Rodney R. Higgins

Prognostic relevance of integrated genetic profiling in acute myeloid leukemia

BACKGROUND Acute myeloid leukemia (AML) is a heterogeneous disease with respect to presentation and clinical outcome. The prognostic value of recently identified somatic mutations has not been systematically evaluated in a phase 3 trial of treatment for AML. METHODS We performed a mutational analysis of 18 genes in 398 patients younger than 60 years of age who had AML and who were randomly assigned to receive induction therapy with high-dose or standard-dose daunorubicin. We validated our prognostic findings in an independent set of 104 patients. RESULTS We identified at least one somatic alteration in 97.3% of the patients. We found that internal tandem duplication in FLT3 (FLT3-ITD), partial tandem duplication in MLL (MLL-PTD), and mutations in ASXL1 and PHF6 were associated with reduced overall survival (P=0.001 for FLT3-ITD, P=0.009 for MLL-PTD, P=0.05 for ASXL1, and P=0.006 for PHF6); CEBPA and IDH2 mutations were associated with improved overall survival (P=0.05 for CEBPA and P=0.01 for IDH2). The favorable effect of NPM1 mutations was restricted to patients with co-occurring NPM1 and IDH1 or IDH2 mutations. We identified genetic predictors of outcome that improved risk stratification among patients with AML, independently of age, white-cell count, induction dose, and post-remission therapy, and validated the significance of these predictors in an independent cohort. High-dose daunorubicin, as compared with standard-dose daunorubicin, improved the rate of survival among patients with DNMT3A or NPM1 mutations or MLL translocations (P=0.001) but not among patients with wild-type DNMT3A, NPM1, and MLL (P=0.67). CONCLUSIONS We found that DNMT3A and NPM1 mutations and MLL translocations predicted an improved outcome with high-dose induction chemotherapy in patients with AML. These findings suggest that mutational profiling could potentially be used for risk stratification and to inform prognostic and therapeutic decisions regarding patients with AML. (Funded by the National Cancer Institute and others.).

Comparison of interphase FISH and metaphase cytogenetics to study myelodysplastic syndrome: an Eastern Cooperative Oncology Group (ECOG) study

Cytogenetic analysis can be important in determining the prognosis and diagnosis of a number of hematological disorders, including myelodysplastic syndromes (MDS). Here, we compared metaphase chromosomal analyses on bone marrow aspirates from MDS patients with interphase fluorescence in situ hybridization (FISH) using probes specific for chromosomes nos. 5, 7, 8, 11, 13 and 20. Forty-three patients enrolled in ECOG protocol E1996 for low risk MDS and five patients enrolled in ECOG protocol E3996 for high risk MDS were studied by both metaphase chromosomal analysis and interphase FISH. Excluding those with a clonal loss of the Y chromosome, an abnormal clone was detected by cytogenetic analysis in 18 of 48 samples (37.5%). In comparison, our FISH panel detected an abnormal clone in 17 of 48 samples (35.4%). Twenty-nine of 30 samples with apparently normal karyotypes, including those with a missing Y chromosome, were also normal by our FISH panel. One patient had an occult deletion of chromosome 11 that was detected by FISH. These results indicate that around 60% of patients with MDS do not have abnormalities that are detectable by either chromosomal or FISH studies. In addition, it appears that interphase FISH studies are nearly as sensitive as cytogenetic analyses and can be a useful tool in studying bone marrow aspirates where cytogenetic analysis is not possible.

A multicenter investigation with interphase fluorescence in situ hybridization using X- and Y-chromosome probes

Twenty-six laboratories used X and Y chromosome probes and the same procedures to process and examine 15,600 metaphases and 49,400 interphases from Phaseolus vulgaris-leucoagglutinin (PHA)-stimulated lymphocytes. In Part I, each laboratory scored 50 metaphases and 200 interphases from a normal male and a normal female from its own practice. In Part II, each laboratory scored 50 metaphases and 200 interphases on slides prepared by a central laboratory from a normal male and a normal female and three mixtures of cells from the male and female. In Part III, each laboratory scored 50 metaphases (in samples of 5, 10, 15, and 20) and 100 interphases (in samples of 5, 10, 15, 20, and 50) on new, coded slides of the same specimens used in Part II. Metaphases from male specimens were scored as 98-99% XY with no XX cells, and 97-98% of interphases were scored as XY with 0.04% XX cells. Metaphases from female specimens were scored as 96-97% XX with 0.03% XY cells, and 94-96% of interphases were scored as XX with 0.05% XY cells. Considering the data as a model for any probe used with fluorescence in situ hybridization (FISH), a statistical approach assessing the impact of analytical sensitivity on the numbers of observations required to assay for potential mosaicisms and chimerisms is discussed. The workload associated with processing slides and scoring 50 metaphases and 200 interphases using FISH averaged 27.1 and 28.6 minutes, respectively. This study indicates that multiple laboratories can test/develop guidelines for the rapid, efficacious, and cost-effective integration of FISH into clinical service.

Toward Quality Assurance for Metaphase FISH: A Multi-Center Experience

Although fluorescent in situ hybridization (FISH) is rapidly becoming a part of clinical cytogenetics, no organization sponsors multi-center determinations of the efficacy of probes. We report on 23 laboratories that volunteered to provide slides and to use a probe for SNRPN and a control locus. Experiences with FISH for these laboratories during 1994 ranged from 0 to 645 utilizations (median = 84) involving blood, amniotic fluid and bone marrow. In an initial study of hybridization efficiency, the median percentage of metaphases from normal individuals showing two SNRPN and 2 control signals for slides prepared at each site was 97.0 (range = 74-100); for slides prepared by a central laboratory, it was 97.8 (range = 81.6-100). In a subsequent blind study, each laboratory attempted to score 5 metaphases from each of 23 specimens [8 with del(15) (q11.2-->q12) and 15 with normal 15 chromosomes]. Of 529 challenges, the correct SNRPN pattern was found in 5 of 5 metaphases in 457 (86%) and in 4 of 5 in 33 (6%). Ambiguous, incomplete or no results were reported for 32 (6%) challenges. Seven (1%) diagnostic errors were made including 6 false positives and 1 false negative: 1 laboratory made 3 errors, 1 made 2, and 2 made 1 each. Most errors and inconsistencies seemed due to inexperience with FISH. The working time to process and analyze slides singly averaged 49.5 minutes; slides processed in batches of 4 and analyzed singly required 36.9 minutes. We conclude that proficiency testing for FISH using an extensive array of challenges is possible and that multiple centers can collaborate to test probes and to evaluate costs.

Pediatric acute lymphoblastic leukemia with a t(8;14)(q11.2;q32): B-cell disease with a high proportion of Down syndrome: a Children's Oncology Group study

The rare translocation t(8;14)(q11.2;q32) has been described in patients with B-cell acute lymphoblastic leukemia (ALL), particularly patients with Down syndrome (DS). We describe patients with the t(8;14)(q11.2;q32) who were identified by the Childrens Oncology Group (COG) ALL cytogenetics database, expanding our previous report of 10 patients with this translocation. Twenty-two such patients were treated with COG protocols. All patients had B-cell ALL, and seven (31.8%) had DS. None of the children with DS had an event; thus, these patients had a superior estimated 5-year event-free survival (EFS) compared to non-DS patients (100% vs. 50.1 ± 17.7%; P = 0.04). Only one patient (4.5%) had a concomitant Philadelphia chromosome t(9;22)(q34;q11.2). The cytogenetics data of two additional patients, who were not eligible for COG protocols, are also included in this report. In conclusion, ALL patients with the recurring translocation t(8;14)(q11.2;q32) have the B-cell phenotype and a high percentage have DS. Children with DS and the t(8;14)(q11.2;q34) have improved EFS using standard COG therapy compared to non-DS patients. We did not find an increased number of patients with a concomitant Philadelphia chromosome in this population.

Duplication 6q22→qter: Definition of the phenotype

We report on a girl with duplication of 6q22.32 --> qter and microcephaly, frontal bossing, facial anomalies, and webbed neck. She has congenital heart disease, renal hypoplasia, and hearing loss along with severe developmental delay. Published reports of seven other patients are reviewed and compared. The most frequent anomalies include microcephaly, abnormal face, webbed neck, congenital heart disease, limb contractures, and developmental delay.

Independent Prognostic Significance of Monosomy 17 and Impact of Karyotype Complexity in Monosomal Karyotype/Complex Karyotype Acute Myeloid Leukemia: Results from Four ECOG-ACRIN Prospective Therapeutic Trials.

The presence of a monosomal karyotype (MK+) and/or a complex karyotype (CK+) identifies subcategories of AML with poor prognosis. The prognostic significance of the most common monosomies (monosomy 5, monosomy 7, and monosomy 17) within MK+/CK+AML is not well defined. We analyzed data from 1,592 AML patients age 17-93 years enrolled on ECOG-ACRIN therapeutic trials. The majority of MK+ patients (182/195; 93%) were MK+/CK+ with 87% (158/182) having ≥5 clonal abnormalities (CK≥5). MK+ patients with karyotype complexity ≤4 had a median overall survival (OS) of 0.4y compared to 1.0y for MK- with complexity ≤4 (p<0.001), whereas no OS difference was seen in MK+vs. MK- patients with CK≥5 (p=0.82). Monosomy 5 (93%; 50/54) typically occurred within a highly complex karyotype and had no impact on OS (0.4y; p=0.95). Monosomy 7 demonstrated no impact on OS in patients with CK≥5 (p=0.39) or CK≤4 (p=0.44). Monosomy 17 appeared in 43% (68/158) of CK≥5 patients and demonstrated statistically significant worse OS (0.4y) compared to CK≥5 patients without monosomy 17 (0.5y; p=0.012). Our data suggest that the prognostic impact of MK+is limited to those with less complex karyotypes and that monosomy 17 may independently predict for worse survival in patients with AML.