<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

Patient characteristics.

<p>Patient characteristics.</p

Genetic profile of 60 CLL samples carrying gene mutations determined by NGS.

<p>Each row represents the variants of one patient, each column summarizes the mutations occurring in one specific gene. Per each gene the number of mutations is given per patient. Dark blue samples indicate patients with aberration on chromosome 11 (del11q) for <i>ATM</i> mutated cases or on chromosome 17 (del17p) for <i>TP53</i> mutated cases, determined by FISH.</p

Algorithm of variant analysis.

<p>A) Variants with an allelic frequency below 5% were discarded, resulting in 4,396 variants. B) Only the 3,322 non-synonymous variants were used for further analysis. The variant count per gene is represented in the bar chart. C) Variants located in areas of high background noise and/or in homopolymeric regions, and single strand variants were visually identified in the Integrative Genomic Viewer (IGV, Broad Institute) and removed. In doubtful cases, Sanger sequencing was performed to prove or disprove an alteration. Furthermore variants without functional impact on the protein determined by at least two of four applied program algorithms as described in material and method were removed. This resulted in 102 final mutations in 60 CLL specimens.</p

Alteration type, number of occurrence and location of detected mutations in <i>TP53</i>, <i>SF3B1</i>, <i>NOTCH1</i> and ATM are shown.

<p><i>TP53</i>: AD activation domain (amino acid 1–50); PD proline-rich domain (amino acid 63–97); TD tetramerization domain (amino acid 323–356); ND negative regulation domain (amino acid 363–393); <i>SF3B1</i>: The majority of <i>SF3B1</i> alterations were clustered in the region encoding the highly conserved HEAT (huntingtin, elongation factor 3, protein phosphatase 2A, target of rapamycin 1) repeats 5–8. Only one alteration occurred in the N-terminal (amino acids 1–450), domain, which is an important docking or binding domain for numerous splicing factor partners like U2AF1/2, and cyclin E. <i>NOTCH1</i>: (EGF)-like epidermal growth factor repeats (amino acid 20–1426), LNR Lin-12 NOTCH repeats (amino acid 1449–1571), HD-N/C heterodimerization domain (N-terminus; C-terminus), RAM RAM domain, ANK ankyrin repeat domain (amino acid 1927–2089); PEST Pro-Glu-Ser-Thr motif for degradation (amino acid 2507–2526); <i>ATM</i>: FAT FRAP-ATM-TRRAP (amino acid 1960–2566), KD protein kinase domain (amino acid 2712–2962), PRD PIKK-regulatory domain (amino acid 2961–3025), FATC FAT-c-term domain (amino acid 3024–3056); aa amino acid</p