Amber C. Donahue

Proteasome Enzymatic Activities in Plasma as Risk Stratification of Patients with Acute Myeloid Leukemia and Advanced-Stage Myelodysplastic Syndrome

Purpose: Cytogenetic abnormalities are currently the most important predictors of response and clinical outcome for patients with acute myeloid leukemia (AML) or advanced-stage myelodysplastic syndrome (MDS). Because clinical outcomes vary markedly within cytogenetic subgroups, additional biological markers are needed for risk stratification. Experimental Design: We assessed the utility of measuring pretreatment proteasome chymotrypsin-like, caspase-like, and trypsin-like activities in plasma to predict response and survival of patients with AML (n = 174) or advanced-stage MDS (n = 52). Results: All three enzymatic activities were significantly (P < 0.001) increased in the plasma of patients with AML and MDS compared with normal controls. Both chymotrypsin-like and caspase-like activities, but not trypsin-like activity, correlated with outcome. Chymotrypsin-like and caspase-like activities, but not trypsin-like activity, predicted response in univariate analysis (P = 0.002). However, only chymotrypsin-like activity was independent predictor of response from age grouping (<70 versus ≥70 years), cytogenetics, and blood urea nitrogen in multivariate analysis. Similarly, both chymotrypsin-like and caspase-like activities, but not trypsin-like activity, were predictors of overall survival in univariate analysis (P < 0.0001), but only chymotrypsin-like activity was independent of cytogenetics, age, performance status, blood urea nitrogen, and β2-microglobulin in multivariate Cox regression models. Chymotrypsin-like activity was also a strong independent predictor of survival in patients with intermediate karyotype (n = 124). Conclusions: Measuring plasma chymotrypsin-like activity may provide a powerful biomarker for risk stratification in patients with AML and advanced-stage MDS, including those with normal karyotype.

Significant association between polymorphism of the erythropoietin gene promoter and myelodysplastic syndrome

BackgroundMyelodysplastic syndrome (MDS) may be induced by certain mutagenic environmental or chemotherapeutic toxins; however, the role of susceptibility genes remains unclear. The G/G genotype of the single-nucleotide polymorphism (SNP) rs1617640 in the erythropoietin (EPO) promoter has been shown to be associated with decreased EPO expression. We examined the association of rs1617640 genotype with MDS.MethodsWe genotyped the EPO rS1617640 SNP in 189 patients with MDS, 257 with acute myeloid leukemia (AML), 106 with acute lymphoblastic leukemia, 97 with chronic lymphocytic leukemia, 353 with chronic myeloid leukemia, and 95 healthy controls.ResultsThe G/G genotype was significantly more common in MDS patients (47/187; 25.1%) than in controls (6/95; 6.3%) or in patients with other leukemias (101/813; 12.4%) (all P < 0.001). Individuals with the G/G genotype were more likely than those with other genotypes to have MDS (odd ratio = 4.98; 95% CI = 2.04-12.13). Clinical and follow up data were available for 112 MDS patients and 186 AML patients. There was no correlation between EPO promoter genotype and response to therapy or overall survival in MDS or AML. In the MDS group, the GG genotype was significantly associated with shorter complete remission duration, as compared with the TT genotype (P = 0.03). Time to neutrophils recovery after therapy was significantly longer in MDS patients with the G/G genotype (P = 0.02).ConclusionsThese findings suggest a strong association between the rs1617640 G/G genotype and MDS. Further studies are warranted to investigate the utility of screening for this marker in individuals exposed to environmental toxins or chemotherapy.

Detection of nucleophosmin gene mutations in plasma from patients with acute myeloid leukemia: Clinical significance and implications

Roughly one-third of acute myeloid leukemia (AML) patients exhibit mutations in the nucleophosmin (NPM1) gene, and multiple studies have linked these mutations with a more favorable clinical outcome. We developed an assay for the detection of NPM1 mutations in peripheral blood plasma, and compared the results with clinical outcomes from a single institution. Analyzing plasma from previously untreated AML patients revealed NPM1 insertion mutations in 24 of 98 (24%) patients, with greater sensitivity than existing peripheral blood cell-based tests which showed positivity in only 22 of the 24 patients. Plasma testing allowed the detection of a novel 4 bp deletion in NPM1 in one patient. Analysis of clinical data corroborated previous data linking NPM1 mutations with better clinical outcome. These data underline the significance of NPM1 in the biology and clinical behavior of AML, and demonstrate the reliability and efficacy of plasma-based testing for NPM1 mutations.

Detection, Analysis and Clinical Validation of Chromosomal Aberrations by Multiplex Ligation- Dependent Probe Amplification in Chronic Leukemia

Current diagnostic screening strategies based on karyotyping or fluorescent in situ hybridization (FISH) for detection of chromosomal abnormalities in chronic lymphocytic leukemia (CLL) are laborious, time-consuming, costly, and have limitations in resolution. Multiplex ligation-dependent probe amplification (MLPA) can simultaneously detect copy number changes of multiple loci in one simple PCR reaction, making it an attractive alternative to FISH. To enhance the clinical robustness and further harness MLPA technology for routine laboratory operations, we have developed and validated a protocol for comprehensive, automatic data analysis and interpretation. A training set of 50 normal samples was used to establish reference ranges for each individual probe, for the calling of statistically significant copy number changes. The maximum normal ranges of 2 and 3 standard deviations (SD) are distributed between 0.82 and 1.18 (Mean ± 2SD, 95% CI, P = 0.05), and between 0.73 and 1.27 (Mean ± 3SD, 99% CI, P = 0.01), respectively. We found an excellent correlation between MLPA and FISH with 93.6% concordance (P<0.0001) from a testing cohort of 100 clinically suspected CLL cases. MLPA analyses done on 94/100 patients showed sensitivity and specificity of 94.2% and 92.9%, respectively. MLPA detected additional copy number gains on 18q21.1 and chromosome 19, and novel micro-deletions at 19q13.43 and 19p13.2 loci in six samples. Three FISH-failed samples were tested positive by MLPA, while three 13q- cases with a low percentage of leukemia cells (7%, 12% and 19%) were not detected by MLPA. The improved CLL MLPA represents a high-throughput, accurate, cost-effective and user-friendly platform that can be used as a first-line screening test in a clinical laboratory.

Multiplex ligation-dependent probe amplification for detection of chromosomal abnormalities in myelodysplastic syndrome and acute myeloid leukemia

Current strategies for detecting chromosome abnormalities in MDS/AML include FISH or traditional cytogenetics. MLPA detects abnormalities in multiple loci simultaneously, with higher resolution and throughput. Peripheral blood from 50 healthy subjects was used to establish probe-specific reference ranges, increasing MLPA sensitivity and specificity. MLPA was then performed on 110 FISH-tested blood or bone marrow samples from suspected leukemia patients. Our novel MLPA analysis system combined maximum stringency with sensitive detection of low-frequency abnormalities. Accuracy/specificity of MLPA were excellent compared to FISH. Our MLPA analysis/interpretation method provides a clinically robust, high-throughput, high-resolution option for detection of abnormalities associated with MDS/AML.

Proteomics-based prediction of clinical response in acute myeloid leukemia

OBJECTIVE Response to chemotherapy is achieved in 60% to 70% of patients with acute myeloid leukemia. The ability to predict responders may help in stratifying patients and exploring different therapeutic approaches for nonresponders. Proteomics methods were used to search for predictive factors or combinations of factors. MATERIALS AND METHODS Peripheral blood plasma samples from 41 patients with confirmed acute myeloid leukemia with intermediate or poor cytogenetics were obtained prior to induction therapy for proteomic analysis. For each plasma sample, four fractions eluted from a strong anion column were applied to 3 different ProteinChip array surfaces and 12 surface-enhanced laser desorption/ionization spectra were generated. Peaks that correlated with response were identified, and decision trees incorporating these peaks along with various clinical and laboratory findings were constructed to predict response. RESULTS Multiple decision trees were constructed. One peak, when combined with age, provided strong positive prediction of responders with 83% accuracy. A second tree, which combined one peak with both cytogenetics and the percent of monocytes in peripheral blood, detected responders with 95% accuracy. A third peak was adequate to predict responders in the intermediate cytogenetic group with 86% accuracy. CONCLUSIONS Proteomic analysis should be further explored to define factors important in predicting clinical response in patients with acute myeloid leukemia.

Circulating Ki-67 index in plasma as a biomarker and prognostic indicator in chronic lymphocytic leukemia.

Ki-67 is a nuclear antigen that is expressed in all stages of the cell cycle, except G(0), and is widely used as a marker of cellular proliferation in human tumors. We recently showed that elevated levels of Ki-67 circulating in plasma (cKi-67) are associated with shorter survival in patients with acute lymphoblastic leukemia. The current study included 194 patients with CLL and 96 healthy control subjects. cKi-67 levels in plasma were determined using an electrochemiluminescent immunoassay. We normalized the cKi-67 level to the absolute number of lymphocytes in the patients peripheral blood to establish the plasma cKi-67 index. The cKi-67 index showed significant correlation with lymph node involvement and Rai stage (P=0.05). Higher cKi-67 index values were significantly associated with shorter survival. Multivariate Cox proportional hazards regression analysis demonstrated that the association of the cKi-67 index with shorter survival was independent of IgV(H) mutation status. In a multivariate model incorporating the cKi-67 index with B2M and IgV(H), only cKi-67 index and B2M levels remained as independent predictors of survival. The results of this study suggest that the plasma cKi-67 index, along with B2M level, is a strong predictor of clinical behavior in CLL.

Antibodies in Biosensing

Biosensing, the detection of biological phenomena with accuracy and precision, is a rapidly growing and increasingly divergent field. The requirement for a stable, adaptable, and highly specific recognition receptor is of paramount importance to the design of a good biosensing assay. Among the receptors currently used in these applications, the antibody is perhaps the only one designed by evolution to be a natural biosensor. A greater understanding of the genetics of antibodies has given researchers the ability to manipulate their structure and to take advantage of the vast range of possible specificities. The platforms that rely on antibodies as recognition receptors, including enzyme-linked immunosorbent assay (ELISA), flow cytometry, radioimmunoassay (RIA), immunohistochemistry and immunocytochemistry, and antibody arrays, have evolved significantly to include new techniques and greater multiplexing opportunities. Here we give an overview of these techniques, and the ways in which the development of these biosensing assays are revolutionizing basic research, diagnostics, and therapeutics.

Principles of Immunological Diagnostic Tests for Cancers

Many of the immunological methods currently used in laboratory testing for cancer are based on the use of antibodies, including many diagnostic and prognostic tests, as well as some tests intended to inform treatment decisions. Unlocking the genetics of antibodies (Abs) has allowed researchers to manipulate their structure and harness the immense array of possible specificities granted to the immune system by evolution. The platforms that rely on Abs for detection are numerous and include enzyme-linked immunosorbent assays (ELISA), flow cytometry, radioimmunoassay (RIA), immunohistochemistry and immunocytochemistry (IHC/ICC), and Ab arrays. Here we will provide a brief background on the ways in which the immunoglobulin protein can be manipulated and used as a tool for the detection of targets of interest. In addition, we will describe the principles behind many of the immunological methods used in testing in patients with cancer.

Abstract 4569: Amplicon-targeted ultra-deep sequencing for the detection of KRAS, BRAF, and PIK3CA actionable mutations

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Background: Next-generation deep sequencing techniques have the potential to revolutionize clinical testing, especially in the arena of mutation detection. Molecular diagnostic testing for KRAS, BRAF, and PIK3CA mutations has become an integral part of clinical practice for the determination of treatment eligibility for anti-EGFR therapy. Purpose: Here we investigate the feasibility of ultra-deep advanced sequencing with the 454 GS Junior instrument (454 Life Sciences/Roche, Branford CT) for the simultaneous detection of actionable mutations in KRAS, BRAF, and PIK3CA in formalin-fixed paraffin-embedded (FFPE) colorectal cancer tissue. Methods: Advanced sequencing with a 4-amplicon panel was performed on 17 de-identified residual FFPE colorectal cancer biopsy specimens originally submitted for mutation analysis of one or more genes by standard sequencing. The 4-amplicon panel was multiplexed for up to 4 samples through “barcoding” with molecular identifier (MID) sequences, for a total of 16 amplicons per run. Results: Sequence analysis with ultra-deep coverage of 3000-5000 reads detected KRAS mutations in 5 (29%) tumor specimens, BRAF mutations in 3 (18%), and PIK3CA mutations in 4 (24%). Ten cases had a single mutation and one had two: PIK3CA E545K and BRAF D594G. Coverage was equivalent for each of the four amplicons, and in both the forward and reverse directions, providing a high level of confidence for each mutation detected. Sequencing data was obtained for each sample tested. When sample quantity allowed, Sanger sequencing and/or castPCR (Life Technologies, Grand Island, NY) were performed as method comparisons, and results were concordant. Conclusions: The use of MIDs with ultra-deep advanced sequencing allowed the simultaneous detection of mutations in multiple amplicons from multiple patients in a single sequencing run. This proof-of-concept study confirmed that using advanced sequencing to simultaneously test for multiple biomarkers in one FFPE sample is feasible, and that amplicon-based advanced sequencing is a robust and high-throughput platform for the detection of clinically actionable mutations. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4569. doi:1538-7445.AM2012-4569