Mutations of the BCR-ABL-kinase domain occur in a minority of patients with stable complete cytogenetic response to imatinib.

Residual leukemia is demonstrable by reverse transcriptase-polymerase chain reaction in most patients with chronic myeloid leukemia who obtain a complete cytogenetic response (CCR) to imatinib. In patients who relapse during imatinib therapy, a high rate of mutations in the kinase domain of BCR-ABL have been identified, but the mechanisms underlying disease persistence in patients with a CCR are poorly characterized. To test whether kinase domain mutations are a common mechanism of disease persistence, we studied patients in stable CCR. Mutations were demonstrated in eight of 42 (19%) patients with successful amplification and sequencing of BCR-ABL. Mutation types were those commonly associated with acquired drug resistance. Four patients with mutations had a concomitant rise of BCR-ABL transcript levels, two of whom subsequently relapsed; the remaining four did not have an increase in transcript levels and follow-up samples, when amplifiable, were wild type. BCR-ABL-kinase domain mutations in patients with a stable CCR are infrequent, and their detection does not consistently predict relapse. Alternative mechanisms must be responsible for disease persistence in the majority of patients.

Overexpression of C-terminally but not N-terminally truncated Myb induces fibrosarcomas: a novel nonhematopoietic target cell for the myb oncogene.

The myb oncogene encodes a DNA-binding transcriptional transactivator which can become a hematopoietic cell-transforming protein following the deletion of amino acid sequences from either its amino or carboxyl terminus. Although a number of hematopoietic tumors express terminally deleted variants of Myb, the involvement of truncated Myb in nonhematopoietic tumors has not been adequately investigated. To assess the full spectrum of Myb's oncogenic capability, a replication-competent retroviral vector (RCAMV) was used to express a full-length protein (C-Myb), an amino-terminally truncated protein (VCC- or delta N-Myb), a carboxyl-terminally truncated protein (T-Myb), or a doubly truncated protein (VCT-Myb) in vivo. These viruses were injected intravenously into 10-day chicken embryos, and the infected chicks were monitored for tumors. Approximately 4 to 8 weeks after hatching, the majority (30 of 39 [77%]) of animals infected with the T-Myb retrovirus (without 214 carboxyl-terminal residues) developed nodular muscle tumors which could be identified by both morphologic and immunohistochemical criteria as fibrosarcomas. Identically appearing tumors could also be found in the kidney of some T-Myb-infected animals. The T-Myb-induced fibrosarcomas expressed the appropriately sized T-Myb protein, contained an unaltered proviral T-myb gene, and showed clonal proviral integration sites. In comparison, no sarcomas were observed in any of the animals infected with the amino-terminally truncated (VCC- and delta N-Myb) or doubly truncated (VCT-Myb) viruses. A loss of carboxyl-terminal but not amino-terminal sequences can thus convert Myb into a potent in vivo transforming protein for nonhematopoietic mesenchymal cells. In comparison, a truncation of either or both ends of the protein can activate Myb into a hematopoietic cell-transforming protein.

Control of the expression of c-sis mRNA in human glioblastoma cells by phorbol ester and transforming growth factor beta 1.

The regulation of c-sis oncogene expression in human glioblastoma cell line A172 has been investigated using a sensitive RNA-RNA solution hybridization method. Enhanced expression of c-sis mRNA was induced by phorbol ester (PMA) and diacylglycerol, each of which activates protein kinase C. c-sis mRNA was also induced by transforming growth factor beta (TGF-beta). The response to PMA and TGF-beta was transient, and in each case the decrease in c-sis mRNA level following maximum stimulation occurred with a half-life similar to the mRNA half-life previously determined. Cycloheximide had no significant effect on the induction of c-sis mRNA by either PMA or TGF-beta. The increases in c-sis mRNA following addition of either PMA or TGF-beta correlated well with increases in c-sis transcription as observed by the nuclear run-on technique. In cells in which protein kinase C had been down-regulated, there was no inhibition of the c-sis mRNA response to TGF-beta. Furthermore in cells pretreated with TGF-beta, induction by PMA was unaffected. Thus the TGF-beta signal pathway does not involve activation of protein kinase C, and at least two initially distinct intracellular signaling routes lead to activation of c-sis gene expression in this glioblastoma cell line. The protein kinase inhibitor H7 abolished the ability of not only PMA but also of TGF-beta to induce c-sis mRNA. The ability of H7 to inhibit the TGF-beta stimulation suggests that a protein kinase other than protein kinase C is involved in the signal transduction by TGF-beta.

Development and evaluation of a secondary reference panel for BCR-ABL1 quantification on the International Scale.

Molecular monitoring of chronic myeloid leukemia patients using robust BCR-ABL1 tests standardized to the International Scale (IS) is key to proper disease management, especially when treatment cessation is considered. Most laboratories currently use a time-consuming sample exchange process with reference laboratories for IS calibration. A World Health Organization (WHO) BCR-ABL1 reference panel was developed (MR(1)-MR(4)), but access to the material is limited. In this study, we describe the development of the first cell-based secondary reference panel that is traceable to and faithfully replicates the WHO panel, with an additional MR(4.5) level. The secondary panel was calibrated to IS using digital PCR with ABL1, BCR and GUSB as reference genes and evaluated by 44 laboratories worldwide. Interestingly, we found that >40% of BCR-ABL1 assays showed signs of inadequate optimization such as poor linearity and suboptimal PCR efficiency. Nonetheless, when optimized sample inputs were used, >60% demonstrated satisfactory IS accuracy, precision and/or MR(4.5) sensitivity, and 58% obtained IS conversion factors from the secondary reference concordant with their current values. Correlation analysis indicated no significant alterations in %BCR-ABL1 results caused by different assay configurations. More assays achieved good precision and/or sensitivity than IS accuracy, indicating the need for better IS calibration mechanisms.

Induction of B cell lymphomas by overexpression of a Myb oncogene truncated at either terminus.

The c-myb oncogene encodes a nuclear transcriptional transactivator that is often terminally truncated in hematopoietic tumors. To directly assess the tumorigenic activity of full length and terminally-truncated variants of c-myb, we have overexpressed several structurally-altered forms of myb within an avian retroviral vector and have shown that overexpression of truncated (but not full length) myb transforms both myeloid cells in vitro and mesenchymal cells in vivo. In vivo infection with these truncated myb viruses is now shown to induce metastatic B cell lymphomas in a significant minority of animals. Evaluation of the lymphomas revealed two distinct mechanisms of myb-induced tumorigenesis. In most of the lymphomas, proviral DNA inserted into the endogenous chicken c-myb gene and promoted the expression of a 5'-truncated myb transcript encoding an amino terminal truncated protein. In comparison, some animals infected with a virus encoding a carboxyl (C) terminal truncated myb (T-myb) developed non-insertional B cell lymphomas that directly expressed the provirally-encoded T-myb gene. The lymphomagenic T-myb protein lacks 214 C terminal amino acids including all of the myb transcription inhibition domain. This novel lymphomagenic activity for a C terminal truncated myb suggests that a loss of regulatory sequences at either end of c-myb is sufficient to create a B cell-specific transforming gene.

Real-time polymerase chain reaction with fluorescent hybridization probes for the detection of prevalent mutations causing common thrombophilic and iron overload phenotypes.

We evaluated more than 450 patients with thrombophilia or iron overload for the presence of a factor V Leiden (R506Q), prothrombin G20210A, or HFE C282Y mutation using a standard method (polymerase chain reaction [PCR]-restriction fragment length polymorphism) and a comparative real-time PCR fluorescent resonance energy transfer (FRET) hybridization probe melting curve method. There was 100% concordance between the genotypes ascertained by the 2 methods (at each loci). In addition, phenotypic biochemical laboratory parameters measured on a subset of referred patients correlated with their respective genotypes. In the iron overload cohort, HFE C282Y homozygotes (n = 74) had significantly higher (P < .0001) transferrin saturation levels (74% +/- 25%) than did nonhomozygotes (n = 340; 51.4% +/- 28%), suggesting a genotype-dependent increase in body iron loads. In the thrombophilic cohort, the degree of activated protein C resistance (APCR), measured by a clotting time-based test, was associated significantly with the presence of 0 (n = 255; APCR = 2.59 +/- 0.26), 1 (n = 84; APCR = 1.61 +/- 0.13), or 2 (n = 5; APCR = 1.16 +/- 0.04) copies of the mutant factor V Leiden allele. As the fluorescent genotyping method required no postamplification manipulation, genotypes could be determined more quickly and with minimized risk of handling errors or amplicon contamination. In addition to these practical advantages, the FRET method is diagnostically accurate and clinically predictive of phenotypic, disease-associated manifestations.

Hepatic iron overload: direct HFE (HLA-H) mutation analysis vs quantitative iron assays for the diagnosis of hereditary hemochromatosis.

Among patients with hepatic iron overload, the distinction between hereditary hemochromatosis (HH), a common yet treatable genetic disease, and other causes of siderosis remains problematic. The recent discovery of a specific homozygous mutation (C282Y) in a novel major histocompatibility complex class I-like gene (named HLA-H or HFE) in 80% to 100% of well-characterized cases of HH suggests that direct DNA-based mutation analysis may help resolve this dilemma. To assess the clinical utility of direct HLA-H mutation analysis in a typical diagnostic setting, we measured genotypic and phenotypic parameters of iron overload in 37 subjects with biopsy-proven hepatic siderosis (2+ or greater) and in 127 healthy control subjects. The prevalence of C282Y homozygotes was significantly greater in the hepatic siderosis group (32%) than in the control group (0%), confirming the association between this homozygous mutation and hepatic iron overload. In the hepatic siderosis group, C282Y homozygotes had significantly higher hepatic iron and ferritin levels, a significantly lower prevalence of hepatitis C virus or alcoholic liver disease, but no significant difference in the saturation of serum transferrin. Of the 20 subjects with a hepatic iron index (HII) in the previously defined "hemochromatosis range" (>1.9), 9 (45%) were C282Y homozygotes. Of the 11 nonhomozygous subjects with an HII greater than 1.9 (presumed false-positive HIIs), 10 (91%) had hepatic cirrhosis compared with 3 of 9 (33%) homozygotes with an HII greater than 1.9 who had cirrhosis (P<.02). The HII thus has poor diagnostic specificity for predicting genotypic HH in patients with cirrhosis. We conclude that direct determination of the HLA-H C282Y genotype may be the single best diagnostic test for HH, particularly in patients with cirrhosis, for whom the HII is quite nonspecific.

Molecular detection of a common mutation in coagulation factor V causing thrombosis via hereditary resistance to activated protein C.

More than half of all patients with familial or recurring venous thrombosis have hereditary resistance to activated protein C (HRAPC) as the result of specific missense mutation in the gene for coagulation factor V. Because the mutant factor Va (with an Arg to Gln substitution at codon 506) cannot be cleaved and inactivated by activated protein C, carriers of this mutation are at significantly increased risk of venous thrombosis. We have recently introduced a direct polymerase chain reaction (PCR)-based clinical diagnostic test for the factor V codon 506 mutation based on the destruction of an Mnl I restriction site by the causative nucleotide substitution. To assess the accuracy of this PCR-based assay, we compared a functional clotting time test for HRAPC with the direct mutation test. Of 47 patients dually tested, only five had discrepant values for the functional test versus the DNA test. Either of these two complementary assays is useful for the accurate diagnosis of HRAPC. The DNA-based test is, however, specifically recommended for evaluation of anticoagulated patients or patients with borderline functional tests and confirmation of genotype in HRAPC families. In an additional analysis of 287 normal individuals, we found an extremely high prevalence of the mutated codon 506 allele-- approximately 4% in each of two different populations. The absence of disease in the majority of heterozygous carriers suggests that symptomatic thrombosis requires the simultaneous presence of both a mutated factor V protein and additional synergistic factors.

Role of a common mutation in the homocysteine regulatory enzyme methylenetetrahydrofolate reductase in ischemic stroke.

A common mutation in methylenetetrahydrofolate reductase (MTHFR), a homocysteine metabolic pathway enzyme, has been associated with increased homocysteine levels and increased risk for premature cardiovascular disease. The purpose of this study was to assess the association between the prevalence of the MTHFR mutation, hyperhomocysteinemia, and subtypes of ischemic stroke in an elderly population comprised of three age-balanced groups of patients. The presence of the C677T MTHFR mutation was determined by a direct polymerase chain reaction-based assay performed on blood samples from 136 patients with acute ischemic stroke, 95 patients with atherosclerotic risk factors for stroke (including some with a history of previous stroke or transient ischemic attack), and 52 healthy control subjects. The prevalence of the homozygous C677T mutation was not significantly higher in the elderly stroke patients (7%) than in the atherosclerotic risk (8%) or healthy elderly control (2%) groups. Plasma homocysteine levels were higher in the acute stroke patient group (14.5+/-4.5 micromol/L) and atherosclerotic risk patient group (14.6+/-6.2 micromol/L) compared with the control subjects (10.3+/-3.1 micromol/ L, P < 0.03). Homozygotes for the C677T MTHFR mutation did not have significantly higher homocysteine levels than non-homozygotes. Moderate hyperhomocysteinemia, though common in older patients with ischemic cerebrovascular disease, is not attributable, at least in this patient group, to a higher prevalence of the C677T MTHFR mutation.

Hereditary hemochromatosis: impact of molecular and iron-based testing on the diagnosis, treatment, and prevention of a common, chronic disease.

OBJECTIVE: To review the current state-of-the-art regarding the role of iron- and DNA-based testing on the detection, treatment, and prevention of hereditary hemochromatosis (HH), the most common single-gene disorder in white people. SOURCES: Review of the medical literature, with particular emphasis on recent reports of the impact of DNA-based testing on the detection of symptomatic and presymptomatic patients with HH. CONCLUSIONS: Hereditary hemochromatosis, a common autosomal recessive iron overload disorder (with a population prevalence of 0.3%-0.8%), is a common cause of preventable liver, heart, joint, and endocrine disease. Since the associated clinical signs and symptoms are nonspecific, an accurate HH diagnosis demands both a high index of suspicion and the direct laboratory demonstration of elevated iron parameters. The substantial public health burden of HH as a common, deadly, detectable, and treatable chronic disease has led the College of American Pathologists to recommend that "systematic screening for hemochromatosis is warranted for all persons over the age of 20 years." The recent discovery that most HH cases are the result of a single well-conserved homozygous missense mutation (C282Y) within a novel transferrin-receptor binding protein (HFE) has given rise to diagnostic clinical tests for the DNA-based detection of this pathologic mutation. This direct HFE mutation test can now be used not only to confirm the diagnosis of HH in those with symptomatic disease, but also, perhaps more importantly, to detect those with presymptomatic iron overload in whom future disease manifestations may be prevented (with phlebotomy therapy).