Can population differences in chemotherapy outcomes be inferred from differences in pharmacogenetic frequencies?

Inter-ethnic differences in drug handling and frequencies of pharmacogenetic variants are increasingly being characterized. In this study, we systematically assessed the feasibility of inferring ethnic trends in chemotherapy outcomes from inter-ethnic differences in pharmacogenetic variant frequencies. Frequencies of 51 variants and chemotherapy outcomes of East Asian and Caucasian colorectal cancer patients on standard chemotherapy regimens were summarized by meta-analyses, and variant frequencies were validated by MassARRAY analysis. Inferences of relative chemotherapy outcomes were made by considering minor allele function and population differences in their frequency. Significant population differences in genotype distributions were observed for 13/23 (60%) and 27/35 (77%) variants in the meta-analyses and validation series, respectively. Across chemotherapy regimens, East Asians had lower rates of grade 3/4 toxicity for diarrhea and stomatitis/mucositis than Caucasians, which was correctly inferred from 13/18 (72%, P=0.018) informative genetic variants. With appropriate variant selection, inferring relative population toxicity rates from population genotype differences may be relevant.

Ultrasonographic evaluation of the caudal vena cava in dogs with right-sided heart disease.

INTRODUCTION/OBJECTIVES: In humans with impaired right-sided cardiac function, the caudal vena cava (CVC) diameter serves as a marker of venous congestion. This study aimed to investigate whether ultrasonographic CVC variables could identify the presence of right-sided congestive heart failure (R-CHF) in dogs with right-sided heart disease (RHD). ANIMALS: Fifty client-owned control dogs and 67 dogs with RHD were enrolled. The dogs with RHD were subdivided into the non-R-CHF (n = 43) and R-CHF (n = 24) groups. MATERIALS AND METHODS: We measured and compared the ultrasonographic CVC variables and echocardiographic variables among the groups. Receiver operating characteristic (ROC) curve analysis was performed to calculate the sensitivity and specificity of the variables at optimal cutoff values. RESULTS: We obtained the highest accuracies of the ratio of the shortest diameter (SD) of the minimal CVC area to the aorta diameter (Ao) during inspiration [SD(min)/Ao] and of the ratio of SD(min) to the longest diameter of the minimal CVC area during inspiration [LD(min),SD/LD(min)], with high sensitivities, specificities, and an area under the ROC curve greater than 0.925. CONCLUSIONS: In addition to the echocardiographic assessment of right-sided cardiac function, the CVC variables in this study, especially SD(min)/Ao and SD/LD(min), would be useful diagnostic indices for identifying R-CHF in dogs with RHD.

Novel lipoprotein density profiling in laminitic, obese, and healthy horses.

Lipoproteins are water-miscible macromolecules enabling the transport of lipids in blood. In humans, altered proportions of lipoproteins are used to detect and classify metabolic diseases. Obesity and obesity-related comorbidities are common in horses. The pathophysiology of obesity is poorly understood and likely multifactorial. Development of new diagnostic tests to identify horses at risk of developing obesity to implement preventative measures is critical; however, a necessary first step to accomplish this goal is to improve our understanding of the pathophysiology of disease. Thus, the objective of this study was to characterize and compare lipoprotein profiles of horses with normal and excess body conditions, with and without laminitis using a novel method of continuous lipoprotein density profiling (CLPDP). Comparisons were made between 4 groups of horses: (1) laminitic, obese horses (n = 66); (2) laminitic, nonobese horses (n = 35); (3) nonlaminitic, obese horses (n = 41); and (4) nonlaminitic, nonobese horses (n = 95). Lipoprotein profiling, including evaluation of triglyceride-rich lipoprotein (TRL), low-density lipoproteins (LDLs), and high-density lipoproteins (HDLs) was performed using CLPDP, and all 4 groups were compared. A significant difference was observed among groups for the subfractions TRL, LDL1, LDL2, HDL2b, HDL2a, HDL3a, HDL3b, HDL3c, and total HDL. This is the first known description of CLPDP to characterize equine lipid profiles and holds promise as a useful method for lipid characterization of horses.

Low expression of gamma-glutamyl hydrolase mRNA in primary colorectal cancer with the CpG island methylator phenotype.

The CpG island methylator phenotype (CIMP+) in colorectal cancer (CRC) is defined as concomitant and frequent hypermethylation of CpG islands within gene promoter regions. We previously demonstrated that CIMP+ was associated with elevated concentrations of folate intermediates in tumour tissues. In the present study, we investigated whether CIMP+ was associated with a specific mRNA expression pattern for folate- and nucleotide-metabolising enzymes. An exploratory study was conducted on 114 CRC samples from Australia. mRNA levels for 17 genes involved in folate and nucleotide metabolism were measured by real-time RT-PCR. CIMP+ was determined by real-time methylation-specific PCR and compared to mRNA expression. Candidate genes showing association with CIMP+ were further investigated in a replication cohort of 150 CRC samples from Japan. In the exploratory study, low expression of gamma-glutamyl hydrolase (GGH) was strongly associated with CIMP+ and CIMP+-related clinicopathological and molecular features. Trends for inverse association between GGH expression and the concentration of folate intermediates were also observed. Analysis of the replication cohort confirmed that GGH expression was significantly lower in CIMP+ CRC. Promoter hypermethylation of GGH was observed in only 5.6% (1 out of 18) CIMP+ tumours and could not account for the low expression level of this gene. CIMP+ CRC is associated with low expression of GGH, suggesting involvement of the folate pathway in the development and/or progression of this phenotype. Further studies of folate metabolism in CIMP+ CRC may help to elucidate the aetiology of these tumours and to predict their response to anti-folates and 5-fluorouracil/leucovorin.

Jun NH2-terminal kinase phosphorylation of p53 on Thr-81 is important for p53 stabilization and transcriptional activities in response to stress.

The p53 tumor suppressor protein plays a key role in the regulation of stress-mediated growth arrest and apoptosis. Stress-induced phosphorylation of p53 tightly regulates its stability and transcriptional activities. Mass spectrometry analysis of p53 phosphorylated in 293T cells by active Jun NH2-terminal kinase (JNK) identified T81 as the JNK phosphorylation site. JNK phosphorylated p53 at T81 in response to DNA damage and stress-inducing agents, as determined by phospho-specific antibodies to T81. Unlike wild-type p53, in response to JNK stimuli p53 mutated on T81 (T81A) did not exhibit increased expression or concomitant activation of transcriptional activity, growth inhibition, and apoptosis. Forced expression of MKP5, a JNK phosphatase, in JNK kinase-expressing cells decreased T81 phosphorylation while reducing p53 transcriptional activity and p53-mediated apoptosis. Similarly transfection of antisense JNK 1 and -2 decreased T81 phosphorylation in response to UV irradiation. More than 180 human tumors have been reported to contain p53 with mutations within the region that encompasses T81 and the JNK binding site (amino acids 81 to 116). Our studies identify an additional mechanism for the regulation of p53 stability and functional activities in response to stress.

Infrequent K-ras activation in superficial-type (flat) colorectal adenomas and adenocarcinomas.

Clinicopathological evidence is accumulating that a superficial-type (flat) colorectal tumor is a distinct neoplastic entity. To clarify the genetic characteristics of this tumor, we investigated the K-ras gene mutations and morphological features of 43 tumors of this type. A mutation of the K-ras codon 12 was detected in only 5 (16%) of 31 adenomas and 2 (17%) of 12 adenocarcinomas. The presence or absence of this mutation was not correlated with the tumor size or stage or with histopathological findings. None of these tumors had a mutation in codon 13 or exon 2, including codon 61. This low incidence of K-ras mutations (16%) suggests that superficial-type colorectal tumors are etiologically distinct from ordinary colorectal polypoid tumors and that there may be an alternative pathway of colorectal tumorigenesis.

Reduction in formation and growth of 1,2-dimethylhydrazine-induced aberrant crypt foci in rat colon by docosahexaenoic acid.

The effect of intragastric gavage administration of docosahexaenoic acid (DHA) on the formation of 1,2-dimethylhydrazine (DMH)-induced aberrant crypt foci in rat colon was investigated. Male F344 rats were treated three times s.c. with 20 mg/kg of DMH and were given either 0.7 ml of DHA or water intragastrically 5 times a week for 4, 8, or 12 weeks from the day before the first carcinogen treatment. The numbers of DMH-induced aberrant crypt foci per colon after 4, 8, and 12 weeks of DHA treatment were approximately 40% of those in the respective control groups, and the differences were statistically significant. The numbers of foci reached plateau levels at 8 weeks in both the DHA-treated and control groups. The mean number of aberrant crypts per focus was also significantly smaller in the group given DHA than that in the control group at each time. These results suggest that DHA suppresses the formation and growth of aberrant crypt foci and has a preventive effect on colon carcinogenesis.

Distinct pattern of p53 phosphorylation in human tumors.

The protein product of the tumor suppressor gene p53 is phosphorylated on multiple residues by several protein kinases. Using a battery of 10 antibodies developed against different phosphorylated and acetylated residues of p53, we compared the pattern of p53 phosphorylation and acetylation in tumor-derived cell lines, tumor samples, and non-neoplastic cells. Irrespective of tumor types or the presence of p53 mutation, phosphorylation and acetylation of p53 was substantially higher in samples obtained from tumor tissues than those found in non-transformed samples. Among the 10 sites analysed, phosphorylation of residues 15, 81, 392, and acetylation were among the more frequent modifications. Analysis of two of the more abundant phosphorylation or acetylation sites on p53 is sufficient to detect 72% of tumor-derived p53 proteins. The distinct pattern of p53 phosphorylation and acetylation in human tumors may offer a new means to monitor the status and activity of p53 in the course of tumor development and progression.

Analysis of JNK, Mdm2 and p14(ARF) contribution to the regulation of mutant p53 stability.

Identification of Mdm2 and JNK as proteins that target degradation of wt p53 prompted us to examine their effect on mutant p53, which exhibits a prolonged half-life. Of five mutant p53 forms studied for association with the targeting molecules, two no longer bound to Mdm2 and JNK. Three mutant forms, which exhibit high expression levels, showed lower affinity for association with Mdm2 and JNK in concordance with greater affinity to p14(ARF), which is among the stabilizing p53 molecules. Monitoring mutant p53 stability in vitro confirmed that, while certain forms of mutant p53 are no longer affected by either JNK or Mdm2, others are targeted for degradation by JNK/Mdm2, albeit at lower efficiency when compared with wt p53. Expression of wt p53 in tumor cells revealed a short half-life, suggesting that the targeting molecules are functional. Forced expression of mutant p53 in p53 null cells confirmed pattern of association with JNK/Mdm2 and prolonged half-life, as found in the tumor cells. Over-expression of Mdm2 in either tumor (which do express endogenous functional Mdm2) or in p53 null cells decreased the stability of mutant p53 suggesting that, despite its expression, Mdm2/JNK are insufficient (amount/affinity) for targeting mutant p53 degradation. Based on both in vitro and in vivo analyses, we conclude that the prolonged half-life of mutant p53 depends on the nature of the mutation, which either alters association with targeting molecules, ratio between p53 and targeting/stabilizing molecules or targeting efficiency.

Combined analysis of microsatellite instability and K-ras mutation increases detection incidence of normal samples from colorectal cancer patients.

Microsatellite instability (MI) and K-ras oncogene mutation have been widely used as biomarkers of genetic changes in colorectal cancer (CRC). Each of these biomarkers was independently found in normal-appearing colonic mucosa at stages preceding the development of CRC, albeit at a relatively low incidence. To assess the potential value of combined MI and K-ras mutation analysis in the detection of normal-appearing colonic mucosa samples taken from patients with CRC, we have chosen to analyze multiple (3-7) normal colonic mucosa samples and the respective colorectal tumor tissues from 20 patients with CRC. As a control, we have used 54 normal mucosa samples obtained from 9 autopsies of patients without CRC. In at least 1 of 5 loci analyzed, MI was found in 8 of 20 patients via analysis of multiple normal-appearing colonic mucosa samples from each patient. Combined analysis of MI and mutant ras alleles in normal-appearing colonic mucosa samples enabled the identification of 11 of 20 patients with CRC. None of the 54 normal colonic mucosa samples obtained from 9 patients without CRC were found to carry mutant ras or MI. The ability to detect 55% of patients with CRC via the analysis of normal mucosa samples provides an important advance in our approach toward early detection of individuals who may be at risk to develop this tumor type.

MiFish, a set of universal PCR primers for metabarcoding environmental DNA from fishes: detection of more than 230 subtropical marine species.

We developed a set of universal PCR primers (MiFish-U/E) for metabarcoding environmental DNA (eDNA) from fishes. Primers were designed using aligned whole mitochondrial genome (mitogenome) sequences from 880 species, supplemented by partial mitogenome sequences from 160 elasmobranchs (sharks and rays). The primers target a hypervariable region of the 12S rRNA gene (163-185 bp), which contains sufficient information to identify fishes to taxonomic family, genus and species except for some closely related congeners. To test versatility of the primers across a diverse range of fishes, we sampled eDNA from four tanks in the Okinawa Churaumi Aquarium with known species compositions, prepared dual-indexed libraries and performed paired-end sequencing of the region using high-throughput next-generation sequencing technologies. Out of the 180 marine fish species contained in the four tanks with reference sequences in a custom database, we detected 168 species (93.3%) distributed across 59 families and 123 genera. These fishes are not only taxonomically diverse, ranging from sharks and rays to higher teleosts, but are also greatly varied in their ecology, including both pelagic and benthic species living in shallow coastal to deep waters. We also sampled natural seawaters around coral reefs near the aquarium and detected 93 fish species using this approach. Of the 93 species, 64 were not detected in the four aquarium tanks, rendering the total number of species detected to 232 (from 70 families and 152 genera). The metabarcoding approach presented here is non-invasive, more efficient, more cost-effective and more sensitive than the traditional survey methods. It has the potential to serve as an alternative (or complementary) tool for biodiversity monitoring that revolutionizes natural resource management and ecological studies of fish communities on larger spatial and temporal scales.

Lineage-specific RUNX3 hypomethylation marks the preneoplastic immune component of gastric cancer.

Runt domain transcription factor 3 (RUNX3) is widely regarded as a tumour-suppressor gene inactivated by DNA hypermethylation of its canonical CpG (cytidine-phosphate-guanidine) island (CGI) promoter in gastric cancer (GC). Absence of RUNX3 expression from normal gastric epithelial cells (GECs), the progenitors to GC, coupled with frequent RUNX3 overexpression in GC progression, challenge this longstanding paradigm. However, epigenetic models to better describe RUNX3 deregulation in GC have not emerged. Here, we identify lineage-specific DNA methylation at an alternate, non-CGI promoter (P1) as a new mechanism of RUNX3 epigenetic control. In normal GECs, P1 was hypermethylated and repressed, whereas in immune lineages P1 was hypomethylated and widely expressed. In human GC development, we detected aberrant P1 hypomethylation signatures associated with the early inflammatory, preneoplastic and tumour stages. Aberrant P1 hypomethylation was fully recapitulated in mouse models of gastric inflammation and tumorigenesis. Cell sorting showed that P1 hypomethylation reflects altered cell-type composition of the gastric epithelium/tumour microenvironment caused by immune cell recruitment, not methylation loss. Finally, via long-term culture of gastric tumour epithelium, we revealed that de novo methylation of the RUNX3 canonical CGI promoter is a bystander effect of oncogenic immortalization and not likely causal in GC pathogenesis as previously argued. We propose a new model of RUNX3 epigenetic control in cancer, based on immune-specific, non-CGI promoter hypomethylation. This novel epigenetic signature may have utility in early detection of GC and possibly other epithelial cancers with premalignant immune involvement.

Gene mutation as a target for early detection in cancer diagnosis.

The increasing number of genetic aberrations implicated in the development of human cancer has prompted a search to detect them at the earliest possible stage of their formation. Of the many such genetic changes identified thus far, relatively few meet the standard for markers in early diagnosis and prognosis, namely that the genetic modifications occur during the early onset phase of cancer development. Parallel to the increasing number of such genes is the growing availability of technologies using more powerful and cost-efficient methods that enable mass screening for genetic alterations. The purpose of this review is to summarize the currently available genes that can serve as markers for early detection of cancers and methods that allow their detection.

Effect of docosahexaenoic acid on azoxymethane-induced colon carcinogenesis in rats.

The effect of intragastric gavage administration of docosahexaenoic acid (DHA) on azoxymethane (AOM)-induced colon carcinogenesis in rats was studied. Male F344 rats were treated s.c. with 15 mg/kg of AOM once a week for 2 weeks and were given either 0.7 ml of DHA or water intragastrically twice a week starting the day before the first carcinogen treatment. The number of crypt multiplicity (number of crypts/focus) of aberrant crypt foci (ACF) in the colon were measured after 4, 12, and 36 weeks. The numbers and average crypt multiplicities of ACF induced by AOM were significantly lower after 12 and 36 weeks in animals given DHA. DHA also reduced the incidence of spontaneous ACF in animals without carcinogen treatment. Colorectal tumor incidence and number of tumors per rat after 36 weeks were slightly, but not significantly, lower in the DHA-treated group. These results suggest that DHA slightly suppresses colon carcinogenesis, and a possibility that warrants further study.

Detection of ki-ras mutation in nonneoplastic mucosa of Japanese patients with colorectal cancers.

About 50% of colorectal adenocarcinoma in humans have been reported to contain mutated Ki-ras gene. To provide a better understanding for the possible role of this mutation and to examine whether its presence can reliably predict a risk of colorectal cancer, we have analyzed the normal appearing mucosa of patients with colorectal adenocarcinoma. With an Enriched PCR procedure, we can detect mutated Ki-ras allele in the presence of 10(3) to 10(4) normal alleles. Only by this procedure was Ki-ras mutation detected in the non-neoplastic colonic mucosa of 9 out of 50 (18%) Japanese patients with colorectal cancer. This analysis indicated that epithelial cells which carry mutated Ki-ras gene were 100- to 1000-fold less frequent in the non-neoplastic mucosa than at the tumor site. The presence of ras gene mutation in normal appearing mucosa points to a previous exposure which had initiated the multistage process of colorectal carcinogenesis.

Analysis of mutant K-ras in multiple sites of normal appearing mucosa of colorectal cancer patients.

While 50% of colorectal tumors were found to harbor K-ms codon 12 mutation, only 18% of the respective patients contain this mutation in the normal appearing tissue when enriched PCR, a sensitive method that enables detection of one mutant allele in 10(4) normal alleles, is used. To determine whether the lower percentage could be attributed to the low incidence of this mutation or to the method of sampling, we have analyzed multiple normal appearing samples obtained from the same patient. Of 90 non-neoplastic mucosal samples collected from 20 patients with colorectal cancer, K-ras codon 12 mutation was identified in 6 samples taken from 5 patients. These results indicate that only one or two of the multiple samples contain mutant ras alleles. The presence of mutant ras alleles in the normal appearing tissues did not always correlate with that in the tumor site with respect to its presence and/or the type of base pair alterations, indicating independent or late events. While pointing to the importance of proper sampling method, the ability to detect ms mutation in normal mucosal tissues suggests it may serve as a useful biomarker of internal/external exposure which precede colorectal cancer development.

Desmoplastic reaction of gastric carcinoma: a light- and electron-microscopic immunohistochemical analysis using collagen type-specific antibodies.

The desmoplastic reaction in ten cases of gastric carcinoma was investigated light and electron immunohistochemically by using monospecific antibodies to collagen types. In addition to type I and III collagens, type V collagen was constantly recognized in the fibrous stroma, increasingly of the scirrhous carcinoma. Type IV collagen delineated the basement membranes of carcinoma nests linearly with occasional discontinuity, whereas in the scirrhous carcinoma, it was present along the thick bundles of collagenous fibers. Immunoelectron microscopic studies revealed that type I and III collagens were distributed on the collagen fibers, and type V collagen was stained in the margin of these fibers. These antibodies also reacted in the rough endoplasmic reticulum of fibroblasts or myofibroblasts in a few cases. Type IV collagen was localized in the periphery of smooth muscle cells, endothelial cells of collapsed capillaries, and myofibroblasts scattered in the stroma of scirrhous carcinoma. Carcinoma cells were not reactive with any antibodies examined. These findings suggest that type V collagen, as well as type I and III collagens, is involved in the formation of desmoplastic stroma, and that these collagens are reactively synthesized by fibroblasts and myofibroblasts in some interaction with invading carcinoma cells.

Biochemical and immunohistochemical studies on the scirrhous carcinoma of human stomach.

Tumor mass of the stomach from patients with scirrhous carcinoma was analyzed biochemically and immunohistochemically to elucidate whether or not infiltrating carcinoma cells are directly responsible for overproductions of collagen in the lesion. Collagen content per unit transverse section of the tumor was two to four times higher than the normal. Of particular interest was that the contents of hyaluronic acid and chondroitin sulfate were five to ten times higher than the normal, suggesting that cells in the lesion of the tumor are in an actively proliferating stage. Immunohistochemical observations using type-specific anti-collagen antibodies and anti-carcinoembryonic antigen antibody revealed that type IV collagen was diffusely distributed through the tumor stroma of submucosa and fragmented regions of muscle layer, along with dense fibrous components composed of type I and type III collagens. Stroma cells in the lesion were often stained with antibody to type IV collagen. In contrast, carcinoma cells were with antibody to type I collagen, but not with antibodies to type III and type IV collagen. Quantitative analysis of the collagen production by isolated stroma cells and undifferentiated (KATO-III) and highly differentiated (MKN-28) carcinoma cells in culture in the presence and absence of a combination of the conditioned medium of these cells has shown that the scirrhous carcinoma of stomach results from the "stroma reaction" of stroma cells induced by infiltrating malignant epithelium.

Altered expression of beta-catenin and c-erbB-2 in early gastric cancer.

To investigate the possible relationship between altered expression (loss of membranous staining or nuclear accumulation) of beta-catenin and invasion/metastasis in early gastric cancer (EGC), beta-catenin was detected immunohistochemically in 116 cases of EGC, including 86 differentiated and 30 undifferentiated carcinomas. In parallel, immunohistochemical expression of c-erbB-2 was analyzed in all EGC cases. Regardless of histological type, altered expression of beta-catenin was found in 47% of mucosal carcinomas and 89% of carcinomas with submucosal invasion (p<0.001). Of particular interest is that beta-catenin alteration was found in almost all EGCs with lymph node metastasis, even though no significant statistical comparison could be made. These results suggest that molecular changes resulting in abnormal beta-catenin expression participate in the process of submucosal invasion and metastasis. While loss of expression was preferentially observed in undifferentiated EGCs, nuclear accumulation was found exclusively in 24% of differentiated EGCs. c-erbB-2 was overexpressed in only 16% of differentiated EGCs but there was no correlation between this overexpression and invasion or metastasis. However, it is intriguing that 12 out of 14 cases with c-erbB-2 overexpression also showed altered beta-catenin expression, suggesting that both molecules are involved in the development of a certain set of differentiated EGCs.