Screening of Pleural Mesotheliomas for DNA-damage Repair Players by Digital Gene Expression Analysis Can Enhance Clinical Management of Patients Receiving Platin-Based Chemotherapy

Background: Malignant pleural mesothelioma (MPM) is a rare, predominantly asbestos-related and biologically highly aggressive tumour leading to a dismal prognosis. Multimodality therapy consisting of platinum-based chemotherapy is the treatment of choice. The reasons for the rather poor efficacy of platinum compounds remain largely unknown. Material and Methods: For this exploratory mRNA study, 24 FFPE tumour specimens were screened by digital gene expression analysis. Based on data from preliminary experiments and recent literature, a total of 366 mRNAs were investigated using a Custom CodeSet from NanoString. All statistical analyses were calculated with the R i386 statistical programming environment. Results: CDC25A and PARP1 gene expression were correlated with lymph node spread, BRCA1 and TP73 expression levels with higher IMIG stage. NTHL1 and XRCC3 expression was associated with TNM stage. CHECK1 as well as XRCC2 expression levels were correlated with tumour progression in the overall cohort of patients. CDKN2A and MLH1 gene expression influenced overall survival in this collective. In the adjuvant treated cohort only, CDKN2A, CHEK1 as well as ERCC1 were significantly associated with overall survival. Furthermore, TP73 expression was associated with progression in this subgroup. Conclusion: DNA-damage response plays a crucial role in response to platin-based chemotherapeutic regimes. In particular, CHEK1, XRCC2 and TP73 are strongly associated with tumour progression. ERCC1, MLH1, CDKN2A and most promising CHEK1 are prognostic markers for OS in MPM. TP73, CDKN2A, CHEK1 and ERCC1 seem to be also predictive markers in adjuvant treated MPMs. After a prospective validation, these markers may improve clinical and pathological practice, finally leading to a patients' benefit by an enhanced clinical management

Massive parallel sequencing and digital gene expression analysis reveals potential mechanisms to overcome therapy resistance in pulmonary neuroendocrine tumors

Background: Lung cancer is the leading cause of cancer-related deaths worldwide. 25% show neuroendocrine differentiation (typical/atypical carcinoids, large-/small-cell neuroendocrine carcinomas). Carcinoids present with long survival rates, but metastatic carcinoids correlate with decreased survival and are commonly insensitive to standard chemotherapy or radiation. Therefore, novel therapeutic strategies are urgently needed. Material and methods: 70 representative tumor specimens were used for next-generation sequencing analysis of 14 genes related to therapy response. Additionally, mRNA-expression profiles of 60 matching samples were determined for 13 selected drug targets by using the NanoString nCounter technology. Results: A number of features known to sensitize tumors for different targeted therapies could be identified, which hopefully improve the clinical management of this subgroup of lung neoplasias. In particular, EGFR expression was observed in the investigated tumors in a noteworthy manner. Additionally, MDM2 was strongly expressed in the majority of all samples whereas the expression of its physiological inhibitor, CDKN2A, was nearly absent in all low-grade tumors. TP53 showed a high frequency of variants in high-grade tumors but mutations were rare in carcinoids. Conclusion: Based on our results, therapeutic approaches with MDM2-inhibitors and monoclonal anti-EGFR antibodies may be promising in pulmonary carcinoid tumors

ACTB, CDKN1B, GAPDH, GRB2, RHOA and SDCBP Were Identified as Reference Genes in Neuroendocrine Lung Cancer via the nCounter Technology

Background Neuroendocrine lung cancer (NELC) represents 25% of all lung cancer cases and large patient collectives exist as formalin-fixed, paraffin-embedded (FFPE) tissue only. FFPE is controversially discussed as source for molecular biological analyses and reference genes for NELC are poorly establishes. Material and methods Forty-three representative FFPE-specimens were used for mRNA expression analysis using the digital nCounter technology (NanoString). Based on recent literature, a total of 91 mRNA targets were investigated as potential tumor markers or reference genes. The geNorm, NormFinder algorithms and coefficient of correlation were used to identify the most stable reference genes. Statistical analysis was performed by using the R programming environment (version 3.1.1) Results RNA integrity (RIN) ranged from 1.8 to 2.6 and concentrations from 34 to 2,109 ng/μl. However, the nCounter technology gave evaluable results for all samples tested. ACTB, CDKN1B, GAPDH, GRB2, RHOA and SDCBP were identified as constantly expressed genes with high stability (M-)values according to geNorm, NormFinder and coefficients of correlation. Conclusion FFPE-derived mRNA is suitable for molecular biological investigations via the nCounter technology, although it is highly degraded. ACTB, CDKN1B, GAPDH, GRB2, RHOA and SDCBP are potent reference genes in neuroendocrine tumors of the lung

<i>ACTB</i>, <i>CDKN1B</i>, <i>GAPDH</i>, <i>GRB2</i>, <i>RHOA</i> and <i>SDCBP</i> Were Identified as Reference Genes in Neuroendocrine Lung Cancer via the nCounter Technology

<div><p>Background</p><p>Neuroendocrine lung cancer (NELC) represents 25% of all lung cancer cases and large patient collectives exist as formalin-fixed, paraffin-embedded (FFPE) tissue only. FFPE is controversially discussed as source for molecular biological analyses and reference genes for NELC are poorly establishes.</p><p>Material and methods</p><p>Forty-three representative FFPE-specimens were used for mRNA expression analysis using the digital nCounter technology (NanoString). Based on recent literature, a total of 91 mRNA targets were investigated as potential tumor markers or reference genes. The geNorm, NormFinder algorithms and coefficient of correlation were used to identify the most stable reference genes. Statistical analysis was performed by using the R programming environment (version 3.1.1)</p><p>Results</p><p>RNA integrity (RIN) ranged from 1.8 to 2.6 and concentrations from 34 to 2,109 ng/μl. However, the nCounter technology gave evaluable results for all samples tested. <i>ACTB</i>, <i>CDKN1B</i>, <i>GAPDH</i>, <i>GRB2</i>, <i>RHOA</i> and <i>SDCBP</i> were identified as constantly expressed genes with high stability (M-)values according to geNorm, NormFinder and coefficients of correlation.</p><p>Conclusion</p><p>FFPE-derived mRNA is suitable for molecular biological investigations via the nCounter technology, although it is highly degraded. <i>ACTB</i>, <i>CDKN1B</i>, <i>GAPDH</i>, <i>GRB2</i>, <i>RHOA</i> and <i>SDCBP</i> are potent reference genes in neuroendocrine tumors of the lung.</p></div

A to D show a correlation matrix for gene expression (A), a heatmap for tumor type versus gene expression (B), scatterplots (C and D) for gene versus gene correlations and R² calculation.

<p><b>Fig 2A</b> depicts a correlation matrix of genes that were identified as potential reference genes by geNorm and NormFinder algorithms and previously identified tumor markers (<i>CDK6</i> and <i>TYMS</i>). High correlations are outlined by red colored squares. Between <i>CDKN1B</i>, <i>GRB2</i> and <i>GAPDH</i> as well as between <i>ACTB</i>, <i>SDCBP</i> and <i>RHOA</i> a high correlation was identified. Low correlations are indicated by blue squares and were found for tumor markers (<i>CDK6</i> and <i>TYMS</i>) versus reference gene. <b>Fig 2B</b> displays a heatmap. On the x-axis the potential reference genes and tumor markers <i>CDK6</i> and <i>TYMS</i> are shown. On the y-axis the investigated tumor types are depicted. Differential expression was found between tumor types. Though, the reference genes show a constant expression cluster (either low or high) between the samples investigated. The tumor markers present with differential expression between all samples without showing a specific cluster. <b>Fig 2C</b> and <b>2D</b> are exemplary scatterplots of gene versus gene correlation, which were created to calculate the coefficient of determination (R²). <b>Fig 2C</b> depicts the highest correlation identified (R² = 0.88) between two potential reference genes (<i>ACTB</i> and <i>SDCBP</i>). In <b>D</b>, the weakest correlation is depicted, which was found between the two tumor markers (<i>CDK6</i> and <i>TYMS</i>).</p

A and B show gel-smear analysis to assess the RNA quantity and quality (RIN) in total RNA derived from formalin-fixed, paraffin-embedded tissue.

<p><b>Fig 1A</b> depicts a representative smear gel analysis of twelve samples. A ladder was included to allow size calculation. The microfluid analysis shows that RNA from FFPE is highly degraded giving no distinct size patterns.<b>Fig 1B</b> depicts the electropherogram of two representative samples. The rRNA Ratio (28s/18s) is used to calculate the RNA quality according to an algorithm supplied by the manufacturer. Neither 28s nor 18s bands can be found for FFPE-derived RNA leading to considerably low RNA integrity numbers (RIN). RNA concentration is calculated from the area under the curve.</p