Prognostic utility of circulating tumor DNA methylation analysis in stage IV colorectal cancer.

BACKGROUND AND OBJECTIVES: Our aim in this study was to investigate the usefulness of circulating tumor (ct) DNA methylation analysis for predicting long-term outcomes after resection in Stage IV colorectal cancer (CRC). METHODS: Methylation analyses were performed on 95 plasma samples from patients with CRC who underwent surgery. The methylation status (relative methylation value: RMV) of CpG within the promoter region of three genes (CHFR, SOX11, and CDO1) was assessed to quantitative methylation-specific PCR (qMSP) analysis. RESULTS: In the patients who had undergone resection of the primary tumor and metastatic organs with curative intent, the CHFR-RMV high group had significantly worse recurrence-free survival (RFS) compared with the CHFR-RMV low group (p = 0.001). Multivariate analysis revealed that CHFR-RMV was a significant independent prognostic factor (hazard ratio = 2.63 (1.29-5.36); p = 0.008). In the patients who had undergone resection of the primary tumor with metastatic organs with curative intent after neoadjuvant systemic chemotherapy, the SOX11-RMV high group had significantly worse RFS compared with the SOX11-RMV low group (p = 0.004). CONCLUSIONS: The current study showed the usefulness of ctDNA methylation analysis for predicting the possibility of curative resection and long-term outcomes after resection in Stage IV CRC. A future prospective study is needed to obtain more conclusive results.

The methylation level of a single cancer risk marker gene reflects methylation burden in gastric mucosa.

BACKGROUND: Gastric cancer risk can be accurately predicted by measuring the methylation level of a single marker gene in gastric mucosa. However, the mechanism is still uncertain. We hypothesized that the methylation level measured reflects methylation alterations in the entire genome (methylation burden), induced by Helicobacter pylori (H. pylori) infection, and thus cancer risk. METHODS: Gastric mucosa of 15 healthy volunteers without H. pylori infection (G1), 98 people with atrophic gastritis (G2), and 133 patients with gastric cancer (G3) after H. pylori eradication were collected. Methylation burden of an individual was obtained by microarray analysis as an inverse of the correlation coefficient between the methylation levels of 265,552 genomic regions in the person's gastric mucosa and those in an entirely healthy mucosa. RESULTS: The methylation burden significantly increased in the order of G1 (n = 4), G2 (n = 18), and G3 (n = 19) and was well correlated with the methylation level of a single marker gene (r = 0.91 for miR124a-3). The average methylation levels of nine driver genes tended to increase according to the risk levels (P = 0.08 between G2 vs G3) and was also correlated with the methylation level of a single marker gene (r = 0.94). Analysis of more samples (14 G1, 97 G2, and 131 G3 samples) yielded significant increases of the average methylation levels between risk groups. CONCLUSIONS: The methylation level of a single marker gene reflects the methylation burden, which includes driver gene methylation, and thus accurately predicts cancer risk.

[A Case of Cecum Cancer with Lymph Node Metastasis and Invasion in the Superior Mesenteric Vein Treated with Combined SMV Resection and Reconstruction].

The patient was a 77-year-old woman. She visited her family doctor with a complaint of bloody stools, and was pointed out a Type 3 colon cancer in the cecum with a colonoscopy. In addition, an enlarged lymph node(#203)was found on the right side of the superior mesenteric vein(SMV). Laparoscopic surgery was initiated, and when the patient was moved to vascular processing, a firm adhesion of the lymph node(#203)was observed on the right side of the SMV. A small laparotomy was added, and a partial combined resection of the SMV was performed en bloc to complete the ileal resection. Histopathological findings showed T4b(transverse colon)N3M0, pStage Ⅲc, and metastatic lymph node(#203)showed evidence of invasion to the SMV. Adjuvant chemotherapy was administered, but lung metastases appeared 4 months and liver metastasis appeared 29 months after surgery. The patient was transferred to a different hospital for best supportive care(BSC)at 34 months after surgery.

[A Case of Recurrence in the Abdominal Wall and Perineal Region of Rectal Cancer Successfully Treated with Minimally Invasive Surgery and Radiation Therapy].

Local recurrence after rectal cancer surgery is often difficult to treat because there are few effective treatments. In this study, we report a case of parastomal and perineal recurrence after Miles' surgery in an elderly patient who had a favorable outcome after laparoscopic surgery and radiation therapy. Our results suggest that a combination of minimally invasive treatment may be effective in elderly patients and after polysurgery.

[A Case Report of a Giant GIST Markedly Responsive to Neoadjuvant Chemotherapy with Imatinib Mesylate].

Male in his 50s complaining of abdominal pain was referred to our hospital. Abdominal CT scan showed a giant tumor which had diameter of approximately 50 mm in lower rectum. A biopsy specimen was positive for CD34 and c-kit. Based on these findings, it was diagnosed as gastrointestinal stromal tumor(GIST). We treated the patient with neoadjuvant therapy using imatinib mesylate(IM)to reduce the tumor size and to avoid the extensive surgery. The patient started to take IM at a daily dose of 400 mg. After 3 months, CT and MRI revealed that the tumor size decreased(40% reduction). We performed the robot assisted intersphincteric resection(ISR). Although it has been 28 months since the surgery, there are no obvious signs of recurrence. A patient diagnosed with giant GIST could avoid an extensive surgery due to neoadjuvant therapy with IM.

[A Case Report for Peritoneal Recurrences by Laparoscopic Surgery after Colorectal Cancer Resection].

We report a cases report of colorectal cancer who underwent repeated resection for peritoneal recurrences by laparoscopic surgery. In 2013, a 70-year-old woman diagnosed with an ascending colon cancer underwent laparoscopic right hemicolectomy. The pathological diagnosis was tub2, pT4aN1M0, Stage Ⅲb. Postoperative adjuvant chemotherapy(uracil and tegafur/Leucovorin)was administered. PET-CT performed at 25 months after the surgery because of CEA elevation. It revealed a peritoneal recurrence in the pouch of Douglas. The following peritoneal recurrences were removed by laparoscopic Hartmann's procedure. Chemotherapy(5-fluorouracil/levofolinate/oxaliplatin/bevacizumab)was administered 11 courses and after that chemotherapy(5-fluorouracil/levofolinate/bevacizumab)was administered 6 courses. PET-CT performed 37 months after the second surgery revealed a peritoneal recurrence near the right ovary in the pouch of Douglas. The following peritoneal recurrences was removed. Chemotherapy(tegafur/gimeracil/oteracil/bevacizumab)was administered 11 courses. The long-term survival has been continued for 7 years and 7 months after first operation. It was considered that laparoscopic surgery for peritoneal recurrence in colorectal cancer is contributed to one of the surgical procedures in selected patients.

[A Case of Metastatic Colorectal Cancer with Hyperammonemic Encephalopathy Induced by mFOLFOX6 and SOX Therapy].

A 77-year-old man was given a diagnosis of pT4aN0M1a(PUL2), stage Ⅳ, RAS mutant type, after the operation for advanced ascending colon cancer. He was administered mFOLFOX6 plus Bmab as first-line chemotherapy. He showed consciousness disturbance on the 2nd day during the 6 cycles. Because of head computed tomography and magnetic resonance imaging showing no abnormal findings, we diagnosed convulsive seizure. His consciousness level gradually improved after intravenous infusion. He showed consciousness disturbance on the 2nd day during the 7 cycles again. Because blood ammonia level were high at 400µg/dL, he was diagnosed as hyperammonemic encephalopathy. His consciousness level rapidly recovered after branched chain amino acid(BCAA)infusion. SOX plus Bmab therapy was started as a post-treatment, he developed hyperammonemia(NH3 288µg/dL)again, on the 4th day during the 3 cycles. After taking of oral administration of BCAA and lactulose, the recurrence of hyperammonemic encephalopathy was not found. Therefore, 3 cycles of SOX plus Bmab therapy and 12 cycles of IRIS plus Bmab therapy were administered.

Rearing in strontium-enriched water induces vaterite otoliths in the Japanese rice fish, Oryzias latipes.

Sagittal otoliths, typically composed of aragonite, are frequently laid down rather as vaterite during growth in hatchery-reared fish populations. Sagittal vateritization is believed to impair individual hearing/balancing abilities, but the causal mechanism remains unclear. Here we experimentally demonstrated that rearing in Sr-rich water induces sagittal vateritization in the HdrR-II1 inbred strain of the Japanese rice fish, Oryzias latipes. Both sagittae were partly vateritized in 70% of individuals subjected to the Sr2+ treatment (n = 10), whereas fish reared in normal tap water showed no sagittal vateritization (n = 8). Our result is consistent with the theoretical prediction that vaterite becomes thermodynamically more stable than aragonite as the Sr2+ concentration in solution increases. A vateritic layer develops surrounding the original aragonitic sagitta in vateritized otoliths, some of which take on a comma-like shape. Electron probe microanalysis demonstrates that the vateritized phase is characterized by lower Sr2+ and higher Mg2+ concentrations than the aragonitic phase. It is unlikely that increased environmental Sr2+ is responsible for the sagittal vateritization in farmed fish. However, our findings likely help to establish an in vivo assay using O. latipes to understand the physiological process underlying the sagittal vateritization in farmed fish.

Positive Circumferential Resection Margin in Rectal Cancer Is a Robust Predictor of Poor Long-term Prognosis With Clinicopathological Bias Between Groups Compensated by Propensity-score Matching Analysis.

BACKGROUND/AIM: Circumferential resection margin (CRM) is the most reliable predictor of local and distant recurrence in locally-advanced rectal cancer (LARC). The present study was conducted to compare the long-term outcomes between CRM (+) and (-) groups using propensity-score (PS) matching analysis to compensate for bias between groups. PATIENTS AND METHODS: Of 563 consecutive patients with Stage II/III rectal cancer who were treated surgically with curative-intent at Juntendo University Hospital between Jan 1989 and Mar 2018, 412 patients were enrolled retrospectively in the study. The patients were divided into a CRM (+) group (n=21; 5.1%) and a CRM (-) group (n=391; 94.9%). RESULTS: In the entire cohort, recurrence-free survival (RFS), local recurrence-free survival (LRFS), non-local recurrence-free survival (NLRFS), and cancer-specific survival (CSS) were significantly worse among patients in the CRM (+) group compared with those in the CRM (-) group. Univariate analysis demonstrated patients in the CRM (+) group had significantly larger primary tumors (p=0.02), more frequently had open surgery (p=0.009), had an abdominoperineal resection (APR) procedure (p=0.01) and a T4 primary tumor (p<0.0001). After PS matching analysis, in the propensity-matched cohort, RFS, LRFS, NLRFS and CSS were significantly worse among patients in the CRM (+) group compared with those in the CRM (-) group. CONCLUSION: PS matching analysis demonstrated that RFS, LRFS, NLRFS, and CSS were significantly worse among patients in the CRM (+) group compared with those in the CRM (-) group. The present results indicate that CRM (+) is a robust predictor of long-term outcome of LARC, independent of tumor size.

Local recurrence of submucosal invasive colorectal cancer after endoscopic submucosal dissection revealed by copy number variation.

We report a case in which analysis of copy number variation revealed local recurrence of submucosal invasive colorectal cancer after curative endoscopic submucosal dissection (ESD). An 86-year-old man with a history of abdominoperineal resection of the rectum for rectal cancer underwent resection with ESD for early-stage sigmoid cancer 5 cm away from the stoma opening. At the same time, ileocecal resection was performed for advanced cecal cancer. Twelve months after ESD, advanced cancer occurred in the area of the ESD lesion. It was unclear if the cancer was a local recurrence after ESD, implantation of cecal cancer, or a new lesion. Copy number variation analysis performed for the three lesions revealed that the new lesion originated from residual tumor cells from ESD and was unlikely to be cecal cancer.

CHFR-Promoter-Methylation Status Is Predictive of Response to Irinotecan-based Systemic Chemotherapy in Advanced Colorectal Cancer.

BACKGROUND/AIM: We investigated whether promoter methylation of the checkpoint-with-forkhead-and-ring-finger-domains (CHFR) gene is a predictor of the efficacy of irinotecan-based systemic chemotherapy for advanced colorectal cancer (CRC) patients. MATERIALS AND METHODS: CHFR-promoter methylation was measured by quantitative methylation-specific PCR (qMSP). The histoculture drug response assay (HDRA) was used in vitro to analyze the correlation between CHFR-promoter methylation and the efficacy of the irinotecan-active-metabolite SN38 in colorectal-cancer tissues from 44 CRC patients. CHFR promoter-methylation was also analyzed for its correlation with clinical response to irinotecan-based systemic chemotherapy of 49 CRC patients. RESULTS: CHFR-promoter methylation significantly-positively correlated with inhibition of colon cancer by SN38 in the HDRA (p=0.002). CHFR-promoter methylation also significantly-positively correlated with clinical response to irinotecan-based systemic chemotherapy (p=0.04 for disease control). CHFR-promoter methylation also significantly-positively correlated (p=0.01) with increased progression-free survival for patients treated with irinotecan-containing FLOFIRI in combination with bevacizumab, the most-frequent regimen in the cohort. CONCLUSION: Sensitivity of advanced CRC patients to irinotecan-based systemic chemotherapy can be predicted by the extent of CHFR-promoter methylation.

Laparoscopic Surgery Is Acceptable for Elderly Patients With Colorectal Cancer: A Propensity Score-matched Study.

BACKGROUND/AIM: The study was performed to examine the suitability of laparoscopic surgery for elderly patients with colorectal cancer. PATIENTS AND METHODS: The subjects were 242 patients aged ≥80 years who underwent primary tumor resection of colorectal cancer using laparoscopic assisted colectomy (LAC, n=145) or open colectomy (OC, n=97). Propensity score matching used to balance the characteristics of the groups resulted in 76 patients being assigned to each group. RESULTS: Before matching, Glasgow Prognostic Score (GPS), American Society of Anesthesiologists physical status (ASA-PS), and previous abdominal surgery differed significantly between the groups (p<0.05), but after matching, all covariates were balanced (p≥0.05). Short-term outcomes were better after LAC (p<0.05), including fewer postoperative complications and less delirium. Regarding long-term outcomes, 5-year overall survival did not differ significantly between the groups (p=0.91). CONCLUSION: In elderly patients with colorectal cancer, short-term results are better after LAC than OC and long-term results are similar. These findings indicate that LAC is acceptable in this patient population.

Decrease in volume and density of foraminiferal shells with progressing ocean acidification.

Rapid increases in anthropogenic atmospheric CO2 partial pressure have led to a decrease in the pH of seawater. Calcifying organisms generally respond negatively to ocean acidification. Foraminifera are one of the major carbonate producers in the ocean; however, whether calcification reduction by ocean acidification affects either foraminiferal shell volume or density, or both, has yet to be investigated. In this study, we cultured asexually reproducing specimens of Amphisorus kudakajimensis, a dinoflagellate endosymbiont-bearing large benthic foraminifera (LBF), under different pH conditions (pH 7.7-8.3, NBS scale). The results suggest that changes in seawater pH would affect not only the quantity (i.e., shell volume) but also the quality (i.e., shell density) of foraminiferal calcification. We proposed that pH and temperature affect these growth parameters differently because (1) they have differences in the contribution to the calcification process (e.g., Ca2+-ATPase and Ω) and (2) pH mainly affects calcification and temperature mainly affects photosynthesis. Our findings also suggest that, under the IPCC RCP8.5 scenario, both ocean acidification and warming will have a significant impact on reef foraminiferal carbonate production by the end of this century, even in the tropics.

The mitochondrial genome of the gold-ringed cowry Monetaria annulus (Mollusca: Gastropoda: Cypraeidae) determined by whole-genome sequencing.

The mitochondrial genome of the cowry snail Monetaria annulus (Caenogastropoda: Cypraeoidea: Cypraeidae) was determined by whole-genome next-generation sequencing. The mitogenome is composed of 13 protein-coding genes (PCGs), 2 ribosomal RNA (12S and 16S) genes, and 22 transfer RNA genes (tRNAs). This gene order is consistent with the previously published mitochondrial genomes of other species belonging to the order Littorinimorpha. The superfamily Cypraeoidea was recovered as a sister clade to the group of Tonnoidea and Neogastropoda.

Convergent evolution of body color between sympatric freshwater fishes via different visual sensory evolution.

Although there are many examples of color evolution potentially driven by sensory drive, only few studies have examined whether distinct species inhabiting the same environments evolve similar body colors via shared sensory mechanisms. In this study, we tested whether two sympatric freshwater fish taxa, halfbeaks of the genus Nomorhamphus and ricefishes of the genus Oryzias in Sulawesi Island, converge in both body color and visual sensitivity. After reconstructing the phylogeny separately for Nomorhamphus and Oryzias using transcriptome-wide sequences, we demonstrated positive correlations of body redness between these two taxa across environments, even after phylogenetic corrections, which support convergent evolution. However, substantial differences were observed in the expression profiles of opsin genes in the eyes between Nomorhamphus and Oryzias. Particularly, the expression levels of the long wavelength-sensitive genes were negatively correlated between the taxa, indicating that they have different visual sensitivities despite living in similar light environments. Thus, the convergence of body colorations between these two freshwater fish taxa was not accompanied by convergence in opsin sensitivities. This system presents a case in which body color convergence can occur between sympatric species via different mechanisms.

Phenotypic plasticity and sexual dimorphism in size at post-juvenile metamorphosis: common-garden rearing of an intertidal gastropod with determinate growth.

Proximate factors of the intraspecific variation in molluscan shell morphology have long received attention in biology. The intertidal gastropod Monetaria annulus (Mollusca; Gastropoda; Cypraeidae) is particularly suitable for the study of variation in body size, because this species is a determinate grower in the sense that soft-body size shows no further increase after the juvenile stage. Cross-sectional field surveys on post-juvenile individuals have indicated that the mean body size varies widely among populations and is larger in females than in males within populations. To examine whether these patterns are due to genetic differences, we conducted a common-garden rearing experiment with juvenile individuals collected from two populations on Okinawa Island. After adjusting for among-individual differences in initial degree of development, statistical analyses revealed that this species exhibits female-biased sexual size dimorphism mediated by a longer development time rather than by faster growth rates in females. Although wild individuals show a remarkable size difference between populations, no size difference was found between the populations in the individuals reared in a common-garden condition. This result suggests that the among-population size difference does not have a genetic basis and is caused by phenotypic plasticity based on environmental heterogeneity among habitats.

Inter- and intrapopulation variation in thermal reaction norms for growth rate: evolution of latitudinal compensation in ectotherms with a genetic constraint.

In ectotherms, lower temperatures in high-latitude environments would theoretically reduce the annual growth rates of individuals. If slower growth and resultant smaller body size reduce fitness, individuals in higher latitudes may evolve compensatory responses. Two alternative models of such latitudinal compensation are possible: Model I: thermal reaction norms for growth rates of high-latitude individuals may be horizontally shifted to a lower range of temperatures, or Model II: reaction norms may be vertically shifted so that high-latitude individuals can grow faster across all temperatures. Model I is expected when annual growth rates in the wild are only a function of environmental temperatures, whereas Model II is expected when individuals in higher latitudes can only grow during a shorter period of a year. A variety of mixed strategies of these two models are also possible, and the magnitude of horizontal versus vertical variation in reaction norms among latitudinal populations will be indicative of the importance of "temperature" versus "seasonality" in the evolution of latitudinal compensation. However, the form of latitudinal compensation may be affected by possible genetic constraints due to the genetic architecture of reaction norms. In this study, we examine the inter- and intrapopulation variations in thermal reaction norms for growth rate of the medaka fish Oryzias latipes. Common-environment experiments revealed that average reaction norms differed primarily in elevation among latitudinal populations in a manner consistent with Model II (adaptation to "seasonality"), suggesting that natural selection in high latitudes prefers individuals that grow faster even within a shorter growing season to individuals that have longer growing seasons by growing at lower temperatures. However, intrapopulation variation in reaction norms was also vertical: some full-sibling families grew faster than others across all temperatures examined. This tendency in intrapopulation genetic variation for thermal reaction norms may have restricted the evolution of latitudinal compensation, irrespective of the underlying selection pressure.

Pharmacological characterization of histamine-gated chloride channels from the housefly Musca domestica.

The biogenic amine histamine (HA) is not only the neurotransmitter of photoreceptors but also has important roles in mechanosensory reception, temperature preference, sleep and olfactory processing in insects. Two cDNAs (MdhclA and MdhclB) that encode HA-gated chloride channel subunits (MdHCLA and MdHCLB) were cloned from the housefly Musca domestica. The cRNAs were injected into Xenopus laevis oocytes to examine the functions and pharmacological characteristics of MdHCLA and MdHCLB channels using a two-electrode voltage clamp method. HA was used to activate MdHCLA and MdHCLB channels to evoke inward currents with EC50s of 33.1µM and 6.28µM, respectively. 2-(3-Trifluoromethylphenyl)histamine, an HA H1 receptor agonist, was a partial agonist of MdHCLB receptors with an EC50 of 49.4µM. MdHCLB channels were also activated by γ-aminobutyric acid (GABA) and monoamines, such as octopamine, serotonin (5-HT) and dopamine (DA); 5-HT and DA also acted as competitive antagonists. GABA acted as a full agonist of MdHCLB receptors with an EC50 of 1.11mM. d-Tubocurarine, cimetidine and picrotoxinin were poor inhibitors of HA- and GABA-evoked currents in MdHCLB channels. Our data show that HCLB channels are more sensitive to agonists when compared with HCLA channels. HCLB channels are also affected by antagonists but insusceptible to known insecticides that target GABA- and glutamate-gated chloride channels.

Optimal growth pattern of defensive organs: the diversity of shell growth among mollusks.

Mollusks show a diversity of shell growth patterns. We develop a model for the dynamic resource allocation to defense organs and analyze it with the Pontryagin maximum principle. A typical optimal growth schedule is composed of the initial phase of soft-body growth without shell followed by a simultaneous growth of shell and soft body and finally the reproductive phase without growth (simultaneous shell growth). If the defensible predation risk is low or if the cost of defense is high, the optimal strategy is to have no shell (shell-less growth). If defensible predation pressure or general mortality differs before and after maturation, an additional three strategies, characteristic of the exclusive growth of shell or soft body, can be optimal (sequential shell growth, additional body-expansion growth, and additional callus-building growth). These optimal strategies are in accord with the patterns observed for mollusks. In particular, the growth strategies with exclusive growth phase of external shells are preferred when durophagous predation pressure after maturation is higher than that before maturation. This result explains the observation that many tropical gastropods with thickened shell lips spend their vulnerable juvenile phase in sheltered habitats.

Increasing costs due to ocean acidification drives phytoplankton to be more heavily calcified: optimal growth strategy of coccolithophores.

Ocean acidification is potentially one of the greatest threats to marine ecosystems and global carbon cycling. Amongst calcifying organisms, coccolithophores have received special attention because their calcite precipitation plays a significant role in alkalinity flux to the deep ocean (i.e., inorganic carbon pump). Currently, empirical effort is devoted to evaluating the plastic responses to acidification, but evolutionary considerations are missing from this approach. We thus constructed an optimality model to evaluate the evolutionary response of coccolithophorid life history, assuming that their exoskeleton (coccolith) serves to reduce the instantaneous mortality rates. Our model predicted that natural selection favors constructing more heavily calcified exoskeleton in response to increased acidification-driven costs. This counter-intuitive response occurs because the fitness benefit of choosing a better-defended, slower growth strategy in more acidic conditions, outweighs that of accelerating the cell cycle, as this occurs by producing less calcified exoskeleton. Contrary to the widely held belief, the evolutionarily optimized population can precipitate larger amounts of CaCO(3) during the bloom in more acidified seawater, depending on parameter values. These findings suggest that ocean acidification may enhance the calcification rates of marine organisms as an adaptive response, possibly accompanied by higher carbon fixation ability. Our theory also provides a compelling explanation for the multispecific fossil time-series record from ∼200 years ago to present, in which mean coccolith size has increased along with rising atmospheric CO(2) concentration.