Circulating metastasis associated in colon cancer 1 transcripts in gastric cancer patient plasma as diagnostic and prognostic biomarker

METHODS: We provide for the first time a blood-based assay for transcript quantification of the metastasis inducer MACC1 in a prospective study of gastric cancer patient plasma. MACC1 is a strong prognostic biomarker for tumor progression and metastasis in a variety of solid cancers. We conducted a study to define the diagnostic and prognostic power of MACC1 transcripts using 76 plasma samples from gastric cancer patients, either newly diagnosed with gastric cancer, newly diagnosed with metachronous metastasis of gastric cancer, as well as follow-up patients. Findings were controlled by using plasma samples from 54 tumor-free volunteers. Plasma was separated, RNA was isolated, and levels of MACC1 as well as S100A4 transcripts were determined by quantitative RT-PCR. RESULTS: Based on the levels of circulating MACC1 transcripts in plasma we significantly discriminated tumor-free volunteers and gastric cancer patients (P < 0.001). Levels of circulating MACC1 transcripts were increased in gastric cancer patients of each disease stage, compared to tumor-free volunteers: patients with tumors without metastasis (P = 0.005), with synchronous metastasis (P = 0.002), with metachronous metastasis (P = 0.005), and patients during follow-up (P = 0.021). Sensitivity was 0.68 (95%CI: 0.45-0.85) and specificity was 0.89 (95%CI: 0.77-0.95), respectively. Importantly, gastric cancer patients with high circulating MACC1 transcript levels in plasma demonstrated significantly shorter survival when compared with patients demonstrating low MACC1 levels (P = 0.0015). Furthermore, gastric cancer patients with high circulating transcript levels of MACC1 as well as of S100A4 in plasma demonstrated significantly shorter survival when compared with patients demonstrating low levels of both biomarkers or with only one biomarker elevated (P = 0.001). CONCLUSION: Levels of circulating MACC1 transcripts in plasma of gastric cancer patients are of diagnostic value and are prognostic for patient survival in a prospective study

Survival of CRC patients based on circulating transcript levels of MACC1 or a combination of MACC1 and S100A4.

<p>A, B. Kaplan-Meier analysis for newly diagnosed (A, n = 49) and all (B, n = 294) CRC patients, based on MACC1 alone (A, low MACC1 n = 24, high MACC1 n = 25; B, low MACC1 n = 147, high MACC1 n = 147). Newly diagnosed CRC patients (A) as well as all CRC patients (B) with high circulating MACC1 transcript levels demonstrated shorter survival when compared with patients demonstrating low MACC1 levels (<i>P</i> = .003 and <i>P</i><.0001, respectively). C, D. Kaplan-Meier analysis for newly diagnosed (C, n = 49) and all (D, n = 294) CRC patients, based on a combination of MACC1 and S100A4. Newly diagnosed CRC patients (C) and all CRC patients (D) were classified into groups of low expressors of both genes (n = 13 and n = 85, respectively), of patients with high S100A4 levels (n = 25 and n = 147, respectively), of patients with high MACC1 levels (n = 25 and n = 147, respectively), or with high expression of both biomarkers (n = 14 and n = 85, respectively). For newly diagnosed patients (C), survival was reduced with increasing levels of circulating transcripts of S100A4, of MACC1, or of both. Expression induction of either MACC1 or S100A4 or of both biomarkers correlated to reduced patients survival.</p

Circulating MACC1 transcript levels in plasma discriminate cancer patients from tumor-free volunteers.

<p>A. Plasma separation conditions for determination of circulating MACC1 transcripts. Plasma separation of samples from tumor-free volunteers (n = 12) was done immediately, 7, 24, 48, and 72 hours after taking blood, either from blood samples kept at 4°C or at room temperature. Subsequently, plasma was generated within the first 7 hours from 4°C-cooled blood. B. MACC1 transcripts in plasma of tumor-free volunteers (n = 54). No difference in MACC1 transcript levels were found in two independently analyzed cohorts of tumor-free volunteers (n = 34 and n = 20, respectively). C. MACC1 transcripts in plasma of all colorectal cancer patients (n = 312). All patient cohorts bearing colorectal, colon (n = 151), or rectal (n = 161) cancer expressed significantly higher MACC1 transcript levels than tumor-free volunteers (<i>P</i><.001 for all comparisons).</p

Colon cancer patients characteristics and circulating MACC1 transcript levels in plasma.

<p>Colon cancer patients characteristics and circulating MACC1 transcript levels in plasma.</p

Colorectal cancer patients characteristics and circulating MACC1 transcript levels in plasma.

<p>Colorectal cancer patients characteristics and circulating MACC1 transcript levels in plasma.</p

Diagnostic value of circulating MACC1 transcript levels in plasma of newly diagnosed colorectal, colon, and rectal cancer patients with a primary tumor with or without synchronous metastasis.

<p>Optimal cut offs were calculated with a fourfold table.</p

Circulating MACC1 transcripts in colorectal, colon, and rectal cancer patient plasma.

<p>A, B, C. All colorectal (A, n = 312), colon (B, n = 151), and rectal (C, n = 161) cancer patient sub-cohorts showed significantly higher circulating MACC1 transcript levels than tumor-free volunteers (n = 54). Higher MACC1 levels were also found for colorectal (<i>P</i> = .006; A) and rectal (<i>P</i> = .041; C) cancer patients with synchronous metastases (n = 20 and n = 10, respectively) compared to patients without distant metastases (n = 51 and n = 39, respectively). Box plot analysis, based on quantitative real-time RT-PCR.</p

Rectal cancer patients characteristics and circulating MACC1 transcript levels in plasma.

<p>Rectal cancer patients characteristics and circulating MACC1 transcript levels in plasma.</p

Diagnostic and Prognostic Value of Metastasis Inducer S100A4 Transcripts in Plasma of Colon, Rectal, and Gastric Cancer Patients

Early detection of tumors and metastases is critical for improving treatment strategies and patient outcomes. The development of molecular markers and simple tests that are clinically applicable for detection, prognostication, and therapy monitoring is strongly needed. The gene S100A4 has long been known to act as a metastasis inducer. High S100A4 levels in the primary tumor are prognostic for metachronous metastasis and correlate with reduced patient survival. We provide, for the first time, a plasma-based assay for transcript quantification of S100A4 in gastrointestinal patients' plasma. We conducted a study to define the diagnostic and prognostic power of S100A4 transcripts using 466 plasma samples from colon, rectal, and gastric cancer patients. Plasma was separated, RNA was isolated, and S100A4 mRNA was determined by quantitative RT-PCR. S100A4 transcripts were increased in cancer patients of each entity (P < 0.0001) and all disease stages (P < 0.05), compared with tumor-free volunteers (sensitivities of 96%, 74%, and 90% and specificities of 59%, 82%, and 71%, for colon, rectal, and gastric cancer patients, respectively). Prospectively analyzed follow-up patients who later experienced metastasis showed higher S100A4 levels than follow-up patients without metastasis. Disease-free survival was decreased in high S100A4-expressing follow-up colorectal cancer patients (P = 0.013). In summary, we developed a method for quantitative S100A4 transcript determination in plasma that allows clinical application routinely. We demonstrated the diagnostic and prognostic potential of this method for early defining cancer staging and patients' risk for metastasis