Xenobiotic-metabolizing enzymes and cancer risk: correspondence between genotype and phenotype.

Metabolic activation and/or inactivation of a carcinogen is usually studied in appropriate in vitro systems but ultimately needs confirmation from in vivo studies, i.e. phenotype studies. It determines what initially happens to a carcinogen to which an organism is exposed. Consequently, it is of major importance to investigate the correspondence between any particular genotype of a carcinogen-metabolizing enzyme and its phenotypic expression, if any. The need to elucidate the relationship between genotype and phenotype is particularly important now, when methods for uncovering changes in genomic DNA are rather easy, even routine. There are several examples where the correspondence between a variant allele and an altered phenotype, measured by a probe drug or by some other means, has been elucidated (e.g. several alleles of CYP2D6). However, there are also cases where this correspondence has either not been studied (sometimes because of a lack of suitable probe substances) or has remained unclear (e.g. CYP1A1 or CYP2E1), despite case-control studies demonstrating an association between a variant allele and cancer risk. In the end one has to address the basic question as to how the genotype determines the phenotype and whether there is any biologically plausible link between the genotypic differences and cancer susceptibility. A knowledge of the complete sequence of events, from the gene to the outcome, would be helpful in unravelling the implications and possible preventive and treatment strategies to be employed in cases where clear associations between carcinogen-metabolizing enzymes and cancer susceptibility have been uncovered.     

Glioblastoma multiforme: the role of DSB repair between genotype and phenotype

Glioblastoma is the most frequent primary brain tumor in adults. The average survival time of less than 1 year did not improve notably over the last three decades. The dismal prognosis of glioblastoma patients is largely due to the striking radioresistance of this tumor. Here, we attempt a combined view on the genetics, the repair mechanisms and the radioresistance of glioblastoma. Specifically, we address the role of DNA-PKcs and the novel potential end-joining factor KUB3 in maintaining the radioresistant phenotype, the interrelationship between genetic lesions and repair mechanisms, and new perspectives that emerge from the identification of glioblastoma stem cells.     

Associations between catalase phenotype and genotype: modification by epidemiologic factors.

Catalase is an endogenous antioxidant enzyme that neutralizes hydrogen peroxide and is induced by oxidative challenge. A -262C --> T polymorphism in the promoter region of the gene (CAT) is associated with risk of several conditions related to oxidative stress. We sought to determine the functional effects of the CAT polymorphism on enzyme activity in erythrocytes and the potential modifying effects of demographic and lifestyle factors on genotype/phenotype relationships, using specimens and data from controls from breast and prostate cancer studies in Arkansas (n = 420). There was a dose-response reduction in catalase activity by genotype, with geometric means of 115.4 units/mg hemoglobin for those with CC genotypes, 82.1 units/mg for those with CT genotypes, and 73.5 units/mg for those with TT genotypes. Associations were only observed among Caucasians (P < 0.0001), with no effects among African Americans (P = 0.91), and were stronger among women than men, although numbers in stratified analyses were small. Differences in catalase activity by genotype were most pronounced among those in the highest tertiles of consumption of fruits and vegetables (-35%, P = 0.003), with weaker relationships among those who were lower consumers (-21.8%, P = 0.16). Among those with CC genotypes, there was no change in activity by consumption, but there were notable decreases in activity by tertiles of consumption for those with at least one T allele. These data indicate that the CAT -262C --> T polymorphism predicts a portion of catalase phenotype, which may be limited to Caucasians. Associations between genotype and phenotype were modified by dietary factors, illustrating the biochemical complexity of studies of genetic polymorphisms and disease risk.     

Mucin phenotype expression of gastric neuroendocrine neoplasms: analysis of histopathology and carcinogenesis

Background

Gastric neuroendocrine neoplasia has been classified as neuroendocrine tumor (NET), a less-malignant type, and neuroendocrine carcinoma (NEC), a more-malignant type. We investigated phenotypic expression profiles to clarify the differences between NET and NEC in terms of histopathology and carcinogenesis.

Methods

We assayed 86 cases of gastric neuroendocrine neoplasms (NET G1, n = 25; NET G2, n = 9; NEC, n = 52), using six exocrine markers (MUC5AC, human gastric mucin, MUC6, M-GGMC-1, MUC2, and CDX2).

Results

NEC frequently coexisted with adenocarcinomatous components (75 %; 39 of 52) and the majority (71.8 %; 28 of 39) showed intraglandular endocrine cell hyperplasia, although no cases of NET showed adenocarcinomatous components. Mucin phenotype significantly differed between NET and NEC; none of NET cases expressed any exocrine markers other than CDX2, although the majority of NEC (86.5 %; 45 of 52) expressed at least one or more exocrine markers with various positive rates for each marker (range, 8.2–74.0 %). Each NEC component showed only the phenotype expressed in the adenocarcinomatous component in the same tumor. Furthermore, double immunohistochemistry revealed dual expression of CDX2 and chromogranin A in half the NEC cases (23 of 46).

Conclusions

These data suggest that gastric NETs (G1 and G2) and NECs have different processes of carcinogenesis, and gastric NECs may be generated from preceding adenocarcinomas.     

The glycolytic phenotype in carcinogenesis and tumor invasion: insights through mathematical models

Malignant cells characteristically exhibit altered metabolic patterns when compared with normal mammalian cells with increased reliance on anaerobic metabolism of glucose to lactic acid even in the presence of abundant oxygen. The inefficiency of the anaerobic pathway is compensated by increased glucose flux, a phenomenon first noted by Otto Warburg approximately 80 years ago and currently exploited for 2-fluoro-2-deoxy-D-glucose-positron emission tomography imaging in clinical radiology. The latter has demonstrated the glycolytic phenotype is a near-universal phenomenon in human cancers. The potential role of the glycolytic phenotype in facilitating tumor invasion has been investigated through mathematical models of the tumor-host interface. Modified cellular automaton and diffusion reaction models demonstrate protons will diffuse from the tumor into peritumoral normal tissue subjecting nontransformed cells adjacent to the tumor edge to an extracellular pH significantly lower than normal. This leads to normal cell death via p53-dependent apoptosis pathways, as well as degradation of the interstitial matrix, loss of intercellular gap junctions, enhanced angiogenesis, and inhibition of the host immune response to tumor antigens. Transformed cells maintain their proliferative capacity in acidic extracellular pH because of mutations in p53 or some other component in the apoptosis pathways. This allows tumor cells to remain proliferative and migrate into the peritumoral normal tissue producing the invasive phenotype. Mathematical models of invasive cancer based on tumor-induced acidification are consistent with extant data on tumor microenvironment and results from clinical positron emission tomography imaging, including the observed correlation between tumor invasiveness and glucose utilization. Novel treatment approaches focused on perturbation of the tumor microenvironment are predicted from the mathematical models and are supported by recent clinical data demonstrating the benefits of azotemia and metabolic acidosis in survival of patients with metastatic renal cancer. The evolutionary basis for adoption of the glycolytic phenotype during carcinogenesis remains unclear because it appears to confer significant competitive disadvantages on the tumor cells due to of inefficient energy production and expenditure of resources to remove the acid byproducts. We propose that the glycolytic phenotype represents a successful adaptation to environmental selection parameters because it confers the ability to invade. That is, the glycolytic phenotype allows the cell to move from the microenvironment of a premalignant lesion to adjacent normal tissue. There it competes with normal cells that are less fit than the populations within the tumor in a microenvironment of relative substrate abundance. The consequent unrestrained proliferation allows the glycolytic phenotype to emerge simultaneous with the transition from a premalignant lesion to an invasive cancer.     

Maintenance of primary tumor phenotype and genotype in glioblastoma stem cells

The clinicopathological heterogeneity of glioblastoma (GBM) and the various genetic and phenotypic subtypes in GBM stem cells (GSCs) are well described. However, the relationship between GSCs and the corresponding primary tumor from which they were isolated is poorly understood. We have established GSC-enriched neurosphere cultures from 15 newly diagnosed GBM specimens and examined the relationship between the histopathological and genomic features of GSC-derived orthotopic xenografts and those of the respective patient tumors. GSC-initiated xenografts recapitulate the distinctive cytological hallmarks and diverse histological variants associated with the corresponding patient GBM, including giant cell and gemistocytic GBM, and primitive neuroectodermal tumor (PNET)-like components. This indicates that GSCs generate tumors that preserve patient-specific disease phenotypes. The majority of GSC-derived intracerebral xenografts (11 of 15) demonstrated a highly invasive behavior crossing the midline, whereas the remainder formed discrete nodular and vascular masses. In some cases, GSC invasiveness correlated with preoperative MRI, but not with the status of PI3-kinase/Akt pathways or O(6)-methylguanine methyltransferase expression. Genome-wide screening by array comparative genomic hybridization and fluorescence in situ hybridization revealed that GSCs harbor unique genetic copy number aberrations. GSCs acquiring amplifications of the myc family genes represent only a minority of tumor cells within the original patient tumors. Thus, GSCs are a genetically distinct subpopulation of neoplastic cells within a GBM. These studies highlight the value of GSCs for preclinical modeling of clinically relevant, patient-specific GBM and, thus, pave the way for testing novel anti-GSC/GBM agents for personalized therapy.     

Fulminant clonal expansion of large granular lymphocytes. Characterization of their morphology, phenotype, genotype, and function

A 39-year-old woman exhibited abrupt malignant transformation of the large granular lymphocytes (LGL) after a chronic course of T gamma-lymphoproliferative disease (T gamma-LPD). The T gamma-lymphocytes were CD2+, CD3-, CD8-, CD16+, Leu7-, and Leu19+ with morphologic characteristics of LGL. Newly appearing LGL were much larger and had more prominent azurophilic granules. Although fundamentally they had the same phenotype as the LGL in chronic stage, they showed increased Ia-like antigen and decreased CD16 antigen expressions. Immunoglobulin (Ig) G-kappa type monoclonal component was detected in the patient's serum. The LGL showed a germ-line configuration for T-cell receptor (TCR) beta and gamma chain genes, whereas the clonal chromosomal abnormalities indicated the neoplastic nature of the LGL. The LGL exhibited competent natural killer (NK), interleukin 2 (IL2) activated killer (AK), and antibody-dependent cell-mediated cytotoxicity (ADCC) activities. The LGL may have derived from NK cells at their mature stage with prethymic phenotype and may have influenced the homeostasis of the patient's humoral immune response.     

Malignant transformation of human melanocytes: induction of a complete melanoma phenotype and genotype.

Human melanoma provides a model to study malignant transformation and tumor progression. Expression of ras oncogenes in cultured normal human diploid melanocytes has induced a subset of phenotypic traits that are characteristic of malignant melanoma cells, including altered morphology, anchorage independence, induction of class II MHC antigens, up-regulation of the ganglioside GD3, and chromosomal abnormalities. However, other characteristics of melanoma, such as loss of expression of adenosine deaminase-binding protein and tumorigenicity, were not observed. We report here that melanocytes infected with a retrovirus containing the viral Ha-ras oncogene underwent complete transformation, acquiring all phenotypic characteristics of malignant melanomas observed in vivo. Transformation occurred in a sequential manner and was associated with spontaneous chromosomal instability. Cytogenetic analysis of transformed melanocytes indicated that the earliest structural chromosomal abnormalities were isochromosomes 6p and 9q followed by complete loss of chromosome 1p, all common karyotypic abnormalities described in human melanomas. The findings suggest that these chromosome regions which are deleted or relatively deficient may contain genes that are critical for the initiation and progression of the melanoma phenotype.     

N-Acetylation phenotype and genotype and risk of bladder cancer in benzidine-exposed workers

Several studies in subjects occupationally exposed to arylaminecarcinogens have shown increased risks for bladder cancer associatedwith the slow acetylator phenotype. To follow up these reports,a case-control study of N-acetylation and bladder cancer riskwas carried out among subjects occupationally exposed to benzidine,in benzidine dye production and use facilities in China. Thirty-eightbladder cancer cases and 43 controls from these factories wereincluded for study of acetylation phenotype, by dapsone administration,and for polymorphisms in the NAT2 gene, by a polymerase chainreaction (PCR)-based test. In contrast to previous studies,no increase in bladder cancer risk was found for the slow N-acetylationphenotype (OR= 0.3; 95% CI = 0.1–1.3) or for slow N-acetylation-associateddouble mutations in NAT2 (OR = 0.5; 95% CI = 0.1–1.8).Examination of specific mutations and adjustment for age, weight,city and tobacco use did not alter the results. When examinedby level of benzidine exposure in the cases, the bladder cancerrisks associated with low (OR = 0.3, 95% CI = 0.0–2.2),medium (OR = 0.7, 95% CI = 0.1–4.5) and high (OR = 0.6,95% CI = 0.1–3.5) exposure showed no interaction betweengenotype and benzidine exposure, within the range of exposuresexperienced by subjects in this study. This study, which isthe first to incorporate phenotypic and genotypic analyses,provides evidence that the NAT2-related slow N-acetylation polymorphismis not associated with an increased risk of bladder cancer inworkers exposed to benzidine, and may have a protective effect.     

The tyrosine phosphatase PTPRJ/DEP-1 genotype affects thyroid carcinogenesis

We recently isolated the r-PTPeta gene, which encodes a receptor-type tyrosine phosphatase protein that suppresses the neoplastic phenotype of retrovirally transformed rat thyroid cells. The human homologue gene PTPRJ/DEP-1 is deleted in various tumors. Moreover, the Gln276Pro polymorphism, located in the extracellular region of the gene, seems to play a critical role in susceptibility to some human neoplasias. Here we report the loss of heterozygosity (LOH) of PTPRJ in 11/76 (14.5%) informative thyroid tumors (including adenomas and carcinomas). We also looked for the Gln276Pro, Arg326Gln and Asp872Glu polymorphisms in exons 5, 6 and 13 of PTPRJ in 88 patients with thyroid tumors and in 54 healthy individuals. We found that the PTPRJ genotypes homozygous for the Gln276Pro and Arg326Gln polymorphisms, and the Asp872 allele were more frequent in thyroid carcinoma patients than in healthy individuals (P=0.032). In addition, PTPRJ LOH was more frequent in thyroid carcinomas of heterozygotes for Gln276Pro and Arg326Gln compared with homozygotes (P=0.006). This suggests that the presence of hemizygosity for these polymorphisms in the tumor facilitates tumor progression. These results indicate that the genotypic profile of PTPRJ affects susceptibility to thyroid carcinomas, and that allelic loss of this gene is involved in thyroid carcinogenesis.     

Renal carcinogenesis, hepatic hemangiomatosis, and embryonic lethality caused by a germ-line Tsc2 mutation in mice

Germ-line mutations of the human TSC2 tumor suppressor gene cause tuberous sclerosis (TSC), a disease characterized by the development of hamartomas in various organs. In the Eker rat, however, a germ-line Tsc2 mutation gives rise to renal cell carcinomas with a complete penetrance. The molecular mechanism for this phenotypic difference between man and rat is currently unknown, and the physiological function of the TSC2/Tsc2 product (tuberin) is not fully understood. To investigate these unsolved problems, we have generated a Tsc2 mutant mouse. Tsc2 heterozygous mutant (Tsc2+/-) mice developed renal carcinomas with a complete penetrance, as seen in the Eker rat, but not the angiomyolipomas characteristic of human TSC, confirming the existence of a species-specific mechanism of tumorigenesis caused by tuberin deficiency. Unexpectedly, approximately 80% of Tsc2+/- mice also developed hepatic hemangiomas that are not observed in either TSC or the Eker rat. Tsc2 homozygous (Tsc2-/-) mutants died around embryonic day 10.5, indicating an essential function for tuberin in mouse embryonic development. Some Tsc2-/- embryos exhibited an unclosed neural tube and/or thickened myocardium. The latter is associated with increased cell density that may be a reflection of loss of a growth-suppressive function of tuberin. The mouse strain described here should provide a valuable experimental model to analyze the function of tuberin and its association with tumorigenesis.     

Distribution and concordance of N-acetyltransferase genotype and phenotype in an American population.

Polymorphic arylamine N-acetyltransferase 2 (NAT2) status varies widely between individuals and ethnic groups and has been associated with susceptibility to several cancers. Few studies have reported the distribution of NAT2 status for Caucasian-American populations or evaluated the concordance between methods of assessment for cancer cases and controls. In our study, distribution of NAT2 status was classified by genotype and phenotype measurements in PANCAN, a population-based case-control study of pancreatic cancer, and concordance between measurements was evaluated for 33 cases and 222 controls. Major genotypes and alleles among controls were *5B/*6A, *5B/*5B, *4/*6A, and *5B/*4. One putative new allele was found in a single individual. Genotypes and phenotypes were classified as rapid or slow, according to a bimodal model. Presence of the *4 (wild-type) allele defined a NAT2 genotype as rapid. The NAT2 phenotype was analyzed by the caffeine assay. Ratios of 5-acetylamino-6-formylamino-3-methyluracil to 1-methylxanthine were determined, and individuals with values of > or =0.66 were identified as having a rapid phenotype. In our population, 58.1 and 59.5% of control subjects were classified as slow acetylators by phenotype and genotype, respectively. Concordance of NAT2 genotype and phenotype classification was 97.8% in the bimodal model. A similar analysis was completed for a trimodal model. Concordance of genotype and phenotype was high in cases (90.9%) and similar to controls; genotyping alone provided an efficient, accurate method of analysis for acetylator status. A comparison with two previous reports revealed subtle differences in genotype and allele distribution but exhibited overall similarity with other Caucasian-American populations.     

Hepatitis C virus 1b is the dominant genotype in HCV-related carcinogenesis: A case-control study

In an ongoing case-control study in Athens on the etiology of hepatocellular carcinoma (HCC), an analysis was made in order to assess whether HCV genotype 1b is associated with hepatocellular carcinoma (HCC). The HCV genotype was determined in 17 HCC patients, 87 patients with chronic hepatitis C (CHC) without cirrhosis (NC-CHC) and 23 patients with CHC and cirrhosis (C-CHC). HCV genotype 1b was detected in 14/17, 16/23 and 23/87 of HCC, C-CHC and NC-CHC respectively, The age- and gender-adjusted odds ratios contrasting HCC with NC-CHC and C-CHC with NC-CHC were 8.3 and 3.8 respectively. These data strongly support the hypothesis that HCV 1b is a stronger liver carcinogen than other HCV genotypes, probably through increased HCV replication and enhanced liver cytopathicity. © 1996 Wiley-Liss, Inc.     

Patients younger than 40 years with gastric carcinoma: Helicobacter pylori genotype and associated gastritis phenotype.

BACKGROUND: In the general population, Helicobacter pylori (H. pylori), particularly the cagA positive strain, has been associated with intestinal-type gastric carcinoma. Gastric carcinomas are rarely observed in patients age < or = 40 years. Host-related factors have been thought to be more important than environmental agents in these early-onset cancers. The aim of this study was to ascertain the possible role of H. pylori infection and that of cagA positive strains in the development of gastric carcinoma in these young patients. METHODS: In this case-control study, 105 gastric carcinoma patients (male-to-female ratio = 1.1; mean age, 34.4 years; range, 16-40 years) and an equal number of controls (matched for gender and age) were retrospectively selected from the same geographic area. The phenotypes of gastritis and H. pylori were histologically assessed, and the presence of the ureC gene, which is indicative of H. pylori infection, and the cagA genotype were determined by polymerase chain reaction. Gastric carcinoma risk was calculated by both univariate and multivariate statistical methods, taking into account the cancer phenotype, the gastritis phenotype detected in both patients and controls, and the H. pylori genotype. RESULTS: For 74 diffuse and 31 intestinal gastric carcinomas, multivariate logistic regression analysis produced results consistent with those of univariate statistical tests, showing a significant association between gastric carcinoma and both H. pylori infection (odds ratio [OR] = 2.79; 95% confidence interval [CI] = 1.52-5.11) and cagA positive status (OR = 2.94; 95% CI = 1.56-5.52). CONCLUSIONS: In young Italian patients with gastric carcinoma, the significant association with cagA positive H. pylori infection suggests that the bacterium has an etiologic role in both diffuse-type and intestinal-type gastric carcinoma.     

Immunoregulation of carcinogenesis: past, present, and future

Twenty years ago the concept that immunological mechanisms might regulate carcinogenesis was highly controversial. Today this concept survives in modified form and continues to evolve as our knowledge of the immune system, the etiology of cancer, and tumor antigens expands. This review illustrates how our ideas about immune surveillance have changed in recent years and considers the significance of these changes for the future directions of cancer immunobiology.     

CYP2A6 genotype, phenotype, and the use of nicotine metabolites as biomarkers during ad libitum smoking.

CYP2A6 inactivates nicotine to cotinine and cotinine to 3-hydroxycotinine. We investigated which of plasma nicotine and metabolites were most related to CYP2A6 genotype and smoking levels. We assessed demographic and smoking histories in 152 Caucasian ad libitum smokers, measured breath carbon monoxide (CO) levels, and determined plasma nicotine, cotinine, and 3-hydroxycotinine by high-performance liquid chromatography and CYP2A6 genotypes by PCR. Cigarettes per day was most closely related to CO (r = 0.60, P < 0.001) followed by plasma cotinine (r = 0.53, P < 0.001), whereas plasma cotinine was most strongly correlated with CO levels (r = 0.74, P < 0.001), confirming that cotinine is a good indicator of smoking levels; this was not limited by CYP2A6 variants. 3-Hydroxycotinine/cotinine is reported to be a good marker of CYP2A6 activity, and we found that the 3-hydroxycotinine/(cotinine + nicotine) ratio was most correlated with CYP2A6 genotype (r = 0.38, P < 0.001). Inclusion of the CYP2A6*12A allele strengthened the correlation (r = 0.46, P < 0.001), suggesting that the identification of novel alleles will continue to improve this relationship. Nicotine metabolism is slower in smokers, and we have shown that CYP2A6 is reduced by nicotine treatment in monkeys. Here, we found that plasma nicotine levels were inversely correlated with CYP2A6 activity (3-hydroxycotinine/cotinine, r = -0.41, P < 0.001) among those without CYP2A6 variants, suggesting a reduction in metabolism with higher nicotine levels. Together, these findings (a) confirm the use of plasma cotinine and CO as indicators of Caucasians' smoking levels, and that this is not limited by CYP2A6 genetic variation; (b) indicate that 3-hydroxycotinine/cotinine and 3-hydroxycotinine/(cotinine + nicotine) are moderately good indicators of the CYP2A6 genotype; and (c) support that nicotine exposure may reduce its own metabolism.