Circulating cell-free DNA methylation patterns as non-invasive biomarkers to monitor colorectal cancer treatment efficacy without referencing primary site mutation profiles.

This study investigates methylation patterns in circulating cell-free DNA (ccfDNA) for their potential role in colorectal cancer (CRC) detection and the monitoring of treatment response. Through methylation microarrays and quantitative PCR assays, we analyzed 440 samples from The Cancer Genome Atlas (TCGA) and an additional 949 CRC samples. We detected partial or extensive methylation in over 85% of cases within three biomarkers: EFEMP1, SFRP2, and UNC5C. A methylation score for at least one of the six candidate regions within these genes' promoters was present in over 95% of CRC cases, suggesting a viable detection method. In evaluating ccfDNA from 97 CRC patients and 62 control subjects, a difference in methylation and recovery signatures was observed. The combined score, integrating both methylation and recovery metrics, showed high diagnostic accuracy, evidenced by an area under the ROC curve of 0.90 (95% CI = 0.86 to 0.94). While correlating with tumor burden, this score gave early insight into disease progression in a small patient cohort. Our results suggest that DNA methylation in ccfDNA could serve as a sensitive biomarker for CRC, offering a less invasive and potentially more cost-effective approach to augment existing cancer detection and monitoring modalities, possibly supporting comprehensive genetic mutation profiling.

Neutrophil­to­lymphocyte ratio before each chemotherapy line predicts clinical outcomes in patients with unresectable gastric cancer.

The neutrophil-to-lymphocyte ratio (NLR) is a well-known prognostic biomarker for patients with gastric cancer (GC). However, for patients with GC treated with palliative chemotherapy, the predictive values of NLR remain obscure. Therefore, the present study evaluated the clinical impact of NLR in patients with GC treated with a series of chemotherapies. The present study retrospectively evaluated 83 patients with unresectable GC who received a series of chemotherapies. NLR in the blood was calculated before each chemotherapy initiation (before 1st-, 2nd- and 3rd-line treatment). Of the 83 patients enrolled, 56 patients (67%) received 2nd-line chemotherapy and 34 patients (41%) received 3rd-line chemotherapy. NLR at 1st-line ranged from 0.72 to 48.9 (median NLR, 3.00). Therefore, the median NLR of 3.00 was used as a definite cut-off value throughout the present study. All patients were dichotomized into NLR-high (>3.00) and NLR-low group (<3.00) by NLR evaluated before each line of chemotherapy. The median overall survival (OS) time of the low-NLR group was better than that of the high-NLR group from 1st-line to 3rd-line treatment (1st-line: 18.1 vs. 8.0 months, P=0.06; 2nd-line: 10.7 vs. 4.5 months, P=0.0001; 3rd-line: 8.7 vs. 4.7 months, P=0.003). Of the 24 patients treated with 3rd-line nivolumab, patients with low NLR exhibited better OS than those with high NLR (8.3 months in low-NLR and 6.6 months in high-NLR, P=0.06). In conclusion, NLR should be performed before each chemotherapy line in the clinical setting and may predict outcomes in patients with unresectable GC, including those treated with nivolumab.

Genomically Stable Gastric Cancer Characterized by Hypomethylation in Wnt Signal Cascade.

INTRODUCTION: Gastric cancer is divided into four subtypes by their molecular features linked with genetic alterations, e.g., Epstein-Barr virus (EBV), microsatellite instability-high (MSI-high), chromosomal instability (CIN), and genomically stable (GS), called as TCGA classification. In this study, we tried to clarify the epigenetic features of the four GC subtypes according to aberrant methylation status in 23 loci. METHODS: A total of 98 gastric cancers and their normal gastric mucosa samples were included in this study. We divided gastric cancers into TCGA subtypes which were determined in line with MSI-high, EBV, CIN, to GS by their molecular features. The 13 loci of polymorphic microsatellite sequences were used to determine loss of heterogeneity for the detection of CIN. The MSI status was determined by three mononucleotide repeat markers. Infection of EBV was determined by recovering EBV BNRF1 sequence from genomic DNA collected from gastric cancers. Methylation status of 23 loci was investigated by the combined bisulfite restriction analysis. Status of other findings, e.g., KRAS mutations, HER2 expression status, and infection of helicobacter pylori were confirmed. RESULTS: Gastric cancers were divided into MSI (13%), EBV (7%), CIN (53%), and GS (27%). By histological classification, poorly differentiated adenocarcinoma was more in tumors categorized in MSI-high, and GS and signet-ring cell carcinoma (sig) were more in GS. Among the 23 loci investigated their methylation status, 18 loci were significantly hypermethylated in caner tissues. An unsupervised clustering divided gastric cancers into two clusters and revealed that most GS tumors clustered together in a cluster that exhibited lower methylation levels, distinct from the other subtypes. The inter-variable clustering revealed that a cluster contained the three loci (SFRP2-region 1/2 and APC) belonging to the Wnt signal cascade (Wnt-associated loci). The mean methylation score of Wnt-associated loci was the lowest in GS tumors (MSI-high: 2.7 [95% confidence interval, 2.3-2.9]; EBV: 2.1 [1.2-3.1]; CIN: 2.4 [2.2-2.7]; GS: 1.3 [0.8-0.7]). In contrast, the mean methylation score of the other 15 loci was significantly higher in MSI-high, while that in GS was as same as that in EBV or CIN (MSI-high: 10.4 [8.3-12.4]; EBV: 5.7 [1.7-9.7]; CIN: 4.4 [3.6-5.1]; GS: 3.4 [2.2-4.6]). Additionally, the lower methylation score of Wnt-associated loci was observed only in sig tumors. CONCLUSIONS: GS subtype tumors have the potential to possess distinct signatures in DNA hypomethylation profiles in Wnt signaling pathway, especially in sig.

Concordance of acquired mutations between metastatic lesions and liquid biopsy in metastatic colorectal cancer.

AIM: To evaluate whether PCR-reverse sequence-specific oligonucleotide can examine the concordance between liquid biopsy and metastatic lesions with acquired resistance. MATERIALS & METHODS: We examined acquired mutations in chemoresistant lesions and blood obtained from four patients with RAS wild-type metastatic colorectal cancer who underwent treatment with anti-epidermal growth factor receptor antibodies. RESULTS: In one patient, metastatic lesions harbored diverse acquired mutations in KRAS in all seven metastases; the two acquired mutations were detectable in blood collected after the patient acquired resistance. None of the other patients exhibited liquid biopsy mutations, except one, with a BRAF mutation confirmed in primary tumor and peritoneal dissemination. CONCLUSION: Liquid biopsy based on PCR-reverse sequence-specific oligonucleotide is a successful procedure for capturing acquired mutations with precise information on the RAS mutational spectrum.

Clinical and epigenetic features of colorectal cancer patients with somatic POLE proofreading mutations.

BACKGROUND: Mutations in the POLE gene result in an ultra-hypermutated phenotype in colorectal cancer (CRC); however, the molecular characterisation of epigenetic alterations remains unclear. We examined the genetic and epigenetic profiles of POLE-mutant CRC to elucidate the clinicopathological features of the associated genetic and epigenetic alterations. RESULTS: Tumour tissues (1,013) obtained from a cohort of patients with CRC were analysed to determine associations between the proofreading domain mutations of POLE with various clinicopathological variables, microsatellite instability (MSI) status, BRAF and KRAS mutations, and the methylation status of key regions of MLH1, MGMT, and SFRP2 promoters by calculating the methylation scores (range 0-6). Only four cases (0.4%) exhibited pathogenic POLE hotspot mutations (two p.P286R [c.857C > G], one p.V411L [c.1231G > C], and p.S459F [c.1376C > T] each), which were mutually exclusive to BRAF and KRAS mutations and MSI. CRC patients were divided into four subgroups: patients with POLE mutations (POLE, 0.4%, n = 4), patients with both MSI and extensive methylation in MLH1 (MSI-M, 2.9%, n = 29), patients with MSI but no extensive methylation in MLH1 (MSI-U, 3.6%, n = 36), and patients without MSI (non-MSI, 93.2%, n = 944). The POLE group was younger at diagnosis (median 52 years, P < 0.0001), with frequent right-sided tumour localisation (frequency of tumours located in the right colon was 100%, 93.1%, 36.1%, and 29.9% in POLE, MSI-M, MSI-U, and non-MSI, respectively; P < 0.0001), and was diagnosed at an earlier stage (frequency of stages I-II was 100%, 72.4%, 77.8%, and 46.6% in POLE, MSI-M, MSI-U, and non-MSI, respectively, P < 0.0001). The mean methylation score in POLE was not different from that in MSI-U and non-MSI, but the methylation signature was distinct from that of the other subgroups. Additionally, although the examined number of POLE-mutant tumours was small, the number of CD8-positive cells increased in tumours with partial methylation in the MLH1 gene. CONCLUSIONS: CRC patients with POLE proofreading mutations are rare. Such mutations are observed in younger individuals, and tumours are primarily located in the right colon. Diagnosis occurs at an earlier stage, and distinct epigenetic alterations may be associated with CD8 cell infiltration.

Molecular Characterization of Second Primary Endometrial Cancer.

BACKGROUND/AIM: The objective of this study was to determine the molecular and clinicopathological features, as well as the prognosis of patients with endometrial cancer (EC) having prior malignancy (second primary EC: SPEC) compared with those without a history of prior malignancy (first primary EC: FPEC). MATERIALS AND METHODS: We enrolled 294 FPEC patients and 32 SPEC patients who had undergone surgical resection with curative intent. EC was divided into four groups according to Cancer Genome Atlas Research Network (TCGA) classification. RESULTS: SPEC patients having greater than a 10-year interval from prior malignancy had risk factors including type II histology, deeper myometrial invasion, cervical invasion, and copy number high (CNH) phenotype compared with patients having less than a 10-year interval (p=0.007, p=0.002, p=0.015 and p=0.001). CONCLUSION: SPEC patients having greater than a 10-year interval from prior malignancy possessed numerous high-risk factors for EC.

Upregulation of microRNA-31 is associated with poor prognosis in patients with advanced colorectal cancer.

Colorectal cancer (CRC) manifests after the accumulation of genetic and epigenetic alterations along with tumor microenvironments. MicroRNA (miRNA/miR) molecules have been revealed to serve in critical roles in the progression various types of cancer, and their expression level is often an important diagnostic, predictive or prognostic biomarker. The aim of the present study was to evaluate the potential of miRNAs as prognostic biomarkers for patients with advanced CRC. miRNA arrays were performed on CRC specimens obtained from tumors with various molecular statuses [e.g. KRAS proto-oncogene, GTPase (KRAS)/B-Raf proto-oncogene, serine/threonine kinase (BRAF)/microsatellite instability (MSI)], and their paired normal mucosal specimens. The miRNA array revealed that miR-31-5p (miR-31) was specifically upregulated in CRCs with the BRAF V600E mutation, the results of which were supported by subsequent analysis of a dataset retrieved from The Cancer Genome Atlas (TCGA) database, which contained information regarding 170 patients with CRC including 51 BRAF-mutant CRCs. Of our cohort of 67 patients with stage IV CRC, 15 (22%) and 4 (6%) showed KRAS and BRAF V600E mutations, respectively. Since the median miR-31 expression was 3.45 (range, 0.004-6330.531), the cut-off value was chosen as 3.5, and all tumors were categorized into two groups accordingly (high-/low-miR-31 expression). The high miR-31 expression group (n=33) was significantly associated with a poorer mortality (univariate hazard ratio=2.12; 95% confidence interval, 0.23-0.95; P=0.03) and exhibited a shorter median survival time (MST; 20.1 months) compared with the low miR-31 expression group (n=34) (MST, 38.3 months; P=0.03), indicating that miR-31 is a promising prognostic biomarker for patients with advanced CRC. Thus, performing a functional analysis of miR-31 expression may lead to the development of new targeted therapies for the various genetic subtypes of CRC.

Heterogeneity of Epigenetic and Epithelial Mesenchymal Transition Marks in Hepatocellular Carcinoma with Keratin 19 Proficiency.

OBJECTIVE: Keratin 19 (K19) expression is a potential predictor of poor prognosis in patients with hepatocellular carcinoma (HCC). To clarify the feature of K19-proficient HCC, we traced epigenetic footprints in cultured cells and clinical materials. PATIENTS AND METHODS: In vitro, KRT19 promoter methylation was analyzed and 5-aza-2'-deoxycytidine with trichostatin A (TSA) treatment was performed. Among 564 surgically resected HCCs, the clinicopathological relevance of K19-proficent HCCs was performed in comparison with hepatocytic (HepPar-1 and arginase-1), epithelial-mesenchymal transition (E-cadherin and vimentin), biliary differentiation-associated (K7 and NOTCH-1) markers, and epigenetic markers (KRT19 promoter/long interspersed nucleotide element-1 [LINE-1] methylation status). RESULTS: KRT19 promoter methylation was clearly associated with K19 deficiency and 5-aza-2'-deoxycytidine with TSA treatment-stimulated K19 re-expression, implicating DNA methylation as a potential epigenetic process for K19 expression. After excluding HCCs with recurrence, TNM stage as IIIB or greater, preoperative therapy, transplantation, and combined hepatocellular cholangiocarcinoma, we assessed 125 of 564 HCC cases. In this cohort, K19 expression was found in 29 HCCs (23.2%) and corresponded with poor survival following surgery (p = 0.025) and extrahepatic recurrence-free survival (p = 0.017). Compared with K19-deficient HCCs, lower KRT19 promoter methylation level was observed in K19-proficient HCCs (p < 0.0001). Conversely, HCC with genome-wide LINE-1 hypermethylation was frequently observed in K19-proficient HCCs (p = 0.0079). Additionally, K19 proficiency was associated with K7 proficiency (p = 0.043), and reduced E-cadherin and HepPar-1 expression (p = 0.043 and p < 0.0001, respectively). CONCLUSIONS: K19-proficient HCC exhibited poor prognosis owing to extrahepatic recurrence, with molecular signatures differing from those in conventional cancer stem cells, providing novel insights of the heterogeneity underlying tumor development.

Detection of circulating microRNAs with Ago2 complexes to monitor the tumor dynamics of colorectal cancer patients during chemotherapy.

Because of the different forms of circulating miRNAs in plasma, Argonaute2 (Ago2)-miRNAs and extracellular vesicles (EV-miRNAs), we examined the two forms of extracellular miRNAs in vitro and developed a unique methodology to detect circulating Ago2-miRNAs in small volumes of plasma. We demonstrated that Ago2-miR-21 could be released into the extracellular fluid by active export from viable cancer cells and cytolysis in vitro. As miR-21 and miR-200c were abundantly expressed in both metastatic liver sites and primary lesions, we evaluated Ago2-miR-21 as a candidate biomarker of both active export and cytolysis while Ago2-miR-200c as a biomarker of cytolysis in plasma obtained from colorectal cancer (CRC) patients before treatment and in a series of plasma obtained from CRC patients with liver metastasis who received systemic chemotherapy. The measurement of Ago2-miR-21 allowed us to distinguish CRC patients from subjects without CRC. The trend in ΔCt values for Ago2-miR-21 and -200c during chemotherapy could predict tumor response to ongoing treatment. Thus, capturing circulating Ago2-miRNAs from active export can screen patients with tumor burdens, while capturing them from passive release by cytolysis can monitor tumor dynamics during chemotherapy treatment.

Clinical outcomes of women with ovarian metastases of colorectal cancer treated with oophorectomy with respect to their somatic mutation profiles.

We clarified the clinical prevalence of ovarian metastases from colorectal cancers (CRCs) in 296 female patients with CRC and evaluated clinical outcomes with relation to their mutational profiles, such as BRAF/KRAS mutation and microsatellite instability (MSI) status. The female CRCs were categorised into three subsets: CRCs with ovarian metastases [6.4% (n = 19), 5-year overall survival (OS) = 24.7%], CRCs with extra-ovarian metastases only [32.4% (n = 96), 5-year OS = 34.5%] and CRCs without any recurrence or metastasis [61.2% (n = 181), 5-year OS = 91.3%]. All patients with ovarian metastases underwent oophorectomy; of these, 9 who received preoperative chemotherapy had measurable metastases to extra-ovarian sites and the ovaries. Although 5 of 9 (56%) achieved partial response or complete response at extra-ovarian sites, no patient archived objective response at ovarian sites. Regarding the mutation profiles, in CRCs with extra-ovarian metastases only, the median survival time (MST) after initial treatments to progression to stage IV or recurrence was 13 [95% confidence interval (CI): 7-16 months] in BRAF-mutant and 34 months (95% CI: 22-58 months) in BRAF wild-type (P = 0.0033). Although ovarian metastases demonstrated poor response to systemic chemotherapy in CRCs with ovarian metastases, the MST after initial treatments to progression to stage IV or recurrence was 22 (95% CI: 21-25 months) in BRAF-mutant and 38 months (95% CI: 24-42 months) in BRAF wild-type (P = 0.0398). The outcomes of patients with ovarian metastases could be improved by oophorectomy regardless of their mutation profiles.

Clinical impact of endometrial cancer stratified by genetic mutational profiles, POLE mutation, and microsatellite instability.

BACKGROUND: The molecular characterization of endometrial cancer (EC) can facilitate identification of various tumor subtypes. Although EC patients with POLE mutations reproducibly demonstrate better prognosis, the outcome of patients with microsatellite instability (MSI) remains controversial. This study attempted to interrogate whether genetic stratification of EC can identify distinct subsets with prognostic significance. MATERIALS AND METHODS: A cohort of 138 EC patients who underwent surgical resection with curative intent was enrolled. Sanger sequencing was used to evaluate mutations in the POLE and KRAS genes. MSI analysis was performed using four mononucleotide repeat markers and methylation status of the MLH1 promoter was measured by a fluorescent bisulfite polymerase chain reaction (PCR). Protein expression for mismatch repair (MMR) proteins was evaluated by immunohistochemistry (IHC). RESULTS: Extensive hypermethylation of the MLH1 promoter was observed in 69.6% ECs with MLH1 deficiency and 3.5% with MMR proficiency, but in none of the ECs with loss of other MMR genes (P < .0001). MSI-positive and POLE mutations were found in 29.0% and 8.7% EC patients, respectively. Our MSI analysis showed a sensitivity of 92.7% for EC patients with MMR deficiency, and a specificity of 97.9% for EC patients with MMR proficiency. In univariate and multivariate analyses, POLE mutations and MSI status was significantly associated with progression-free survival (P = 0.0129 and 0.0064, respectively) but not with endometrial cancer-specific survival. CONCLUSIONS: This study provides significant evidence that analyses of proofreading POLE mutations and MSI status based on mononucleotide repeat markers are potentially useful biomarkers to identify EC patients with better prognosis.

Accuracy of four mononucleotide-repeat markers for the identification of DNA mismatch-repair deficiency in solid tumors.

BACKGROUND: To screen tumors with microsatellite instability (MSI) arising due to DNA mismatch repair deficiency (dMMR), a panel of five quasi-monomorphic mononucleotide-repeat markers amplified in a multiplex PCR (Pentaplex) are commonly used. In spite of its several strengths, the pentaplex assay is not robust at detecting the loss of MSH6-deficiency (dMSH6). In order to overcome this challenge, we designed this study to develop and optimize a panel of four quasi-monomorphic mononucleotide-repeat markers (Tetraplex) for identifying solid tumors with dMMR, especially dMSH6. METHODS: To improve the sensitivity for tumors with dMMR, we established a quasi-monomorphic variant range (QMVR) of 3-4 bp for the four Tetraplex markers. Thereafter, to confirm the accuracy of this assay, we examined 317 colorectal cancer (CRC) specimens, comprising of 105 dMMR [45 MutL homolog (MLH)1-deficient, 45 MutS protein homolog (MSH)2-deficient, and 15 MSH6-deficient tumors] and 212 MMR-proficient (pMMR) tumors as a test set. In addition, we analyzed a cohort of 138 endometrial cancers (EC) by immunohistochemistry to determine MMR protein expression and validation of our new MSI assay. RESULTS: Using the criteria of ≥ 1 unstable markers as MSI-positive tumor, our assay resulted in a sensitivity of 97.1% [95% confidence interval (CI) = 91.9-99.0%] for dMMR, and a specificity of 95.3% (95% CI = 91.5-97.4%) for pMMR CRC specimens. Among the 138 EC specimens, 41 were dMMR according to immunohistochemistry. Herein, our Tetraplex assay detected dMMR tumors with a sensitivity of 92.7% (95% CI = 80.6-97.5%) and a specificity of 97.9% (95% CI = 92.8-99.4%) for pMMR tumors. With respect to tumors with dMSH6, in the CRC-validation set, Tetraplex detected dMSH6 tumors with a sensitivity of 86.7% (13 of 15 dMSH6 CRCs), which was subsequently validated in the EC test set as well (sensitivity, 75.0%; 6 of 8 dMSH6 ECs). CONCLUSIONS: Our newly optimized Tetraplex system will help offer a robust and highly sensitive assay for the identification of dMMR in solid tumors.