more aggressive disease. Fibroblast growth factor receptor 1-amplified tumors could potentially comprise a subset of head and neck squamous cell carcinoma against which targeted small-molecule inhibitors hold therapeutic efficacy.

Relationship of microRNA expression profiles and recurrence in advanced serous ovarian carcinoma.

5557 Background: Most patients with epithelial ovarian cancer eventually succumb to chemo-resistant disease. Although microRNAs have been recognized as important regulators of gene expression, little is known about microRNA expression profiles in recurrent serous ovarian carcinoma. We assessed the microRNA expression profiles which contribute to recurrence in advanced serous ovarian carcinoma. Methods: Eight pairs of primary and recurrent tumor samples from 8 patients with advanced serous ovarian carcinoma and 4 normal ovarian samples from patients treated for benign uterine disease between May 2006 and Dec 2012 were examined using miRNA microarray. microRNA profiling were validated using real-time reverse transcription-polymerase chain reaction (RT-PCR). Results: Alterations of microRNA expression profiles in primary and recurrent tumor samples have similar patterns when compared with normal ovarian tissues. Among 31 up-regulated microRNAs more than 4-fold in primary tumors, 27 microRNAs were also significantly up-regulated in recurrent tumor samples. Likewise, Among 35 down-regulated microRNAs more than 4-fold in primary tumors, 34 microRNAs were also significantly down-regulated in recurrent tumor samples. Comparing to primary tumor, we found 60 microRNAs which were significantly up-regulated, including miR-630, 370, and 575, and 52 microRNAs which were significantly down-regulated, including miR-509-3p, 514a-3p, and 506-3p in recurrent serous ovarian carcinoma. Conclusions: Our results indicate that dysregulation of microRNAs may play a role in recurrence of serous ovarian carcinoma.

Abstract 58: Differences in genomic alterations revealed by sequencing of 182 genes in recurrent ovarian cancer specimens compared to TCGA analysis: rearrangements in PTCH1 and FLT3; high frequency of RAS pathway alteration.

Abstract Recent comprehensive genomic analyses of advanced stage, high grade serous ovarian carcinomas carried out by TCGA (Nature 274: 609-615, 2011) have revealed their heterogeneous nature and provide an explanation for the failure of molecularly targeted drugs evaluated without consideration of the tumor molecular characteristics of the patients treated. Copy number (CN) alterations or mutations in RB, RAS-MAPK, and PI3K-AKT pathway genes occur in ∼35%, 20%, and 35% of the tumors, respectively. Activation of these pathways may confer sensitivity to drugs that target them (e.g., PI3K or AKT inhibitors, MEK inhibitors, CDK inhibitors) as well as resistance to drugs (e.g., HER2 or EGFR inhibitors) that have failed in ovarian cancer clinical trials. A comprehensive evaluation of the genetic aberrations in targetable pathways can therefore inform the selection of appropriate drugs and combinations for each patient in the context of clinical trials. As part of molecular profiling analyses performed for recurrent ovarian cancer patients to inform selection of their next treatment, the presence of mutations or alterations (e.g., CN changes) in 182 genes that encode proteins that are key mediators in oncogenic and tumor suppressive pathways were determined using a validated exon-capture sequencing platform provided in a CLIA-certified laboratory setting. 20 serous (4 primary/16 recurrent) and 4 recurrent mixed or pure clear cell ovarian tumor samples were analyzed. A median of 2.5 (range 0-7) alterations were detected in the serous histology specimens; frequencies of p53 mutations (16; 80%) and amplifications in CCND1 (1; 5%), CCND2 (1; 5%), CCNE1 (2; 10%) are consistent with those observed in the TCGA tumors. In contrast, more frequent RAS pathway aberrations (45% vs. 20%) corresponding to KRAS amplification (3;15%) and mutations/truncations in NF1 (5; 20%) or NF2 (1; 5%) were observed, possibly reflecting the recurrent nature of these samples (TCGA used primary tumors). Only one PIK3CA amplification was detected (i.e., 5%) in contrast to the 18% observed in the TCGA dataset. Amplifications of IGF1R (3; 15%) and ERBB2 (1;5%) were also detected in the serous histology tumors, while PIK3CA and KRAS mutations were only detected in the tumors with mixed cc and endometrioid histologies. Interestingly, a predicted protein-truncating rearrangement in PTCH1 and a kinase-activating exon duplication in FLT3 were detected in tumors from single patients suggesting drugs targeting the HH pathway and FLT3 as options for ovarian cancer patients. While it is important to increase the number of samples analyzed, these data suggest that selection of patients for clinical trials should be informed by comprehensive molecular characterization of recurrent tumor specimens. Citation Format: Deborah A. Zajchowski, Doron Lipson, Gary Palmer, Laura K. Shawver. Differences in genomic alterations revealed by sequencing of 182 genes in recurrent ovarian cancer specimens compared to TCGA analysis: rearrangements in PTCH1 and FLT3; high frequency of RAS pathway alteration. [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 58. doi:10.1158/1538-7445.AM2013-58