Circulating cell free DNA cfDNA has

Circulating cell free DNA (cfDNA) has been investigated in patients with solid tumors and circulating tumor DNA (ctDNA) is reported to be detectable in a wide range of malignancies (Bettegowda et al., 2014). In solid tumors there is evidence that ctDNA is an effective biomarker at predicting relapse following surgery (Diehl et al., 2008) and progression during chemotherapy and targeted therapy (Diaz et al., 2012). In prenatal screening for aneuploidy, fetal cfDNA obtained from maternal plasma, has proven to be highly accurate with a detection rate for trisomy 21 of up to 100% (Norton et al., 2015). Since GTN is both a malignancy and pregnancy related we would predict cfDNA from trophoblastic Tozadenant to be present in the plasma of patients with these tumors. Due to the unique genetics of GTN i.e. the presence of non-maternal DNA in the tumor, the DNA signature of these tumors may be easily detectable. cfDNA may therefore provide unique genetic information about a patients’ disease hereto unavailable.
Methods
Results
Discussion Analysis of ctDNA is currently being introduced as a non-invasive method for detecting and monitoring the progress of a number of cancers (Bettegowda et al., 2014; Crowley et al., 2013). In this study we have demonstrated that ctDNA can be detected in the plasma of women with trophoblastic tumors and can facilitate diagnosis. Screening using fetal cfDNA from maternal blood samples is increasingly used for prenatal diagnosis (Bianchi, 2012; Nicolaides et al., 2014). While cfDNA is usually cleared rapidly after delivery (Lo et al., 1999) we speculated that cfDNA would persist in women with invasive molar disease, as trophoblastic tissue continues to grow following termination of the pregnancy in these cases. This study has shown that for the majority of women who develop trophoblastic tumors, there is sufficient ctDNA in patients\' blood to be detected by molecular genotyping, a technique routinely used in clinical diagnosis of trophoblastic disease (Fisher et al., 2007, 2014). Both the total cfDNA extracted from 3mL of plasma, 3.2–24.2ng, and the proportion of total cfDNA that derived from the molar pregnancy, 0–42%, were highly variable. In other tumor types the level of detectable ctDNA has been reported to relate to the extent of disease (Diehl et al., 2008; Dawson et al., 2013; Kim et al., 2014). Since serum hCG levels reflect tumor burden in GTN (Seckl et al., 2010, 2013), the yield of ctDNA might be expected to parallel serum hCG levels. The yield of ctDNA reflected tumor burden in that ctDNA was detectable in all patients with a serum hCG of 66,861IU/L and above but undetectable where serum hCG was 14,884IU/L or below. While Spearman\'s correlation coefficient for serum hCG levels and yields of ctDNA was significant in women with GTN overall, there were a group of women with serum levels between 16,326IU/L and 53,046IU/L where detection of ctDNA was variable, suggesting that the release of ctDNA from tumors following chemotherapy may reflect other aspects of tumor biology and not simply tumor burden. Further studies analyzing ctDNA levels pre- and post-chemotherapy at specific time points are needed to investigate the exact relationship between serum hCG levels and ctDNA yields. With the rare exception of a small number of PSTT and ETT, all GTN produce hCG. While hCG production is a characteristic of all GTN, a number of other tumor subtypes have been shown to secrete hCG ranging from 4% in prostate cancer to as high as 76% of bladder tumors (Iles et al., 2010). For most of these tumors other clinicopathological features are likely to lead to a diagnosis but for women of reproductive age with raised serum hCG and no pathological diagnosis, the differential diagnosis may be between a GTN and a non-gestational, hCG-secreting malignancy (Fisher et al., 2007). In two of five cases of women with hCG-secreting tumors of unknown origin in the present series, genotyping of cfDNA was helpful in establishing a diagnosis. In one patient the presence of ctDNA enabled a diagnosis of a GTN originating in a female pregnancy. In the other genotyping of ctDNA showed gross microsatellite instability consistent with ctDNA from a non-gestational tumor. The same microsatellite instability was demonstrated in the liver metastasis that was later biopsied indicating the unequivocal origin of the cfDNA from the tumor. These two cases demonstrate the utility of cfDNA to provide a ‘liquid biopsy’ to aid diagnosis when histology is unavailable and that genotyping can potentially diagnose both gestational and non-gestational trophoblastic tumors.