Saskia Ting

Different micro-RNA expression profiles distinguish subtypes of neuroendocrine tumors of the lung: results of a profiling study

MicroRNAs (miRNAs) are a class of small (∼22 nucleotides), non-coding, highly conserved single-stranded RNAs with posttranscriptional regulatory features, including the regulation of cell proliferation, differentiation, survival, and apoptosis. They are deregulated in a broad variety of tumors showing characteristic expression patterns and can, thus, be used as a diagnostic tool. In contrast to non-small cell carcinoma of the lung neuroendocrine lung tumors, encompassing typical and atypical carcinoids, small cell lung cancer and large cell neuroendocrine lung cancer, no data about deregulation of tumor entity-specific miRNAs are available to date. miRNA expression differences might give useful information about the biological characteristics of these tumors, as well as serve as helpful markers.In 12 pulmonary neuroendocrine tumors classified as either typical carcinoid, atypical, large cell neuroendocrine or small cell lung cancer, screening for 763 miRNAs known to be involved in pulmonary cancerogenesis was conducted by performing 384-well TaqMan low-density array real-time qPCR. In the entire cohort, 44 miRNAs were identified, which showed a significantly different miRNA expression. For 12 miRNAs, the difference was highly significant (P<0.01). Eight miRNAs showed a negative (miR-22, miR-29a, miR-29b, miR-29c, miR-367*; miR-504, miR-513C, miR-1200) and four miRNAs a positive (miR-18a, miR-15b*, miR-335*, miR-1201) correlation to the grade of tumor biology. The miRNAs let-7d; miR-19; miR-576-5p; miR-340*; miR-1286 are significantly associated with survival. Members of the miR-29 family seem to be extremely important in this group of tumors. We found a number of miRNAs, which showed a highly significant deregulation in pulmonary neuroendocrine tumors. Moreover, some of these deregulated miRNAs seem to allow discrimination of the various subtypes of pulmonary neuroendocrine tumors. Thus, the analysis of specific sets of miRNAs can be proposed as diagnostic and/or predictive markers in this group of neoplasias.

FFPE tissue as a feasible source for gene expression analysis--a comparison of three reference genes and one tumor marker.

BACKGROUND Formalin-fixation, paraffin-embedding is the standard processing technique for tumor tissue in modern pathology. New techniques such as cryo-conservation allow rapid fixation and long-time storage but come along with increased costs and enlarged storage complexity. However, formalin-fixed, paraffin-embedded (FFPE) tissue is available in a large quantity, making it the ideal material for retrospective studies. The following study was designed to investigate the influence of formalin-fixation on the quality of mRNA and applicability of FFPE-derived mRNA for gene expression analysis. Three potential reference genes for pulmonary tumors with neuroendocrine differentiation were included and tested for their robust expression. MATERIALS AND METHODS Eighty specimens collected from 2005 to 2012 at the Institute of Pathology and Neuropathology at the University Hospital Essen were analyzed for their gene expression by using TaqMan(®) gene expression assays on demand (AoD). Three distinct potential reference genes (ACTB, GAPDH, HPRT1) were evaluated for their expression, and a proteasome subunit (PSMA1) was included in the analysis as tumor marker and functioned as an internal technical control. CONCLUSION For GAPDH and ACTB, a highly significant correlation and consistent expression between the investigated entities was found, making them reliable reference genes for further research. Additionally, the feasibility for a FFPE tissue-based gene expression analysis was verified by showing that the mRNA quality is sufficient. When standardized FFPE preparation is performed carefully, sufficient mRNA can be isolated for reliable and successful gene expression analysis. That provides the basis the door for large, retrospective studies that correlate molecular and clinical follow-up data.

High Prevalence of Concomitant Oncogene Mutations in Prospectively Identified Patients with ROS1-Positive Metastatic Lung Cancer

Objectives: Chromosomal rearrangements involving ROS1 define a rare entity of lung adenocarcinomas with exquisite sensitivity to molecularly targeted therapy. We report clinical outcomes and genomic findings of patients with ROS1‐positive lung cancer who were prospectively identified within a multiplex biomarker profiling program at the West German Cancer Center. Methods: Standardized immunohistochemical (IHC) analysis, fluorescence in situ hybridization (FISH), and hotspot mutation analyses were performed in 1345 patients with advanced cancer, including 805 patients with metastatic lung adenocarcinoma. Clinical and epidemiological data were retrieved from the institutional database. Results: ROS1 positivity by IHC analysis was detected in 25 patients with lung cancer (4.8% of lung adenocarcinomas), including 13 patients (2.5%) with ROS1 FISH positivity with a cutoff of at least 15% of events. Of the ROS1 IHC analysis–positive cases, 36% presented with concomitant oncogenic driver mutations involving EGFR (six cases, five of which were clinically validated by response to EGFR‐targeting agents), KRAS (two cases), phosphatidylinositol‐4,5‐bisphosphate 3‐kinase catalytic subunit alpha gene (PIK3CA), and BRAF. Three cases initially classified as ROS1 FISH–negative passed the threshold of 15% positive events when repeat biopsies were analyzed at progression. The median overall survival of the ROS1‐positive patients (104 months) was significantly superior to that of the 261 patients with EGFR/anaplastic lymphoma kinase/ROS1–negative lung adenocarcinoma (24.4 months, p = 0.044). Interestingly, the overall survival of the 13 ROS1‐positive patients with lung cancer from initiation of pemetrexed‐based chemotherapy was significantly prolonged when compared with that of 169 pemetrexed‐treated patients with EGFR/anaplastic lymphoma kinase/ROS1–negative adenocarcinoma (p = 0.01). Conclusions: ROS1‐positive metastatic lung adenocarcinomas frequently harbor concomitant oncogenic driver mutations. Levels of ROS1 FISH–positive events are variable over time. This heterogeneity provides additional therapeutic options if discovered by multiplex biomarker testing and repeat biopsies.

Impact of human papilloma virus infection on the response of head and neck cancers to anti-epidermal growth factor receptor antibody therapy

Infection with human papillomaviruses (HPVs) characterizes a distinct subset of head and neck squamous cell cancers (HNSCCs). HPV-positive HNSCC preferentially affect the oropharynx and tonsils. Localized HPV-positive HNSCCs have a favorable prognosis and treatment outcome. However, the impact of HPV in advanced or metastatic HNSCC remains to be defined. In particular, it is unclear whether HPV modulates the response to cetuximab, an antibody targeting the epidermal growth factor receptor (EGFR), which is a mainstay of treatment of advanced HNSCC. To this end, we have examined the sensitivity of HPV-positive and -negative HNSCC models to cetuximab and cytotoxic drugs in vitro and in vivo. In addition, we have stably expressed the HPV oncogenes E6 and E7 in cetuximab-sensitive cancer cell lines to specifically investigate their role in the antibody response. The endogenous HPV status or the expression of HPV oncogenes had no significant impact on cetuximab-mediated suppression of EGFR signaling and proliferation in vitro. Cetuximab effectively inhibited the growth of E6- and E7-expressing tumors grafted in NOD/SCID mice. In support, formalin-fixed, paraffin-embedded tumor samples from cetuximab-treated patients with recurrent or metastatic HNSCC were probed for p16INK4a expression, an established biomarker of HPV infection. Response rates (45.5% versus 45.5%) and median progression-free survival (97 versus 92 days) following cetuximab-based therapy were similar in patients with p16INK4A-positive and p16INK4A-negative tumors. In conclusion, HPV oncogenes do not modulate the anti-EGFR antibody response in HSNCC. Cetuximab treatment should be administered independently of HPV status.

SOX4, SOX11 and PAX6 mRNA expression was identified as a (prognostic) marker for the aggressiveness of neuroendocrine tumors of the lung by using next-generation expression analysis (NanoString)

BACKGROUND Neuroendocrine tumors of the lung (NELC) account for 25% of all lung cancer cases and transcription factors may drive dedifferentiation of these tumors. This study was conducted to identify supportive diagnostic and prognostic biomarkers. MATERIALS & METHODS A total of 16 TC, 13 AC, 16 large cell neuroendocrine carcinomas and 15 small cell lung cancer were investigated for the mRNA expression of 11 transcription factors and related genes (MYB, MYBBP1A, OCT4, PAX6, PCDHB, RBP1, SDCBP, SOX2, SOX4, SOX11, TEAD2). RESULTS SOX4 (p = 0.0002), SOX11 (p < 0.0001) and PAX6 (p = 0.0002) were significant for tumor type. Elevated PAX6 and SOX11 expression correlated with poor outcome in large cell neuroendocrine carcinomas and small cell lung cancer (p < 0.0001 and p = 0.0232, respectively) based on survival data of 34 patients (57%). CONCLUSION Aggressiveness of NELC correlated with increasing expression of transcription factors. SOX11 seems to be a highly valuable diagnostic and prognostic marker for aggressive NELC.

Gene Expression Analysis of the 26S Proteasome Subunit PSMB4 Reveals Significant Upregulation, Different Expression and Association with Proliferation in Human Pulmonary Neuroendocrine Tumours.

Background: Proteasomal subunit PSMB4 was suggested to be a survival gene in an animal model of hepatocellular carcinoma and in glioblastoma cell lines. In pulmonary adenocarcinoma, a high expression of these genes was found to be associated with poor differentiation and survival. This study investigates the gene expression levels of 26S proteasome subunits in human pulmonary neuroendocrine tumours including typical (TC) and atypical (AC) carcinoid tumours as well as small cell (SCLC) and large cell (LCNEC) neuroendocrine carcinomas. Material and methods: Gene expression levels of proteasomal subunits (PSMA1, PSMA5, PSMB4, PSMB5 and PSMD1) were investigated in 80 neuroendocrine pulmonary tumours (each 20 TC, AC, LCNLC and SCLC) and compared to controls. mRNA levels were determined by using TaqMan assays. Immunohistochemistry on tissue microarrays (TMA) was performed to determine the expression of ki67, cleaved caspase 3 and PSMB4. Results: All proteasomal subunit gene expressions were significantly upregulated in TC, AC, SCLC and LCNEC compared to controls. PSMB4 mRNA is differently expressed between all neuroendocrine tumour subtypes demonstrating the highest expression and greatest range in LCNEC (p=0.043), and is significantly associated with proliferative activity (p=0.039). Conclusion: In line with other 26S proteasomal subunits PSMB4 is significantly increased, but differently expressed between pulmonary neuroendocrine tumours and is associated with the proliferative activity. Unlike in pulmonary adenocarcinomas, no association with biological behaviour was observed, suggesting that increased proteasomal subunit gene expression is a common and probably early event in the tumorigenesis of pulmonary neuroendocrine tumours regardless of their differentiation.

Identification of deregulation of apoptosis and cell cycle in neuroendocrine tumors of the lung via NanoString nCounter expression analysis

Background Neuroendocrine tumors of the lung comprise typical (TC) and atypical carcinoids (AC), large-cell neuroendocrine cancer (LCNEC) and small-cell lung cancer (SCLC). Cell cycle and apoptosis are key pathways of multicellular homeostasis and deregulation of these pathways is associated with cancerogenesis. Materials and Methods Sixty representative FFPE-specimens (16 TC, 13 AC, 16 LCNEC and 15 SCLC) were used for mRNA expression analysis using the NanoString technique. Eight genes related to apoptosis and ten genes regulating key points of cell cycle were investigated. Results ASCL1, BCL2, CASP8, CCNE1, CDK1, CDK2, CDKN1A and CDKN2A showed lower expression in carcinoids compared to carcinomas. In contrast, CCNE1 and CDK6 showed elevated expression in carcinoids compared to carcinomas. The calculated BCL2/BAX ratio showed increasing values from TC to SCLC. Between SCLC and LCNEC CDK2, CDKN1B, CDKN2A and PNN expression was significantly different with higher expression in SCLC. Conclusion Carcinoids have increased CDK4/6 and CCND1 expression controlling RB1 phosphorylation via this signaling cascade. CDK2 and CCNE1 were increased in carcinomas showing that these use the opposite way to control RB1. BAX and BCL2 are antagonists in regulating apoptosis. BCL2 expression increased over BAX expression with increasing malignancy of the tumor from TC to SCLC.

Mdm2 protein expression is strongly associated with survival in malignant pleural mesothelioma

AIMS TP53 mutations are extremely rare in malignant pleural mesothelioma (MPM). In TP53 wild-type tumors, the functional p53 protein can be inactivated by MDM2. MATERIALS & METHODS A total of 61 patient samples were tested for their Mdm2 and p53 protein expression levels via immunohistochemistry. RESULTS This study demonstrates nuclear Mdm2 expression in three out of four mesothelioma cell lines and 21.3% of the MPM specimens investigated. After silencing of the MDM2 gene by siRNA in MPM cell lines, Mdm2 immunoexpression is lost and cells show changes indicative of severe damage. Mdm2 protein expression in MPM is detected in epithelioid and biphasic subtypes only and is significantly associated with poor survival compared with Mdm2-negative tumors. This may be explained by increased Mdm2 levels possibly leading to an increased ubiquitilation and proteasomal degradation of functional p53 protein. CONCLUSION Expression of Mdm2 is a strong prognostic factor associated with shortened overall survival in MPM.

Targeted next-generation sequencing for TP53, RAS, BRAF, ALK and NF1 mutations in anaplastic thyroid cancer

Anaplastic thyroid carcinoma (ATC) is the most aggressive thyroid cancer with a median survival of 4–6 months. Identification of mutations contributing to aberrant activation of signaling cascades in ATC may provide novel opportunities for targeted therapy. Thirty-nine ATC samples were studied by next-generation sequencing (NGS) with an established gene panel. High quality readout was obtained in 30/39 ATC. Twenty-eight ATC harbored a mutation in at least one of the studied genes: TP53 (18/30), NF1 (11/30), ALK (6/30), NRAS (4/30), ATRX (3/30), BRAF (2/30), HRAS (2/30), KRAS (1/30). In 17/30 ATC (54 %) mutations were found in two or more genes. Twenty-one of the identified variants are listed in COSMIC as somatic mutations reported in other cancer entities. In three ATC samples no mutations were detected and none of the ATCs was positive for BRAFV600E. The most frequent mutations were found in TP53 (60 %), followed by NF1 (37 %). ALK mutations were detected in 20 % of ATC and were more frequent than RAS or BRAF mutations. ATRX mutations were identified in 10 % of the ATC samples. These sequencing data from 30 ATC samples demonstrate the accumulation of genetic alterations in ATC because in 90 % of samples mutations were already found in the investigated nine genes alone. Mutations were found with high prevalence in established tumor suppressor and oncogenes in ATC, such as TP53 and H/K/NRAS, but also, although less frequent, in genes that may harbor the potential for targeted treatment in a subset of ATC patients, such as ALK and NF1.

HER2 expression and markers of phosphoinositide-3-kinase pathway activation define a favorable subgroup of metastatic pulmonary adenocarcinomas

OBJECTIVES Pulmonary adenocarcinomas (ADC) can be sub-grouped based on dominant oncogenic drivers. EGFR mutations define an entity of metastatic ADC with favorable prognosis and high susceptibility to EGFR tyrosine kinase inhibition. In contrast, the clinical impact of additional ERBB family members in ADC is less defined. To this end we prospectively studied HER2 expression, gene amplification, and mutation in relation to outcome of patients with advanced or metastatic ADC. MATERIALS AND METHODS Diagnostic tumor biopsies from 193 sequential patients with stage III/IV ADC were prospectively studied for HER2 expression by immunohistochemistry (IHC). Cases with IHC scores 2+ or 3+ were analyzed by HER2 chromogenic in situ hybridization (CISH), and sequencing of HER2 exons 20 and 23. Additional prospectively determined biomarkers included PTEN, cMET, pAKT, and pERK expression, KRAS, EGFR, BRAF and PIK3CA mutations, and ALK fluorescence ISH (FISH). RESULTS AND CONCLUSION HER2-IHC was feasible in 176 (91.2%) cases. Of 53 (30%) cases with IHC scores 2+/3+, 45 (85%) could be studied by CISH and 34 (64%) by sequencing. The lower number of HER2-mutational analyses resulted from exhaustion of tumor tissue and DNA following mutational analysis of KRAS, EGFR, BRAF and PIK3CA. HER2 amplification was detected in 4 cases (2.3%), while no mutation was found. HER2 expression correlated with expression of pAKT and cMET. Expression of HER2 and pAKT was associated with favorable overall survival in stage IV disease. HER2-expressing ADC more frequently harbored KRAS mutations, while HER2 expression was absent in all 4 cases with BRAF mutation. HER2-IHC was not predictive of HER2 gene amplification or mutation, which both were rare events in prospectively studied patients with advanced or metastatic ADC. Expression of HER2 and pAKT define a population of patients with stage IV ADC with a distinct disease course, who could benefit from specifically tailored pharmacotherapies.