BackgroundLiquid biopsies enable the detection of circulating tumor DNA (ctDNA). However, the clinical significance of KRAS-mutated ctDNA for pancreatic cancer has been inconsistent with respect to its prognostic and predictive potential.Methods and findingsA total of 422 blood samples were collected from 78 patients undergoing treatments for localized and metastatic pancreatic ductal adenocarcinoma. KRAS mutation in tissues and KRAS ctDNA levels in plasma were determined by RASKET and droplet digital polymerase chain reaction. Longitudinal monitoring of KRAS ctDNA was performed to assess its significance for predicting recurrence and prognosis and for evaluating therapeutic responses to chemotherapy compared with carbohydrate antigen 19–9 (CA19-9). In 67 tumor tissues, discrepancies in point mutations of KRAS were rarely observed among individual patients, implying that one targeted point mutation of KRAS can be determined in tumor tissues prior to longitudinal blood monitoring. One-time blood assessment of KRAS-mutated ctDNA before surgery or chemotherapy was not clearly associated with recurrence and prognosis. Sequential blood monitoring was performed in 39 patients who underwent surgery for potentially resectable tumors. Increased CA19-9 levels were significantly associated with recurrence, but not prognosis (P<0.001, P = 1.0, respectively), whereas emergence of KRAS ctDNA was significantly associated with prognosis (P<0.001) regardless of recurrence. Furthermore, in 39 patients who did not undergo surgery, detection of KRAS ctDNA was a predictive factor for prognosis (P = 0.005). Multivariate analysis revealed that detection of KRAS ctDNA was the only independent prognostic factor regardless of tumor resection (hazard ratios = 54.5 for patients who underwent surgery and 10.1 for patients who did not undergo surgery; P<0.001 for both). Patients without emergence of KRAS ctDNA within 1 year after surgery showed significantly better prognosis irrespective of recurrence (P<0.001). No detection or disappearance of KRAS ctDNA within 6 months of treatment was significantly correlated with therapeutic responses to first-line chemotherapy (P<0.001). Changes in KRAS status provided critical information for the prediction of therapeutic responses.ConclusionsOur study showed for the first time that detection of KRAS ctDNA levels within a short period enables the prediction of prognosis and therapeutic responses in patients with pancreatic cancer.

The impairment of the stability of the chromosomal structure facilitates the abnormal segregation of chromosomes, thus increasing the risk of carcinogenesis. Chromosomal stability during segregation is managed by appropriate methylation at the centromere of chromosomes. Insufficient methylation, or hypomethylation, results in chromosomal instability. The centromere consists of satellite alpha repetitive sequences, which are ideal targets for DNA hypomethylation, resulting in the overexpression of satellite alpha transcript (SAT). The overexpression of SAT has been reported to induce the abnormal segregation of chromosomes. In this study, we verified the oncogenic pathway via chromosomal instability involving DNA hypomethylation and the overexpression of SAT. For this purpose, we constructed lentiviral vectors expressing SAT and control viruses and then infected human mammary epithelial cells with these vectors. The copy number alterations and segregation errors of chromosomes were evaluated by microarray-based comparative genomic hybridization (array CGH) and immunocytochemistry, respectively. The levels of hypomethylation of satellite alpha sequences were determined by MethyLight polymerase chain reaction. Clinical specimens from 45 patients with breast cancer were recruited to verify the data in vitro. The results of immunocytochemistry revealed that the incidence of segregation errors was significantly higher in the cells overexpressing SAT than in the controls. An array CGH identified the specific chromosomes of 8q and 20q as frequent sites of copy number alterations in cells with SAT overexpression, although no such sites were noted in the controls, which was consistent with the data from clinical specimens. A regression analysis revealed that the expression of SAT was significantly associated with the levels of hypomethylation of satellite alpha sequences. On the whole, the overexpression of SAT led to chromosomal instability via segregation errors at specific chromosomes in connection with DNA hypomethylation, which was also recognized in clinical specimens of patients with breast cancer. Thus, this oncogenic pathway may be involved in the development of breast cancer.

KRAS mutated circulating tumor DNA (MctDNA) can be monitored in the blood of patients with metastatic colorectal cancer (mCRC), but dynamic changes have not been determined. Four hundred and fifty-seven plasma samples were collected prospectively from 85 mCRC patients who underwent chemotherapy. MctDNA in plasma was detected by droplet digital PCR, and the percentage of MctDNA in total circulating cell-free DNA was calculated. KRAS assessment in tumor tissues showed 29 patients with the mutant-type (MT) and 56 patients with the wild-type (WT). Twenty-three of 29 MT patients (79.3%) and 28 of 56 WT patients (50.0%) showed MctDNA. Emergence of MctDNA was recognized during treatments with various drugs. Regardless of KRAS status in tumor tissues, patients with MctDNA in blood showed poor progression-free survival with first-line treatment. Median percentage of MctDNA accounted for 10.10% in MT patients and 0.22% in WT patients. These differences between MT and WT likely affected patterns of changes in MctDNA. KRAS monitoring identified dynamic changes in MctDNA, such as continuous, intermittent, and transient changes (quick elevation and disappearance). Emergence of MctDNA involved drug resistance, except for transient changes, which were seen in WT patients and likely corresponded with the drug response. Transient changes could be involved in recovery of sensitivity to anti-EGFR antibody in WT patients. Monitoring MctDNA during various treatments showed dynamic changes in KRAS status and could provide useful information for determining treatments for patients with mCRC.

Regorafenib has shown survival benefits in metastatic colorectal cancer patients who were exacerbated after all standard therapies. Some patients, however, exhibit severe adverse events (AEs) resulting in treatment discontinuation. Therefore, the selection of patients likely to benefit from regorafenib is crucial. Twenty patients were treated with regorafenib for metastatic colorectal cancer; 122 plasma samples were taken from 16 of these patients for monitoring of circulating tumor DNA (ctDNA) in the blood. The treatment response, AEs, overall survival (OS), progression-free survival (PFS) and tumor morphologic changes on CT images were evaluated. KRAS mutant ctDNA was determined using digital PCR. Median PFS and OS were 2.5 and 5.9 months, respectively. Treatment was discontinued because of disease progression (PD) in 10 patients, and AEs in another 10 patients. AEs included hyperbilirubinemia, severe fatigue and skin rash. Hyperbilirubinemia was seen in two patients with multiple bilateral liver metastases, and severe fatigue in another 2 patients with poor performance status (PS). These severe AEs resulted in treatment discontinuation. Ten patients had a median PFS of 2.1 months with AE related discontinuation; PD occurred at 3.5 months (p=0.00334). Four patients exhibited a morphologic response, achieving better PFS times of 3.5, 5.3, 5.6 and 14.2 months. Emergence of the KRAS mutation in ctDNA was observed during anti-EGFR antibody treatment in 3 patients among 11 with KRAS wild-type tumors; it was detectable in the blood prior to radiographic detection of PD. Moreover, the KRAS mutation declined in two patients during regorafenib monotherapy. These patients were re-challenged with anti-EGFR antibody. Patients with extensive multiple liver metastases or poor PS are unlikely to benefit from regorafenib. Patients with a morphologic response will probably benefit from regorafenib with adequate management of other AEs. KRAS monitoring in ctDNA could be useful regarding treatment response and in determining treatment strategy.

Although epithelial-mesenchymal transition (EMT) has been implicated as the pivotal event in metastasis, there is insufficient evidence related to EMT in clinical settings. Intratumor heterogeneity may lead to underestimation of gene expression representing EMT. In the present study, we investigated the expression of EMT-associated genes and microRNAs in primary colorectal cancer while considering intratumor heterogeneity. One-hundred and thirty-three multiple spatially separated samples were obtained from 8 patients with metastatic colorectal cancers and 8 with non-metastatic colorectal cancers, from the tumor center (TC), invasive front (IF) and metastasis. Differences in gene and microRNA expression were investigated by microarray and quantitative reverse-transcription PCR. Gene expression microarray analysis detected 7920 sites showing differing levels of gene expression among the TC, IF and metastasis. Expression of the EMT-associated gene zinc-finger E-box-binding homeobox 1 (ZEB1) significantly increased in the IF (p<0.01). To exclude individual differences, the expression ratio between TC and IF in each tumor was applied to analysis. This approach enabled recognition of the activation of the VEGF and Wnt signaling pathways, which were involved in metastasis via promotion of EMT. While no activation of these pathways was seen at the TC, regardless of whether tumors were metastatic or non-metastatic, they were preferentially activated at the IF in metastatic tumors, where high ZEB1 expression was seen in connection with decreased miR-200c expression. Multiple sampling in a tumor revealed that heterogeneous ZEB1 expression induced by EMT-associated signaling pathways played a pivotal role in metastasis via regulation of miR-200c.