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Technical Briefs |
Departments of1 Obstetrics and Gynecology, and 2 Human Genetics, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan;3 CREST, Japan Science and Technology Agency (JST), Kawaguchi, Japan;
aaddress correspondence to this author at: Department of Obstetrics and Gynecology, Nagasaki University Graduate School of Biomedical Sciences, 1-7-1 Sakamoto, Nagasaki 852-8501, Japan; fax 81-95-849-7365, e-mail kiyonori{at}net.nagasaki-u.ac.jp
The finding of tumor-related mRNA (tyrosinase mRNA, telomerase component mRNA, various tumor-derived mRNAs, and viral RNAs) in plasma from a patient with cancer suggests that real-time quantitative reverse transcription-PCR (RT-PCR) study of circulating plasma mRNAs may be used as a very simple assay for residual tumor after surgery or therapy (1)(2)(3).
Gestational trophoblastic diseases (GTDs) are a heterogeneous group of diseases that include partial and complete hydatidiform moles (CHMs), invasive moles, choriocarcinomas, placental site trophoblastic tumors, and epithelioid trophoblastic tumors. Human chorionic gonadotropin (hCG) is generally used as a tumor marker in the clinical management of all GTDs. Recently, hCG-ß mRNA from placenta has been detected in maternal plasma by real-time quantitative RT-PCR (4), suggesting that this assay may be useful in the management of GTDs.
In this study, we measured plasma mRNA concentrations by real-time RT-PCR in 3 GTD patients with CHMs and 1 with choriocarcinoma.
Blood samples (10 mL) were collected before and after suction evacuation or chemotherapy. Plasma mRNA was extracted, and a 1-step real-time RT-PCR assay was performed as described by Ng et al. (4). We selected hCG-ß mRNA as a tumor marker of GTD and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA as a housekeeping gene. Primer sets and TaqMan probes for the 2 genes selected were prepared as described previously (4). We prepared calibration curves for quantification of each mRNA by assaying serial dilutions of HPLC-purified single-strand synthetic DNA oligonucleotides from each PCR amplicon (R2 = 0.99; slope, –2.9 to 3.3). The absolute concentration of each mRNA is reported as copies/mL of maternal plasma, based on the formula described by Farina et al. (5). Interpolation of obtained results on the calibration curves indicated that hCG-ß mRNA concentrations ranged from 1 x 107 to 1 x 101 copies/mL, and GAPDH mRNA concentrations ranged from 1x 1010 to 1 x 104 copies/mL. Each sample was analyzed in triplicate under thermal conditions as described previously (4).
All study protocols were approved by the Committee for the Ethical Issues on Human Genome and Gene Analysis in Nagasaki University, and written informed consent was obtained from all of the women. None of the data regarding plasma mRNA concentrations influenced the clinical management of patients.
Three cases with CHM, diagnosed by pathology examinations, were treated twice with suction evacuation. Chest x-rays indicated no pulmonary lesion. The day of the first evacuation was called day 0, and the second evacuation was on day 7. The plasma hCG-ß mRNA in all 3 cases decreased rapidly to undetectable (<10 copies/mL) by day 7 (Fig. 1A
), and the corresponding hCG protein in serum, as measured by an IRMA, also decreased to <1000 IU/L until 35 days after the first evacuation (Fig. 1B
) (6).
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A patient with clinical choriocarcinoma, which was diagnosed by the diagnostic score used in Japan (6), showed a <3 cm pulmonary lesion detected by chest x-ray and a biphasic pattern of basal body temperature (BBT). Etoposide and actinomycin D therapy was given on days 0–3, 15–18, 29–32, and 43–46. Her plasma hCG-ß mRNA concentration showed a decreasing tendency similar to the pattern for hCG protein in serum, but exhibited a transient increase from 901 to 954 copies/mL after the first course of therapy and then decreased to 44 copies/mL (Fig. 1D
). The same transient tendency was detected after all subsequent courses of therapy (Table 1
).
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The cases with CHM had no metastatic lesions, whereas the case with choriocarcinoma had lung metastasis. Rapid clearance of plasma hCG-ß mRNA may reflect complete elimination of CHM, and the transiently increased concentration of plasma hCG-ß mRNA after every each course of therapy may reflect apoptotic activity induced by the therapy (3)(7).
The concentration of plasma GAPDH mRNA in all 3 cases with CHM also increased after both evacuations (Fig. 1C
), and that in a case with choriocarcinoma also increased after therapy with etoposide and actinomycin D (Fig. 1E
and Table 1
). Thus, cell/tissue damage by both evacuations and chemotherapy may be associated with increased circulating concentrations of GAPDH mRNA (3)(7).
Our results suggest that measurement of plasma mRNA by real-time quantitative RT-PCR can be used as a noninvasive diagnostic, prognostic, and follow-up test for GTD (1)(2)(3). The mRNA is cleared rapidly and provides information different from that obtained with hCG immunoassays. The measurements may be particularly useful in patients whose serum contains substances (such as human anti-mouse antibodies) that interfere in immunoassays. As our data were based on only a few cases, it remains to be seen in additional studies whether the method is sensitive enough to monitor changes in mRNA concentrations in GTD.
Acknowledgments
We thank Dr. Joseph Wagstaff for assistance and valuable advice. This work was supported in part by Grants-in-Aid for Scientific Research (15591761, 16591670, and 13854024) from the Ministry of Education, Sports, Culture, Science and Technology of Japan (to H.M., K.M., and N.N.) and CREST from the Japan Science and Technology Agency (to N.N.)
Footnotes
1 these authors contributed equally to this work; 
References
-globin gene expression suggests that fetal hematopoietic cells contribute to the pool of circulating cell-free fetal nucleic acids during pregnancy. Clin Chem 2004;50:689-693.
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, •, and 
in panels A, B, and C indicate cases 1, 2, and 3, respectively. Arrows indicate days 0 and 7, the days on which suction evacuations were performed. (D), plasma hCG-ß mRNA (