Quantitative reverse transcription-polymerase chain reaction measurement of HASH1 (ASCL1), a marker for small cell lung carcinomas with neuroendocrine features.
OBJECTIVE
The Human Achaete-Scute homologue 1 (HASH1, ASCL1), a lineage-specific basic helix-loop-helix member of the achaete-scute family, is essential for the generation of pulmonary neuroendocrine (NE) cells during lung development. In small cell lung cancer (SCLC), the most lethal form of lung cancer, the gene is highly expressed and the expression of HASH1 correlates with NE features found in SCLCs. Here we describe a highly sensitive reverse transcription-PCR method for quantifying HASH1 mRNA in clinical samples, using real-time fluorescence resonance energy transfer technology (LightCycler).


METHODS
The HASH1-positive NE cell line NCI-H187 was compared with the non-NE cell line NCI-N417 by quantitative reverse transcription-PCR. Signals were normalized using the housekeeping gene PBGD, which is pseudogene free. Subsequently, HASH1 expression in RNA isolated from biopsies from SCLC patients (n = 4) was compared with biopsies from non-SCLC (NSCLC) patients (n = 2) or normal bronchus (n = 2).


RESULTS
The HASH1-positive NE cell line NCI-H187 showed 50,000-fold higher normalized expression of HASH1 than did the non-NE cell line NCI-N417, indicating that the method is applicable over a wide dynamic range. Normalized average mRNA expression levels in SCLC clinical samples were 1,000-fold higher than in the NSCLC samples. Expression in normal bronchus was comparable to the expression levels in the NSCLC.


CONCLUSIONS
These results show that marked and measurable differences exist between SCLCs and other lung tissues (either NSCLC or normal bronchus). We show that the method is applicable to small biopsy samples and can discriminate SCLC from NSCLC. This method could contribute to diagnosis based on molecular profiling of tumors.
Westerman
Bart A
BA
Molecular Biology Laboratory, Department of Clinical Chemistry, VU University Medical Center, 1081 HV Amsterdam, the Netherlands.
Neijenhuis
Sari
S
Poutsma
Ankie
A
Steenbergen
Renske D M
RD
Breuer
Roderick H J
RH
Egging
Monique
M
van Wijk
Inge J
IJ
Oudejans
Cees B M
CB
D051792:Basic Helix-Loop-Helix Proteins; D014408:Biomarkers, Tumor / Q000235:genetics; D018278:Carcinoma, Neuroendocrine / Q000235:genetics* / Q000473:pathology; D002289:Carcinoma, Non-Small-Cell Lung / Q000235:genetics; D018288:Carcinoma, Small Cell / Q000235:genetics* / Q000473:pathology; D004268:DNA-Binding Proteins / Q000235:genetics*; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D011163:Hydroxymethylbilane Synthase / Q000235:genetics; D008175:Lung Neoplasms / Q000235:genetics* / Q000473:pathology; D018075:RNA, Complementary / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012334:RNA, Neoplasm / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014157:Transcription Factors / Q000235:genetics*; D014407:Tumor Cells, Cultured; D015854:Up-Regulation
D051792:Basic Helix-Loop-Helix Proteins; D014408:Biomarkers, Tumor; D004268:DNA-Binding Proteins; D011163:Hydroxymethylbilane Synthase; D018075:RNA, Complementary; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D014157:Transcription Factors; C065500:ASCL1 protein, human