Results of circulating tumor cell (CTC) enumeration in clinical study.

<p>(A) Comparison between lung cancer patients and healthy volunteers (<i>n</i> = 50 and 10, respectively) in the number of CTCs (median = 2.5 and 0, respectively); ****<i>p</i> < 0.0001. (B) Distribution of CTC count in all patients. (C) CTC count in 37 NSCLC patients.</p

Comparison of circulating tumor cell counts in SCLC samples detected by the microcavity array (MCA) and CellSearch systems.

<p>Comparison of circulating tumor cell counts in SCLC samples detected by the microcavity array (MCA) and CellSearch systems.</p

Patient characteristics.

<p>Patient characteristics.</p

Comparison between the microcavity array (MCA) system and CellSearch system in circulating tumor cell (CTC) count.

<p>(A) Counts in NSCLC patients (<i>n</i> = 17) (median, 11 versus 0); ***<i>p</i> < 0.0001. (B) Counts in SCLC patients (<i>n</i> = 5) (median, 15 versus 6).</p

Spike-in experiment for evaluation of the cell recovery rate by the microcavity array (MCA) system.

<p>(A) Representative staining images of detected cancer cells. Scale bars: 10 μm. (B) Recovery rates of the lung cancer cell lines by the MCA system.</p

Clinicopathologic correlation of circulating tumor cell (CTC) counts by MCA in lung cancer patients.

<p>(A) Never smokers (n = 18) versus smokers (n = 32). (B) Patients with no previous treatment (n = 35) versus patients with previous treatment (n = 15). (C) Performance status (PS) <2 (n = 40) versus PS ≥2 (n = 10). (D) NSCLC (n = 39) versus SCLC (n = 10). (E) Stage III (n = 7) versus stage IV (n = 32) in NSCLC patients. (F) EGFR wild-type adenocarcinoma (n = 18) versus EGFR-mutated adenocarcinoma (n = 12).</p

Comparison of circulating tumor cell counts in NSCLC samples detected by the microcavity array (MCA) and CellSearch systems.

<p>Comparison of circulating tumor cell counts in NSCLC samples detected by the microcavity array (MCA) and CellSearch systems.</p

The automated microcavity array (MCA) system.

<p>(A) Overview of the MCA system. The system is composed of a blood reservoir, cartridge, and tube. (B) Scanning electron microscope images of a rectangular (8 μm×100 μm) micro metal filter. Scale bar: 100 μm (yellow line). (C) Overview of the cartridge. The cartridge is fabricated from acrylic frames and silicon gaskets. (D) Diagram of the MCA system. The filtration cartridge is connected to two inlets and one outlet. The outlet line is connected to a peristaltic pump to facilitate injection of the blood and each reagent into the cartridge. A bubble trap cartridge is connected to the reagent line to prevent air from entering the filtration cartridge.</p

Allele-specific bisulfite pyrosequencing.

<p>To analyze the allele-specific DNA methylation, we individually measured the DNA methylation of each allele and compared methylation between the two alleles. Thus, the allele-specific polymerase chain reaction (PCR) was performed and the methylation levels of each of the two alleles, given allele-specific amplicons, were measured separately using pyrosequencing. The allele-specific PCR primers were designed such that each primer of 3′ end was a pair of base of heterozygous SNP. (a) The reference sequence of rs36221701 before and after bisulfite conversion. Rs36221701 contained two CpG sites denoted as CpG1 and CpG2 in 5′ to 3′ direction. By bisulfite conversion, unmethylated C is converted to U (U is converted to T by PCR). Rs362210701 (C/T) is converted to (T/T); it was impossible to distinguish the converted T and original T. Thus, we were unable to detect the origin of the allele for this SNP by PCR primers. (b) Allele-specific PCR. We designed PCR primer for rs13239907 (A/G), which is located 297 bp downstream of rs36221701 and was in complete linkage disequilibrium with rs36221701 (A→C, G→T: r² = 1.0) in JPT data from 1,000 genome project. The forward primer was common to both alleles; reverse primers were designed such that each primer of 3′ end was a pair of bases of genotypes (A or G: complementary strand). (c) Allele-specific pyrosequencing. The two given allele-specific amplicons were separately analyzed using pyrosequencing. Sequence primer 1 targeted rs13239907 and was used to check the accuracy of allele-specific PCR, whereas sequence primer 2 targeted CpG1 and CpG2 was used to analyze the ratio of methylation around rs36221701. (d) Primer details. The 3′ end of PCR primer R was a pair of base of heterozygous SNP. 5′ end of PCR primer R was biotinylated.</p

The relation between genotype of rs36221701 and the expression levels of genes around rs36221701.

<p>The relation between genotype of rs36221701 and the expression levels of genes around rs36221701.</p