Richard D. Press

Desirable performance characteristics for BCR-ABL measurement on an international reporting scale to allow consistent interpretation of individual patient response and comparison of response rates between clinical trials

An international basis for comparison of BCR-ABL mRNA levels is required for the common interpretation of data derived from individual laboratories. This will aid clinical decisions for individual patients with chronic myeloid leukemia (CML) and assist interpretation of results from clinical studies. We aligned BCR-ABL values generated by 38 laboratories to an international scale (IS) where a major molecular response (MMR) is 0.1% or less. Alignment was achieved by application of laboratory-specific conversion factors calculated by comparisons performed with patient samples against a reference method. A validation procedure was completed for 19 methods. We determined performance characteristics (bias and precision) for consistent interpretation of MMR after IS conversion. When methods achieved an average BCR-ABL difference of plus or minus 1.2-fold from the reference method and 95% limits of agreement within plus or minus 5-fold, the MMR concordance was 91%. These criteria were met by 58% of methods. When not met, the MMR concordance was 74% or less. However, irrespective of precision, when the bias was plus or minus 1.2-fold as achieved by 89% of methods, there was good agreement between the overall MMR rates. This indicates that the IS can deliver accurate comparison of molecular response rates between clinical trials when measured by different laboratories.

Common Mutation in Methylenetetrahydrofolate Reductase Correlation With Homocysteine Metabolism and Late-Onset Vascular Disease

BACKGROUNDnIncreased homocysteine levels are a risk factor for atherosclerosis and its sequelae. A common genetic mutation in methylenetetrahydrofolate reductase (MTHFR), an enzyme required for efficient homocysteine metabolism, creates a thermolabile enzyme with reduced activity. We determined the prevalence of this mutation in many subjects with and without vascular disease and related it to homocysteine and folate levels.nnnMETHODS AND RESULTSnDNA from 247 older subjects with vascular disease and 594 healthy subjects without vascular disease (in three different control groups) was screened for the MTHFR 677 C-to-T mutation. Within each group, 9% to 17% of the subjects were homozygous for this mutation, and the mutant allele frequency was 31% to 39%. The genotype distributions, homozygote frequencies, and allele frequencies did not differ significantly between the study groups. In the vascular disease subjects, despite significantly lower folate levels in MTHFR homozygotes, there was no significant difference in homocysteine levels among the MTHFR genotype groups. The negative slope of the regression line relating homocysteine and folate was significantly steeper for those with a homozygous MTHFR mutation compared with those without this mutation.nnnCONCLUSIONSnAlthough the thermolabile MTHFR mutation is very common, it does not appear to be a significant genetic risk factor for typical late-onset vascular disease. Because MTHFR homozygotes have increased homocysteine with low folate levels, this mutation may contribute to early-onset or familial vascular disease. The genotype dependence of the folate-homocysteine correlation further suggests that homozygotes for this mutation may have both an exaggerated hyperhomocysteinemic response to folic acid depletion and a better response to folic acid therapy.

BCR-ABL1 compound mutations combining key kinase domain positions confer clinical resistance to ponatinib in Ph chromosome-positive leukemia.

Ponatinib is the only currently approved tyrosine kinase inhibitor (TKI) that suppresses all BCR-ABL1 single mutants in Philadelphia chromosome-positive (Ph(+)) leukemia, including the recalcitrant BCR-ABL1(T315I) mutant. However, emergence of compound mutations in a BCR-ABL1 allele may confer ponatinib resistance. We found that clinically reported BCR-ABL1 compound mutants center on 12 key positions and confer varying resistance to imatinib, nilotinib, dasatinib, ponatinib, rebastinib, and bosutinib. T315I-inclusive compound mutants confer high-level resistance to TKIs, including ponatinib. Inxa0vitro resistance profiling was predictive of treatment outcomes in Ph(+) leukemia patients. Structural explanations for compound mutation-based resistance were obtained through molecular dynamics simulations. Our findings demonstrate that BCR-ABL1 compound mutants confer different levels of TKI resistance, necessitating rational treatment selection to optimize clinical outcome.

A half-log increase in BCR-ABL RNA predicts a higher risk of relapse in patients with chronic myeloid leukemia with an imatinib-induced complete cytogenetic response.

Purpose: Imatinib induces a complete cytogenetic response (CCR) in most chronic myeloid leukemia patients in chronic phase. Although CCR is usually durable, a minority of patients relapse. Biomarkers capable of predicting those CCR patients with a higher risk of relapse would improve therapeutic management. Experimental Design: To assess whether changes in BCR-ABL RNA levels are a prognostic biomarker predictive of relapse, we regularly monitored transcript levels [every 3 months (median)] in 90 patients with CCR during 49 months (median) of imatinib therapy. Results: Throughout follow-up, the 20 patients with eventual relapse had higher transcript levels than the durable responders. Major molecular response (MMR; >3-log reduction of BCR-ABL RNA) was attained by 76 patients (12 with subsequent relapse) and was a significant predictor of prolonged relapse-free survival (P = 0.0008). A minimal 0.5-log increase in transcripts (before relapse; experienced by 42 patients, 16 with subsequent relapse) conveyed a significantly shorter relapse-free survival (P = 0.0017). Loss of MMR (transcript increase to <2.5-log reduction, before relapse; experienced by 33 patients, 11 with subsequent relapse) was also predictive of shortened relapse-free survival (P = 0.0003). A complete molecular response (undetectable transcripts by nested PCR) was attained by 28 MMR patients (one with subsequent relapse) and conveyed a significantly prolonged relapse-free survival (P = 0.0052). Conclusions: In chronic myeloid leukemia patients with an imatinib-induced CCR, a minimal half-log increase in BCR-ABL RNA (including loss of MMR) is a significant risk factor for future relapse. The achievement of a complete molecular response imparts longer progression-free survival than the achievement of an MMR.

TNFα facilitates clonal expansion of JAK2V617F positive cells in myeloproliferative neoplasms

Proinflammatory cytokines such as TNFα are elevated in patients with myeloproliferative neoplasms (MPN), but their contribution to disease pathogenesis is unknown. Here we reveal a central role for TNFα in promoting clonal dominance of JAK2(V617F) expressing cells in MPN. We show that JAK2(V617F) kinase regulates TNFα expression in cell lines and primary MPN cells and TNFα expression is correlated with JAK2(V617F) allele burden. In clonogenic assays, normal controls show reduced colony formation in the presence of TNFα while colony formation by JAK2(V617F)-positive progenitor cells is resistant or stimulated by exposure to TNFα. Ectopic JAK2(V617F) expression confers TNFα resistance to normal murine progenitor cells and overcomes inherent TNFα hypersensitivity of Fanconi anemia complementation group C deficient progenitors. Lastly, absence of TNFα limits clonal expansion and attenuates disease in a murine model of JAK2(V617F)-positive MPN. Altogether our data are consistent with a model where JAK2(V617F) promotes clonal selection by conferring TNFα resistance to a preneoplastic TNFα sensitive cell, while simultaneously generating a TNFα-rich environment. Mutations that confer resistance to environmental stem cell stressors are a recognized mechanism of clonal selection and leukemogenesis in bone marrow failure syndromes and our data suggest that this mechanism is also critical to clonal selection in MPN.

Establishment of the first World Health Organization International Genetic Reference Panel for quantitation of BCR-ABL mRNA.

Serial quantitation of BCR-ABL mRNA levels is an important indicator of therapeutic response for patients with chronic myelogenous leukemia and Philadelphia chromosome-positive acute lymphoblastic leukemia, but there is substantial variation in the real-time quantitative polymerase chain reaction methodologies used by different testing laboratories. To help improve the comparability of results between centers we sought to develop accredited reference reagents that are directly linked to the BCR-ABL international scale. After assessment of candidate cell lines, a reference material panel comprising 4 different dilution levels of freeze-dried preparations of K562 cells diluted in HL60 cells was prepared. After performance evaluation, the materials were assigned fixed percent BCR-ABL/control gene values according to the International Scale. A recommendation that the 4 materials be established as the first World Health Organization International Genetic Reference Panel for quantitation of BCR-ABL translocation by real-time quantitative polymerase chain reaction was approved by the Expert Committee on Biological Standardization of the World Health Organization in November 2009. We consider that the development of these reagents is a significant milestone in the standardization of this clinically important test, but because they are a limited resource we suggest that their availability is restricted to manufacturers of secondary reference materials.

Clinical Utility of Factor V Leiden (R506Q) Testing for the Diagnosis and Management of Thromboembolic Disorders

OBJECTIVEnTo review the current state of the art regarding the role of the clinical laboratory in diagnostic testing for the factor V Leiden (FVL) thrombophilic mutation (and other protein C resistance disorders), and to generate, through literature reviews and opinions of recognized thought-leaders, expert consensus recommendations on methodology and diagnostic, prognostic, and management issues pertaining to clinical FVL testing.nnnDATA SOURCES, EXTRACTION, AND SYNTHESISnAn initial thorough review of the medical literature and of current best clinical practices by a panel of 4 experts followed by a consensus conference review, editing, and ultimate approval by the majority of a panel of 28 additional coagulation laboratory experts.nnnCONCLUSIONSnConsensus recommendations were generated for topics of direct clinical relevance, including (1) defining those patients (and family members) who should (and should not) be tested for FVL; (2) defining the preferred FVL laboratory testing methods; and (3) defining the therapeutic, prophylactic, and management ramifications of FVL testing in affected individuals and their family members. As FVL is currently the most common recognized familial thrombophilia, it is hoped that these recommendations will assist laboratorians and clinicians caring for patients (and families) with this common mutation.

Ischemic Stroke in the Elderly Role of the Common Factor V Mutation Causing Resistance to Activated Protein C

BACKGROUND AND PURPOSEnA common missense mutation in coagulation factor V (Arg 506 Gln) creates phenotypic resistance to the anticoagulant effects of activated protein C and predisposes carriers to venous thrombosis. To assess a correlation between this common hypercoagulable state and ischemic cerebrovascular disease, we have compared the prevalence of this mutation in a group of stroke patients with that in several control patient groups.nnnMETHODSnThe presence of the factor V Arg 506 Gln mutation was determined by a direct polymerase chain reaction-based assay on peripheral blood leukocytes from 161 elderly patients with acute ischemic stroke, 116 elderly patients with stroke risk factors but without acute stroke, 54 healthy elderly control subjects, and 287 younger control individuals (197 blood donors and 90 neonates).nnnRESULTSnThe prevalence of the heterozygous Arg 506 Gln factor V mutation was not significantly different in the elderly stroke patients (2.5%) compared with either of the age-matched control groups (2% to 4%). The prevalence of this mutation was significantly higher in each of two younger control groups (approximately 8%) than in the elderly stroke patients (2.5%).nnnCONCLUSIONSnThe common factor V Arg 506 Gln mutation predisposing to venous thrombosis is not a significant genetic risk factor for ischemic stroke in the elderly.

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.

End-stage liver disease without hemochromatosis associated with elevated hepatic iron index

BACKGROUND/AIMSnThe utility of standard diagnostic tests for hereditary hemochromatosis in end-stage liver disease is unknown. A homozygous mutation (Cys 282 Tyr) has been identified in most patients with hereditary hemochromatosis. We examined whether serum iron studies and hepatic iron measurement distinguish end-stage liver disease patients with Cys 282 Tyr-associated hereditary hemochromatosis.nnnMETHODSnSerum iron, total iron binding capacity, and ferritin were measured in 106 cirrhotic patients prior to liver transplantation. Hepatic iron concentration and hepatic iron index were measured from explant liver tissue. Genotyping was performed on explant liver tissue in patients with an elevated hepatic iron index (>1.9).nnnRESULTSnThirty-three of 106 (31%) patients had elevated serum iron studies suggestive of hereditary hemochromatosis. Only four of 33 (12%) had a mean hepatic iron index >1.9, and none of the four patients was homozygous for Cys 282 Tyr. All four had liver disease due to hepatitis C and/or alcohol.nnnCONCLUSIONSn(i) Serum transferrin saturation and hepatic iron index lack specificity for hereditary hemochromatosis in end-stage liver disease. (ii) Genotyping for Cys 282 Tyr may provide the best method to identify hereditary hemochromatosis in the setting of end-stage liver disease.