High-level Gene Amplification Research Articles

Abstract RF01-04: Final results from the phase 2, open-label FOENIX-MBC2 study: efficacy and safety of futibatinib in adult patients with locally advanced/metastatic HR+/HER2− breast cancer harboring high-level FGFR1 gene amplification

Abstract Background: Fibroblast growth factor receptor 1 (FGFR1) gene amplification and overexpression is associated with an adverse prognosis in hormone receptor–positive (HR+)/human epidermal growth factor receptor 2–negative (HER2−) breast cancer and is observed in ~10% of all invasive breast cancers. The phase 2 FOENIX-MBC2 study (NCT04024436) was designed to evaluate the effect of futibatinib, a highly selective and potent irreversible covalent inhibitor of FGFR1–4 (FDA-approved for intrahepatic cholangiocarcinoma), used either alone or in combination with fulvestrant in patients with metastatic breast cancer. Here, we report final efficacy and safety data for the cohort of patients receiving futibatinib plus fulvestrant for HR+/HER2− breast cancer harboring high-level FGFR1 gene amplification. Methods: Patients were eligible if they had disease progression after prior therapy for advanced/metastatic disease, had measurable disease per RECIST v1.1, had an ECOG performance status of 0 or 1, were fulvestrant-naïve, and had previously received 1–2 endocrine-containing therapies, ≤1 chemotherapy regimen, and a cyclin-dependent kinase 4/6 (CDK4/6) inhibitor for advanced cancer (unless ineligible). High-level FGFR1 gene amplification, determined in tumor tissue using next-generation sequencing, fluorescence in situ hybridization (FISH), or similar assays, was defined as FGFR1/centromere 8 ratio ≥5 or FGFR1 copy number ≥10 signals per cell. Local FGFR1 determination results were confirmed in tumor tissue by a central laboratory using FISH. Patients received oral futibatinib 20 mg once daily and standard fulvestrant dosing until disease progression, unacceptable toxicity, or other discontinuation criteria were met. The primary endpoint was the 6-month progression-free survival (PFS) rate. Key secondary endpoints included objective response rate (ORR), PFS, and overall safety. Results: Overall, 22 female patients were enrolled in this cohort. Patients were a median age of 58 years, had received a median of 3 lines of any prior systemic anticancer therapy, and had all received CDK4/6 inhibitor pretreatment. PFS at 6 months was observed in 10 (45.5%) patients (95% CI: 24.4, 67.8) and the median PFS was 7.2 (95% confidence interval [CI]: 2.1, 7.6) months. Four patients had a confirmed partial response (ORR: 18.2%; 95% CI: 5.2, 40.3). The median duration of response was 6.3 (range: 3.3–12.8) months. All patients had ≥1 treatment-related adverse event (TRAE), the most common being hyperphosphatemia (95.5%), alopecia (54.5%), constipation (45.5%), and dry mouth (40.9%). There were 5 (22.7%) patients with a Grade 3 TRAE (no Grade 4 or 5). TRAEs leading to study treatment interruption or reduction were seen in 9 (40.9%) and 15 (68.2%) patients, respectively. There were 2 (9.1%) patients who had TRAEs leading to study treatment discontinuation. No treatment-related serious adverse events were reported. Conclusions: Futibatinib plus fulvestrant showed antitumor activity in patients with advanced HR+/HER2− breast cancer with FGFR1 amplification progressing on prior CDK4/6 inhibitors, with a numerically higher ORR and doubling in PFS relative to historical fulvestrant results in post-CDK4/6 patients. The safety profile was consistent with those of the individual study drugs. Further biomarker work is ongoing. Citation Format: Senthil Damodaran, Fabrice André, Nisha Unni, Marta Ferreira, Karthik Giridhar, Brooke Daniel, Marco Colleoni, Luis Costa, Thomas Bachelot, Ciara O’Brien, Gail Wright, Masashi Shimura, Gareth Tomlinson, Maciej Gil, Nicholas Turner. Final results from the phase 2, open-label FOENIX-MBC2 study: efficacy and safety of futibatinib in adult patients with locally advanced/metastatic HR+/HER2− breast cancer harboring high-level FGFR1 gene amplification [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr RF01-04.

Urokinase-Type Plasminogen Activator Receptor (uPAR) Cooperates with Mutated KRAS in Regulating Cellular Plasticity and Gemcitabine Response in Pancreatic Adenocarcinomas.

Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal cancers. Given the currently limited therapeutic options, the definition of molecular subgroups with the development of tailored therapies remains the most promising strategy. Patients with high-level gene amplification of urokinase plasminogen activator receptor (uPAR/PLAUR) have an inferior prognosis. We analyzed the uPAR function in PDAC to understand this understudied PDAC subgroup's biology better. A total of 67 PDAC samples with clinical follow-up and TCGA gene expression data from 316 patients were used for prognostic correlations. Gene silencing by CRISPR/Cas9, as well as transfection of uPAR and mutated KRAS, were used in PDAC cell lines (AsPC-1, PANC-1, BxPC3) treated with gemcitabine to study the impact of these two molecules on cellular function and chemoresponse. HNF1A and KRT81 were surrogate markers for the exocrine-like and quasi-mesenchymal subgroup of PDAC, respectively. High levels of uPAR were correlated with significantly shorter survival in PDAC, especially in the subgroup of HNF1A-positive exocrine-like tumors. uPAR knockout by CRISPR/Cas9 resulted in activation of FAK, CDC42, and p38, upregulation of epithelial makers, decreased cell growth and motility, and resistance against gemcitabine that could be reversed by re-expression of uPAR. Silencing of KRAS in AsPC1 using siRNAs reduced uPAR levels significantly, and transfection of mutated KRAS in BxPC-3 cells rendered the cell more mesenchymal and increased sensitivity towards gemcitabine. Activation of uPAR is a potent negative prognostic factor in PDAC. uPAR and KRAS cooperate in switching the tumor from a dormant epithelial to an active mesenchymal state, which likely explains the poor prognosis of PDAC with high uPAR. At the same time, the active mesenchymal state is more vulnerable to gemcitabine. Strategies targeting either KRAS or uPAR should consider this potential tumor-escape mechanism.

EP08.02-145 An Efficacy Analysis of Advanced Non Small Cell Lung Cancer With MET Exon 14 Skipping in the Real World

Lung cancers harboring mesenchymal-to-epithelial transition factor (MET) genetic alterations, such as exon 14 skipping mutations or high-level gene amplification, respond well to METselective tyrosine kinase inhibitors (TKI). Crizotinib is approved for use in patients with non-small cell lung cancer with MET14 mutations. However, there is a lack of real-world data to support the efficacy of crizotinib.

A Biparatopic Antibody-Drug Conjugate to Treat MET-Expressing Cancers, Including Those that Are Unresponsive to MET Pathway Blockade.

Lung cancers harboring mesenchymal-to-epithelial transition factor (MET) genetic alterations, such as exon 14 skipping mutations or high-level gene amplification, respond well to MET-selective tyrosine kinase inhibitors (TKI). However, these agents benefit a relatively small group of patients (4%-5% of lung cancers), and acquired resistance limits response durability. An antibody-drug conjugate (ADC) targeting MET might enable effective treatment of MET-overexpressing tumors (approximately 25% of lung cancers) that do not respond to MET targeted therapies. Using a protease-cleavable linker, we conjugated a biparatopic METxMET antibody to a maytansinoid payload to generate a MET ADC (METxMET-M114). METxMET-M114 promotes substantial and durable tumor regression in xenografts with moderate to high MET expression, including models that exhibit innate or acquired resistance to MET blockers. Positron emission tomography (PET) studies show that tumor uptake of radiolabeled METxMET antibody correlates with MET expression levels and METxMET-M114 efficacy. In a cynomolgus monkey toxicology study, METxMET-M114 was well tolerated at a dose that provides circulating drug concentrations that are sufficient for maximal antitumor activity in mouse models. Our findings suggest that METxMET-M114, which takes advantage of the unique trafficking properties of our METxMET antibody, is a promising candidate for the treatment of MET-overexpressing tumors, with the potential to address some of the limitations faced by the MET function blockers currently in clinical use.

High-level gain of mesenchymal-epithelial transition factor (MET) copy number using next-generation sequencing as a predictive biomarker for MET inhibitor efficacy.

BackgroundIn clinical oncology, targeted next-generation sequencing (NGS) has become an integral part of the routine molecular diagnostics repertoire. However, a consensus is yet to be agreed on the optimal mesenchymal-epithelial transition factor (MET) copy number (CN) cut-off value based on NGS data that could predict the MET-amplified non-small cell lung cancer (NSCLC) patients who could benefit from MET tyrosine kinase inhibitor (TKI) therapy. In this study, we aimed to identify the criteria to define MET amplification derived from NGS data.MethodsSequencing data from matched plasma and tissue samples from 40 MET-amplified NSCLC patients were used to derive a normalization method, referred to as adjusted copy number (adCN). Clinical outcomes from an additional 18 MET TKI-treated NSCLC patients with solely MET-amplified cancers were analyzed to validate the adCN cut-offs.ResultsAdCN, calculated as the absolute CN generated from NGS relative to the maximum mutant allele fraction (maxMAF) per sample, was demonstrated to have a high correlation with MET CN in tissue and plasma samples (R2=0.73). Using a cut-off value of 5.5 and 13, tertile stratification of adCN was able to distinguish patients with high-level MET amplification. The MET TKI-treated patients with adCN >13, categorized as high-level amplification, had significantly longer progression-free survival (PFS) than those with adCN <13 (P=0.009), suggesting that adCN positively correlated with the response to MET TKI.ConclusionsWe derived a normalization method that could reflect the relative CN and distinguish MET-amplified NSCLC patients with high-level gene amplification who were sensitive to crizotinib, suggesting adCN could potentially serve as a predictive biomarker for MET TKI response.

Recommendations for the clinical interpretation and reporting of copy number gains using gene panel NGS analysis in routine diagnostics

Next-generation sequencing (NGS) panel analysis on DNA from formalin-fixed paraffin-embedded (FFPE) tissue is increasingly used to also identify actionable copy number gains (gene amplifications) in addition to sequence variants. While guidelines for the reporting of sequence variants are available, guidance with respect to reporting copy number gains from gene-panel NGS data is limited. Here, we report on Dutch consensus recommendations obtained in the context of the national Predictive Analysis for THerapy (PATH) project, which aims to optimize and harmonize routine diagnostics in molecular pathology. We briefly discuss two common approaches to detect gene copy number gains from NGS data, i.e., the relative coverage and B-allele frequencies. In addition, we provide recommendations for reporting gene copy gains for clinical purposes. In addition to general QC metrics associated with NGS in routine diagnostics, it is recommended to include clinically relevant quantitative parameters of copy number gains in the clinical report, such as (i) relative coverage and estimated copy numbers in neoplastic cells, (ii) statistical scores to show significance (e.g., z-scores), and (iii) the sensitivity of the assay and restrictions of NGS-based detection of copy number gains. Collectively, this information can guide clinical and analytical decisions such as the reliable detection of high-level gene amplifications and the requirement for additional in situ assays in case of borderline results or limited sensitivity.

Abstract P2-05-03: Novel driver genetic alterations in MYB-NFIB-negative breast adenoid cystic carcinomas

Abstract Introduction: Breast adenoid cystic carcinoma (AdCC) is a rare type of triple-negative breast cancer associated with an indolent clinical behavior. AdCCs provide a clear example of genotypic-phenotypic correlation with the majority harboring the MYB-NFIB fusion gene. In this study, we sought to identify alternative driver genetic alterations in breast AdCCs lacking the MYB-NFIB fusion gene. Methods: Nucleic acids obtained from four breast AdCCs lacking the MYB-NFIB fusion gene as defined by reverse transcription (RT)-PCR and/or fluorescence in situ hybridization (FISH) were subjected to RNA-sequencing (n=3), whole-genome (n=2) and/or targeted (n=1) massively parallel sequencing. Sequencing data were analyzed using state-of-the-art bioinformatics algorithms, and potential alternative driver genetic alterations were validated using orthogonal sequencing and molecular pathology methods. Results: RNA-sequencing revealed the presence of MYBL1-ACTN1 or MYBL1-NFIB fusion genes in two breast AdCCs, which were validated by whole-genome sequencing and/or MYBL1 FISH analysis. Both MYBL1 fusion gene-positive cases were found to overexpress MYBL1 as defined by quantitative RT-PCR analysis. In the third MYB-NFIB-negative breast AdCC studied, a high-level MYB gene amplification coupled with overexpression of MYB at the mRNA and protein levels was identified. In the fourth breast AdCC, which expressed high levels of MYB, whole-genome and RNA-sequencing revealed no definite alternative driver alteration, however, a MYBL2 intronic mutation was found in this case, which was associated with high levels of MYBL2 mRNA expression. In this case, single sample gene set enrichment analysis revealed activation of pathways similar to those activated in AdCCs harboring the MYB-NFIB or MYBL1 fusions genes. Conclusion: We demonstrate that in breast AdCCs lacking the MYB-NFIB fusion gene MYBL1 rearrangements and MYB amplification are likely alternative driver genetic events. Given that activation of MYB/MYBL1 and their downstream targets can be driven by the MYB-NFIB fusion gene, MYBL1 rearrangements, MYB amplification or other yet to be validated mechanisms (e.g. MYBL2 non-coding mutations), our findings further suggest that breast AdCCs constitute a convergent phenotype. Citation Format: Kim J, Geyer FC, Martelotto LG, Ng CKY, Lim RS, Selenica P, Li A, Pareja F, Fusco N, Edelweiss M, Mariani O, Badve S, Vincent-Salomon A, Norton L, Reis-Filho JS, Weigelt B. Novel driver genetic alterations in MYB-NFIB-negative breast adenoid cystic carcinomas [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-05-03.

The frequency and clinical impact of HER2 alterations in lung adenocarcinoma.

Human epidermal growth factor receptor 2 (HER2 or ErbB2) can be overexpressed, amplified and/or mutated in malignant tumors, and is a candidate for therapeutic targeting. However, molecular associations and clinical significances of these alterations were controversial in lung cancer. In this study, we investigated the frequency and clinicopathological significance of HER2 dysregulation in patients with lung adenocarcinoma. HER2 protein overexpression, gene amplification, and gene mutation were evaluated by immunohistochemistry (IHC), silver in situ hybridization, and direct sequencing, respectively. The H-scoring method and American Society of Clinical Oncology/College of American Pathologists breast cancer guidelines were used to interpret IHC results. Genetic analyses of EGFR and KRAS mutations, and of ALK and ROS1 rearrangements, were also performed. Of the 321 adenocarcinoma patients identified, HER2 overexpression (H-score ≥200) and gene amplification were found in 6 (1.9%) and 46 (14.3%), respectively. HER2 overexpression was correlated with papillary predominant histology; furthermore, it indicated poor overall survival and was an independent prognostic factor. HER2 amplification was associated with pleural invasion and showed a tendency towards shorter overall and disease-free survival. High-level gene amplification (HER2/CEP17 ratio ≥5 or copy number ≥10) was a poor prognostic factor for disease-free survival. HER2 mutations were detected in 6.7% (7 of 104) of driver oncogene-negative adenocarcinomas. Our study suggests that HER2 overexpression or amplification is a poor prognostic factor in lung adenocarcinoma, although the frequency of such events is low. Since molecular targeted agents are being tested in clinical trials, awareness of the specific HER2 status can influence the prognostic stratification and treatment of patients with molecularly defined subsets of lung adenocarcinoma.

CNS germinomas are characterized by global demethylation, chromosomal instability and mutational activation of the Kit-, Ras/Raf/Erk- and Akt-pathways.

CNS germinomas represent a unique germ cell tumor entity characterized by undifferentiated tumor cells and a high response rate to current treatment protocols. Limited information is available on their underlying genomic, epigenetic and biological alterations. We performed a genome-wide analysis of genomic copy number alterations in 49 CNS germinomas by molecular inversion profiling. In addition, CpG dinucleotide methylation was studied by immunohistochemistry for methylated cytosine residues. Mutational analysis was performed by resequencing of candidate genes including KIT and RAS family members. Ras/Erk and Akt pathway activation was analyzed by immunostaining with antibodies against phospho-Erk, phosho-Akt, phospho-mTOR and phospho-S6. All germinomas coexpressed Oct4 and Kit but showed an extensive global DNA demethylation compared to other tumors and normal tissues. Molecular inversion profiling showed predominant genomic instability in all tumors with a high frequency of regional gains and losses including high level gene amplifications. Activating mutations of KIT exons 11, 13, and 17 as well as a case with genomic KIT amplification and activating mutations or amplifications of RAS gene family members including KRAS, NRAS and RRAS2 indicated mutational activation of crucial signaling pathways. Co-activation of Ras/Erk and Akt pathways was present in 83% of germinomas. These data suggest that CNS germinoma cells display a demethylated nuclear DNA similar to primordial germ cells in early development. This finding has a striking coincidence with extensive genomic instability. In addition, mutational activation of Kit-, Ras/Raf/Erk- and Akt- pathways indicate the biological importance of these pathways and their components as potential targets for therapy.

Reduced cohesin destabilizes high-level gene amplification by disrupting pre-replication complex bindings in human cancers with chromosomal instability.

Gene amplification is a hallmark of cancer with chromosomal instability although the underlying mechanism by which altered copy numbers are maintained is largely unclear. Cohesin, involved in sister chromatid cohesion, DNA repair, cell cycle progression and transcriptional regulation of key developmental genes, is frequently overexpressed in human cancer. Here we show that cohesin-dependent change in DNA replication controls the copy numbers of amplified genes in cancer cells with chromosomal instability. We found that the down-regulation of elevated cohesin leads to copy number-associated gene expression changes without disturbing chromosomal segregation. Highly amplified genes form typical long-range chromatin interactions, which are stabilized by enriched cohesin. The spatial proximities among cohesin binding sites within amplified genes are decreased by RAD21-knockdown, resulting in the rapid decline of amplified gene expression. After several passages, cohesin depletion inhibits DNA replication initiation by reducing the recruitment of pre-replication complexes such as minichromosome maintenance subunits 7 (MCM7), DNA polymerase α, and CDC45 at replication origins near the amplified regions, and as a result, decreases the DNA copy numbers of highly amplified genes. Collectively, our data demonstrate that cohesin-mediated chromatin organization and DNA replication are important for stabilizing gene amplification in cancer cells with chromosomal instability.

Abstract P5-10-18: The chromosomal genomic landscape and targetable co-amplified genes in HER2 positive breast cancer patients who relapsed on an adjuvant trastuzumab chemotherapy trial

Abstract Background Early stage HER2 amplified breast cancer has a generally favorable prognosis with over 95% of patients showing 2-year disease free survival (DFS) when treated with adjuvant trastuzumab. However, a subset of these tumors are refractory to treatment and present a challenge for the oncologist, particularly when clinical and histologic parameters, including the patient’s nodal and hormonal status, are indicative of lower-risk HER2 positive disease. In this study we describe the genomic landscape of three clinically lower-risk patients with HER2 amplified tumors who relapsed on adjuvant docetaxel and cyclophosphamide plus 1 year of trastuzumab in a phase 2 study (Jones et al, Lancet Oncology 14: 1121, 2013). All patients’ tumors showed high-level HER2 amplification ratios by FISH (8.51-14.46) and were analyzed in parallel with a fourth clinically matched 2-year DFS patient’s tumor from the same trial with high HER2 gene amplification (FISH ratio 11.38). Methods Primary tumor genomic DNA analysis was performed from archival tissue by next generation sequencing (NGS) on the Illumina HiSeq 2500 platform in a CLIA certified laboratory. Tumors were screened for point mutations and copy number alterations (CNAs) by NGS using a targeted-whole exome 613 gene panel. CNAs detected by NGS were confirmed on a DNA microarray featuring high-density probe coverage of the same 613 genes on the targeted panel. CGH chromosome ratio plots were overlaid with algorithmically derived NGS copy number data to generate a map of the chromosomal genomic landscape for each patient’s tumor. Results High-level HER2 gene amplification status was confirmed, and co-amplified chromosome 17 genomic regions were detected in all three relapsed patients’ tumors. High-level HER2 amplification was also confirmed in the non-relapsed patient’s tumor but co-amplified regions were not detected on chromosome 17 or elsewhere in this patient’s tumor genome. Two of the relapsed ER negative tumors shared focal high-level CCNE1 gene amplifications on chromosome 19. High-level MAP3K3 gene amplification on chromosome 17 was detected in the one ER positive tumor from a relapsed patient. Focal PIK3CA gene amplifications were not identified in any of these tumors, but two tumors (one from the relapse group and one non-relapse) were positive for activating H1047R mutations. Conclusions Combined NGS and CGH analysis of HER2 positive early stage breast cancer can be performed in the clinical laboratory to reveal the tumor’s chromosomal genomic landscape. Combined with other test results, this tumor map can help identify patients at high-risk for relapse and reveal alternative predictive biomarkers of therapeutic response. Citation Format: Shelly Gunn, Chris McCaskill, Linda Daley, Agnes Puskas, Lina Asmar, Yunfei Wang, Stephen Jones. The chromosomal genomic landscape and targetable co-amplified genes in HER2 positive breast cancer patients who relapsed on an adjuvant trastuzumab chemotherapy trial [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 P5-10-18.

Non-small cell lung cancer: HER2 oncogene status

to study HER2 protein and HER2 gene, their heterogeneity in non-small cell lung cancer. 218 intraoperative non-small cell lung samples were examined using tissue matrix methods. HER2 protein was determined by immunohistochemistry (clone 4B5, <<Ventana>> and HER2 gene and CEP1 7 were evaluated by in situ hybridization (SISH, <<Ventana>>). Positive and indefinite statuses were found in 59 (27%) and 47 (22%) cases, respectively; intratumor heterogeneity was detected in 32 (30%) cases. Amplification of the HER-2 gene was found in 12 (6%) cases; that of the HER2 gene along with an increase in CEPI 7 was observed in 7 (3%) cases; elevated CEP1 7 levels were seen in 19 (9%) cases. Intratumor heterogeneity of HER2 gene amplification was not found; however, one case of adenocarcinoma showed high-level HER2 gene amplification in the gland-like areas and low-level HER2 gene amplification in the solid areas. HER2-positive status and amplification were more common in adenocarcinoma than in squamous cell carcinoma (p<0.001). There was a moderate correlation between HER2 immunohistochemical status and amplification (r=0.38; p<0.001). Thus, in non-small cell lung cancer, there is an elevated HER2 protein level and, well less frequently, altered activity in the HER2 gene (amplification) as a cause of enhanced protein synthesis.

Analysis of gene copy number alterations by multiplex ligation-dependent probe amplification in columnar cell lesions of the breast

Columnar cell lesions (CCLs) are possible precursors of breast cancer, but little is known about the role of breast cancer-related genes in the progression of CCL to invasive breast cancer. Gene copy numbers of 17 breast cancer-related genes were analyzed using Multiplex Ligation-dependent Probe Amplification (MLPA) in CCL (N = 28), ductal carcinoma in situ (DCIS) grade I likely originating from CCL (N = 5), and paired CCL (N = 14/28) with DCIS (N = 7) and/or invasive carcinoma (N = 13). The genes included were BIRC5, C11orf30, CCND1, CCNE1, CDH1, CPD, EGFR, ERBB2, ESR1, FGFR1, IKBKB, MAPT, MED1, MTDH, MYC, TOP2A and TRAF4. No high level gene amplifications were observed in CCL, but copy number gains were encountered for the C11orf30 (3/28), MYC, CPD, MTDH (2/28), and CCND1, CCNE1, ESR1 and TOP2A genes (1/28). In addition, CDH1 showed loss in 2/28 and TOP2A in 1/28 cases. CCLs with or without atypia exhibited comparable numbers of copy number changes (p = 0.312). Overall, the frequency of gene copy number changes increased from CCL towards DCIS and invasive carcinoma (p = 0.004). Also in the cases with synchronous lesions, the CCLs exhibited fewer copy number changes than the DCIS/invasive carcinomas. CCLs carry copy number changes of several known breast cancer-related genes, thereby substantiating their role in breast carcinogenesis. Among them, CCND1 and ESR1 copy number gains and CDH1 copy number losses are of particular interest. Since the copy number changes observed were more prevalent in DCIS and invasive carcinoma than in CCL, the corresponding gene alterations may represent rather late occurring events in low nuclear grade breast carcinogenesis.

MYC amplification and overexpression in primary cutaneous angiosarcoma: a fluorescence in-situ hybridization and immunohistochemical study

MYC, a proto-oncogene located on chromosome 8q24, is involved in the control of cell proliferation and differentiation. Previous studies have documented high-level MYC gene amplification and MYC overexpression by immunohistochemistry (IHC) in post-irradiation angiosarcomas, but not in primary cutaneous angiosarcoma (AS-C) or in other radiation-associated vascular proliferations, such as atypical vascular lesions. Prompted by our recent finding of MYC amplification in a primary hepatic AS, we analyzed a large number of well-characterized AS-C for MYC amplification and protein overexpression. Formalin-fixed, paraffin-embedded blocks from 38 AS-C were retrieved from our archives and were examined by IHC analysis and fluorescence in-situ hybridization (FISH), using a commercially available antibody and probe. For FISH analysis, the number of copies of MYC was compared with the control gene, CEN8 (MYC/CEN8 ratio). All cases occurred on sun-exposed skin; no patient was known to have a history of therapeutic irradiation. Possible associations between survival and a wide variety of clinicopathological variables were evaluated using the log-rank test. By IHC analysis, MYC overexpression was present in 9/38 (24%) AS-C (2–3+: 6 cases, 16%; 1+: 3 cases, 8%). By FISH analysis, 2/5 (40%) informative cases with 2–3+ immunostaining showed high-level gene amplification. One additional case with 3+ immunostaining showed higher level aneusomy of chromosome 8 (5–8 MYC and CEN8). Two out of fourteen (14%) IHC-negative cases also carried MYC amplification (one high level and one lower level). Low copy number gain of chromosome 8 (3–5 MYC and CEN8) was observed in AS-C with or without MYC expression. MYC amplification and MYC protein overexpression were not correlated with clinical outcome. We have shown, for the first time, MYC gene amplification and protein overexpression in primary (non-radiation-associated) AS of the skin. MYC protein overexpression in cases lacking gene amplification likely reflects other mechanisms of MYC activation. The study of a larger number of AS-C showing MYC amplification may be necessary to determine whether the behavior of such cases differs from their more common non-amplified counterparts.

DDR2 mutations in lung cancer in adenocarcinoma and squamous cell carcinoma and association with MET activation.

8115 Background: The discoidin domain receptor tyrosine kinase 2 (DDR2) gene is mutated in a small subset of squamous cell carcinomas (SQCAs) of the lung. Targeting of DDR2 with the inhibitor dasatinib inhibits DDR2-mutated cell line growth and is being investigated in clinical trials for treatment of DDR2-mutated lung tumors. We investigated the frequency and type of DDR2 mutations and their association with other targetable growth factor pathway alterations in non-small cell lung carcinoma (NSCLC). Methods: DNA was extracted from macro-dissected FFPE sections of 105 advanced stage lung carcinomas. Next-generation sequencing was performed using a 36-amplicon panel including the coding regions of DDR2 and exons 11 and 15 of BRAF. DDR2 mutations were confirmed by bidirectional Sanger sequencing. Lung tumors were classified using a standard immunohistochemistry panel. MET pathway activation was assessed with immunohistochemistry using an activation-specific phospho-antibody (pMET, Try1234/1235). MET amplification and ALK rearrangement were assessed using FISH. Mutational analysis for EGFR (exon 18-21), PIK3CA (exons 9 and 20), and KRAS(exons 1-2) was performed using PCR-based DNA sequencing. Results: Heterozygous DDR2 point mutations were identified in 7/105 (6.7%) NSCLCs, including 3/29 (10.3%) SQCAs and 4/69 (5.8%) ACAs, but not in neuroendocrine/large cell carcinomas (0/7). DDR2 mutations were scattered throughout the gene, with 4 present in the discoidin domain and 3 in the kinase domain. MET pathway activation, seen in 25% of DDR2-unmutated cases, was found in 3/6 (50%) DDR2-mutated cases, including 1 SQCA with high-level MET gene amplification. DDR2 mutations were mutually exclusive with BRAF or EGFR mutation or ALK rearrangement, with 1 KRAS-mutated case. Conclusions: DDR2 mutations occur in lung carcinomas with a range of histologic features and in association with MET activation. Dual targeting of the MET and DDR2 kinases in such cases may warrant further investigation.

Oligodendrogliomas

Since the discovery, in 1994, of recurrent codeletion of chromosome regions 1p36/19q13 in oligodendrogliomas, genetics has accomplished significant advances improving our knowledge in biology of this tumor type and our clinical management of oligodendroglioma patients. Indeed, 1p36/19q13 has been shown successively to predict increased chemosensitivity and better prognosis, to be associated with frontal location in brain and classic oligodendroglioma morphology, to be mutually exclusive with high-level gene amplification, to be actually whole chromosome arms 1p/19q codeletion, to mediate a t(1;19)(q10;p10) and to be associated with IDH mutations. More recently, pivotal studies, using high-throughput approaches, have provided significant novel insights in the molecular oncogenesis of oligodendrogliomas. Capicua homolog (Drosophila) (CIC) and Far Upstream element Binding Protein 1 (FUBP1) have been shown to be frequently mutated in 70 and 40% of 1p/19q codeleted oligodendrogliomas, respectively. The biological and clinical significance of these mutations remains unsettled. Additional recent studies have also demonstrated that 1p/19q codeleted oligodendrogliomas exhibit a proneural transcriptomic profile including overexpression of internexin alpha, a neuronal intermediate filament. Finally, 1p/19q codeleted and IDH-mutated tumors have been shown to be hypermethylated, suggesting a strong link between these both molecular alterations detected in the subgroup of oligodendrogliomas with better prognosis. Next-generation molecular biology technologies have recently identified recurrent CIC and FUBP1 point mutations in 1p/19q codeleted and IDH-mutated oligodendrogliomas. Their clinical and biological values are under investigation.

Rapid Detection of high-level oncogene amplifications in ultrasonic surgical aspirations of brain tumors

BackgroundGenomic tumor information, such as identification of amplified oncogenes, can be used to plan treatment. The two sources of a brain tumor that are commonly available include formalin-fixed, paraffin-embedded (FFPE) sections from the small diagnostic biopsy and the ultrasonic surgical aspiration that contains the bulk of the tumor. In research centers, frozen tissue of a brain tumor may also be available. This study compared ultrasonic surgical aspiration and FFPE specimens from the same brain tumors for retrieval of DNA and molecular assessment of amplified oncogenes.MethodsSurgical aspirations were centrifuged to separate erythrocytes from the tumor cells that predominantly formed large, overlying buffy coats. These were sampled to harvest nuclear pellets for DNA purification. Four glioblastomas, 2 lung carcinoma metastases, and an ependymoma were tested. An inexpensive PCR technique, multiplex ligation-dependent probe amplification (MLPA), quantified 79 oncogenes using 3 kits. Copy number (CN) results were normalized to DNA from non-neoplastic brain (NB) in calculated ratios, [tumor DNA]/[NB DNA]. Bland-Altman and Spearman rank correlative comparisons were determined. Regression analysis identified outliers.ResultsPurification of DNA from ultrasonic surgical aspirations was rapid (<3 days) versus FFPE (weeks) and yielded greater amounts in 6 of 7 tumors. Gene amplifications up to 15-fold corresponded closely between ultrasonic aspiration and FFPE assays in Bland-Altman analysis. Correlation coefficients ranged from 0.71 to 0.99 using 3 kit assays per tumor. Although normalized CN ratios greater than 2.0 were more numerous in FFPE specimens, some were found only in the ultrasonic surgical aspirations, consistent with tumor heterogeneity. Additionally, CN ratios revealed 9 high-level (≥ 6.0) gene amplifications in FFPE of which 8 were also detected in the ultrasonic aspirations at increased levels. The ultrasonic aspiration levels of these amplified genes were also greater than 6.0 CN ratio, except in one case (3.53 CN ratio). Ten of 17 mid-level (≥3.0 & <6.0 CN ratio) amplifications detected in FFPE were also detected as being increased (≥ 2.0 CN ratio) in the aspirations.ConclusionsBuffy coats of centrifuged ultrasonic aspirations contained abundant tumor cells whose DNA permitted rapid, multiplex detection of high-level oncogene amplifications that were confirmed in FFPE.Virtual slideshttp://www.diagnosticpathology.diagnomx.eu/vs/1883718801686466

Gene amplification ofESR1in breast cancers—fact or fiction? A fluorescencein situhybridization and multiplex ligation‐dependent probe amplification study

Oestrogen receptor-alpha (ERα), encoded by the ESR1 gene located on 6q25, is a nuclear transcription factor. Since it was reported in 2007 that more than 20% of breast cancers show ESR1 gene amplification, there has been considerable controversy about its frequency and clinical significance. We set out to assess the frequency and levels of ESR1 amplification in breast cancers. In a total of 106 breast needle biopsy specimens examined by immunohistochemistry, 78 tumours contained more than 10% ERα-positive cancer cells. In fluorescence in situ hybridization (FISH) analysis with an ESR1-specific probe, variously extended ESR1 signals were found in ERα-expressing cells. Some of these were indistinguishable from large clustered signals generally accepted to mean high-level gene amplification in homogeneously staining regions (HSRs), and could be considered to represent gene amplification. However, with RNase treatment, the 'HSR-like' signals changed to small compact signals, and are thus thought to represent concentrated RNA. FISH using two differently labelled probes corresponding to the non-overlapping 5'- and 3'-end portions of the ESR1 gene on touch smears showed a preserved spatial relationship of the 3' to 5' sequence of ESR1, therefore strongly suggesting that the RNA consisted of primary transcripts. Using touch smears obtained from 51 fresh tumours, precise enumeration of ESR1 signals with a correction by the number of centromere 6 on FISH after RNase A treatment revealed that three tumours (5.9%) had tumour cells with one to three additional copies of ESR1 as predominant subpopulations. This infrequent and low level of gene amplification of ESR1 was also detected as a 'gain' of the gene by analysis with multiplex ligation-dependent probe amplification (MLPA). The consistent results from immunohistochemistry, FISH, and MLPA in the present study settle the long-standing debate concerning gene amplification of ESR1 in breast carcinoma.

Gene amplification and protein overexpression of MET are common events in ovarian clear-cell adenocarcinoma: their roles in tumor progression and prognostication of the patient

The aim of this study was to assess protein overexpression and gene copy number alterations of MET in ovarian clear-cell adenocarcinoma, and to assess its potential as a novel therapeutic target. Ninety cases of clear-cell adenocarcinoma were analyzed for MET protein overexpression and copy number alterations of the MET gene by immunohistochemistry and brightfield double in situ hybridization, respectively. In addition, 101 cases of the non-clear-cell type ovarian carcinomas at advanced stages were also evaluated for comparison. MET overexpression was assigned when complete membrane staining with moderate or strong intensity was observed in at least 10% of the tumor cells examined. Double in situ hybridization was determined as positive when the tumor exhibited high-level polysomy (≥4 copies in ≥40% of tumor cells) or MET gene amplification. MET overexpression was detected in 20 of 90 clear-cell adenocarcinomas (22%) and none of 111 non-clear-cell type ovarian carcinomas. Double in situ hybridization was positive in 21 of 89 informative clear-cell adenocarcinomas (24%) and only 3 non-clear-cell type ovarian carcinomas (3%). In the whole population, true amplification of the MET gene was detected only in the clear-cell adenocarcinoma histology (five cases, 6%). In clear-cell adenocarcinomas, double in situ hybridization positivity was highly correlated with the presence of MET overexpression and a poorly differentiated histology of tumors (P=0.0105 and 0.00038, respectively). For the patients with clear-cell adenocarcinomas, MET overexpression, as well as advanced clinical stage and the poorly differentiated histology of tumors, was identified as an independent unfavorable prognostic factor for overall survival. In conclusion, among ovarian carcinomas, the amplification of the MET proto-oncogene is highly selective and commonly occurs in clear-cell adenocarcinoma. MET could serve as a biomarker for the prognostication of patients with clear-cell adenocarcinoma and tumor progression, and has potential as a novel therapeutic target for this carcinoma.

Exome Sequencing Reveals Comprehensive Genomic Alterations across Eight Cancer Cell Lines

It is well established that genomic alterations play an essential role in oncogenesis, disease progression, and response of tumors to therapeutic intervention. The advances of next-generation sequencing technologies (NGS) provide unprecedented capabilities to scan genomes for changes such as mutations, deletions, and alterations of chromosomal copy number. However, the cost of full-genome sequencing still prevents the routine application of NGS in many areas. Capturing and sequencing the coding exons of genes (the “exome”) can be a cost-effective approach for identifying changes that result in alteration of protein sequences. We applied an exome-sequencing technology (Roche Nimblegen capture paired with 454 sequencing) to identify sequence variation and mutations in eight commonly used cancer cell lines from a variety of tissue origins (A2780, A549, Colo205, GTL16, NCI-H661, MDA-MB468, PC3, and RD). We showed that this technology can accurately identify sequence variation, providing ∼95% concordance with Affymetrix SNP Array 6.0 performed on the same cell lines. Furthermore, we detected 19 of the 21 mutations reported in Sanger COSMIC database for these cell lines. We identified an average of 2,779 potential novel sequence variations/mutations per cell line, of which 1,904 were non-synonymous. Many non-synonymous changes were identified in kinases and known cancer-related genes. In addition we confirmed that the read-depth of exome sequence data can be used to estimate high-level gene amplifications and identify homologous deletions. In summary, we demonstrate that exome sequencing can be a reliable and cost-effective way for identifying alterations in cancer genomes, and we have generated a comprehensive catalogue of genomic alterations in coding regions of eight cancer cell lines. These findings could provide important insights into cancer pathways and mechanisms of resistance to anti-cancer therapies.