CD74 is expressed in a subset of pediatric acute myeloid leukemia patients and is a promising target for therapy: a report from the Children's Oncology Group.

As curative therapies for pediatric AML remain elusive, identifying potential new treatment targets is vital. We assessed the cell surface expression of CD74, also known as the MHC-II invariant chain, by multidimensional flow cytometry in 973 patients enrolled in the Children's Oncology Group AAML1031 clinical trial. 38% of pediatric AML patients expressed CD74 at any level and a comparison to normal hematopoietic cells revealed a subset with increased expression relative to normal myeloid progenitor cells. Pediatric AML patients expressing high intensity CD74 typically had an immature immunophenotype and an increased frequency of lymphoid antigen expression. Increased CD74 expression was associated with older patients with lower WBC and peripheral blood blast counts, and was enriched for t(8;21), trisomy 8, and CEBPA mutations. Overall, high CD74 expression was associated with low-risk status, however 26% of patients were allocated to high-risk protocol status and 5-year event free survival was 53%, indicating that a significant number of high expressing patients had poor outcomes. In vitro pre-clinical studies indicate that anti-CD74 therapy demonstrates efficacy against AML cells but has little impact on normal CD34+ cells. Together, we demonstrate that CD74 is expressed on a subset of pediatric AMLs at increased levels compared to normal hematopoietic cells and is a promising target for therapy in expressing patients. Given that nearly half of patients expressing CD74 at high levels experience an adverse event within 5 years, and the availability of CD74 targeting drugs, this represents a promising line of therapy worthy of additional investigation.

The impact of Down syndrome-specific non-malignant hematopoietic regeneration in the bone marrow on the detection of leukemic measurable residual disease.

BACKGROUND: Detection of measurable residual disease detection (MRD) by flow cytometry after the first course of chemotherapy is a standard measure of early response in patients with acute myeloid leukemia (AML). Myeloid leukemia associated with Down Syndrome (ML-DS) is a distinct form of AML. Differences in steady-state and regenerating hematopoiesis between patients with or without DS are not well understood. This understanding is essential to accurately determine the presence of residual leukemia in patients with ML-DS. METHODS: A standardized antibody panel defined quantitative antigen expression in 115 follow-up bone marrow (BM) aspirates from 45 patients following chemotherapy for ML-DS or DS precursor B-cell acute lymphoblastic leukemia (B-ALL-DS) with the "difference from normal (ΔN)" technique. When possible, FISH and SNP/CGH microarray studies were performed on sorted cell fractions. RESULTS: 93% of BM specimens submitted post chemotherapy had a clearly identifiable CD34+ CD56+ population present between 0.06% and 2.6% of total non-erythroid cells. An overlapping CD34+ HLA-DRheterogeneous population was observed among 92% of patients at a lower frequency (0.04%-0.8% of total non-erythroid cells). In B-ALL-DS patients, the same CD34+ CD56+ HLA-DRheterogeneous expression was observed. FACS-FISH/Array studies demonstrated no residual genetic clones in the DS-specific myeloid progenitor cells. CONCLUSIONS: Non-malignant myeloid progenitors in the regenerating BM of patients who have undergone chemotherapy for either ML-DS or B-ALL-DS express an immunophenotype that is different from normal BM of non-DS patients. Awareness of this DS-specific non-malignant myeloid progenitor is essential to the interpretation of MRD by flow cytometry in patients with ML-DS.

Comprehensive molecular and clinical characterization of NUP98 fusions in pediatric acute myeloid leukemia.

NUP98 fusions comprise a family of rare recurrent alterations in AML, associated with adverse outcomes. In order to define the underlying biology and clinical implications of this family of fusions, we performed comprehensive transcriptome, epigenome, and immunophenotypic profiling of 2,235 children and young adults with AML and identified 160 NUP98 rearrangements (7.2%), including 108 NUP98-NSD1 (4.8%), 32 NUP98-KDM5A (1.4%) and 20 NUP98-X cases (0.9%) with 13 different fusion partners. Fusion partners defined disease characteristics and biology; patients with NUP98-NSD1 or NUP98-KDM5A had distinct immunophenotypic, transcriptomic, and epigenomic profiles. Unlike the two most prevalent NUP98 fusions, NUP98-X variants are typically not cryptic. Furthermore, NUP98-X cases are associated with WT1 mutations, and have epigenomic profiles that resemble either NUP98-NSD1 or NUP98-KDM5A. Cooperating FLT3-ITD and WT1 mutations define NUP98-NSD1, and chromosome 13 aberrations are highly enriched in NUP98-KDM5A. Importantly, we demonstrate that NUP98 fusions portend dismal overall survival, with the noteworthy exception of patients bearing abnormal chromosome 13 (clinicaltrials gov. Identifiers: NCT00002798, NCT00070174, NCT00372593, NCT01371981).

Multicenter prospective evaluation of diagnostic potential of flow cytometric aberrancies in myelodysplastic syndromes by the ELN iMDS flow working group.

BACKGROUND: Myelodysplastic syndromes (MDS) represent a diagnostic challenge. This prospective multicenter study was conducted to evaluate pre-defined flow cytometric markers in the diagnostic work-up of MDS and chronic myelomonocytic leukemia (CMML). METHODS: Thousand six hundred and eighty-two patients with suspected MDS/CMML were analyzed by both cytomorphology according to WHO 2016 criteria and flow cytometry according to ELN recommendations. Flow cytometric readout was categorized 'non-MDS' (i.e. no signs of MDS/CMML and limited signs of MDS/CMML) and 'in agreement with MDS' (i.e., in agreement with MDS/CMML). RESULTS: Flow cytometric readout categorized 60% of patients in agreement with MDS, 28% showed limited signs of MDS and 12% had no signs of MDS. In 81% of cases flow cytometric readouts and cytomorphologic diagnosis correlated. For high-risk MDS, the level of concordance was 92%. A total of 17 immunophenotypic aberrancies were found independently related to MDS/CMML in ≥1 of the subgroups of low-risk MDS, high-risk MDS, CMML. A cut-off of ≥3 of these aberrancies resulted in 80% agreement with cytomorphology (20% cases concordantly negative, 60% positive). Moreover, >3% myeloid progenitor cells were significantly associated with MDS (286/293 such cases, 98%). CONCLUSION: Data from this prospective multicenter study led to recognition of 17 immunophenotypic markers allowing to identify cases 'in agreement with MDS'. Moreover, data emphasizes the clinical utility of immunophenotyping in MDS diagnostics, given the high concordance between cytomorphology and the flow cytometric readout. Results from the current study challenge the application of the cytomorphologically defined cut-off of 5% blasts for flow cytometry and rather suggest a 3% cut-off for the latter.

Integrated analysis of genotype and phenotype reveals clonal evolution and cytogenetically driven disruption of myeloid cell maturation in myelodysplastic syndromes.

BACKGROUND: Myelodysplastic syndromes (MDS) are a heterogenous collection of clonal bone marrow diseases characterized by cytopenias, abnormal karyotypes, molecular abnormalities, and dysplasia by flow cytometry and/or morphology. The progression of MDS to severe cytopenias and/or overt leukemia is associated with the accumulation of additional cytogenetic abnormalities, suggesting clonal evolution. The impact of these accumulated abnormalities on myeloid maturation and the severity of the disease is poorly understood. METHODS: Bone marrow specimens from 16 patients with cytogenetic abnormalities were flow cytometrically sorted into three myeloid populations: progenitors, immature myeloid cells, and mature myeloid cells. Fluorescence in situ hybridization analysis was performed on each to determine the distribution of chromosomal abnormalities during myeloid maturation. RESULTS: Our findings revealed three distinct distributions of cytogenetic abnormalities across myeloid maturation, each of which corresponded to specific cytogenetic abnormalities. Group 1 had continuous distribution across all maturational stages and contained patients with a single cytogenetic aberration associated with good-to-intermediate prognosis; Group 2 had accumulation of abnormalities in immature cells and contained patients with high-risk monosomy 7; and Group 3 had abnormalities defining the founding clone equally distributed across maturational stages while subclonal abnormalities were enriched in progenitor cells and contained patients with multiple, non-monosomy 7, abnormalities with evidence of clonal evolution. CONCLUSIONS: Our findings demonstrate that low-risk abnormalities (e.g., del(20q) and trisomy 8) occurring in the founding clone display a markedly different disease etiology, with respect to myeloid maturation, than monosomy 7 or abnormalities acquired in subclones, which result in a disruption of myeloid cell maturation in MDS.

Therapeutic targeting of PRAME with mTCRCAR T cells in acute myeloid leukemia.

Preferentially Expressed Antigen in Melanoma (PRAME), a cancer-testis antigen, provides an ideal target for immunotherapy in acute myeloid leukemia (AML). We have shown expression of PRAME in a significant subset of childhood and adult AML and lack of expression in normal hematopoiesis. Although an intracellular antigen, we developed a novel approach to target PRAME using a chimeric antigen receptor (CAR) construct encoding a targeting domain based on T-cell receptor (TCR) mimic antibodies that target the peptide-HLA complex. We used the antibody sequence from a previously designed TCR mimic (mTCR) antibody, Pr20, that recognizes the PRAME ALY peptide in complex with HLA-A∗02 and verified expression of PRAME in AML cell lines and primary AML blasts. Using the Pr20 antibody sequence, we developed CAR T cells (PRAME mTCRCAR T) to be tested against primary samples from patients with AML and AML cell lines that express the PRAME antigen in the context of HLA-A2 expression. In contrast to appropriate controls, PRAME mTCRCAR T cells demonstrate target-specific and HLA-mediated in vitro activity in OCI-AML2 and THP-1 cell lines, HLA-A2 cell lines expressing the PRAME antigen, and against primary AML patient samples. In vivo cell-derived xenograft models treated with PRAME mTCRCAR T cells demonstrated potent leukemia clearance and improved survival compared with unmodified T-cell controls. Furthermore, the cytolytic activity of PRAME mTCRCAR T cells was enhanced by treating the target cells with interferon gamma, which increases PRAME antigen expression. These results demonstrate the feasibility and efficacy of targeting PRAME with novel PRAME mTCRCAR T cells.

Sorafenib in Combination With Standard Chemotherapy for Children With High Allelic Ratio FLT3/ITD+ Acute Myeloid Leukemia: A Report From the Children's Oncology Group Protocol AAML1031.

PURPOSE: High allelic ratio (HAR) FLT3/ITD (AR > 0.4) mutations confer poor prognosis in pediatric acute myeloid leukemia (AML). COG AAML1031 studied the feasibility and efficacy of adding sorafenib, a multikinase tyrosine kinase inhibitor to standard chemotherapy and as single-agent maintenance therapy in this population. MATERIALS AND METHODS: Patients were treated in three cohorts. The initial safety phase defined the maximum tolerated dose of sorafenib starting in induction 2. Cohorts 2 and 3 added sorafenib in induction and as single-agent maintenance. Clinical outcome analysis was limited to n = 72 patients in cohorts 2/3 and compared with n = 76 HAR FLT3/ITD+ AML patients who received identical chemotherapy without sorafenib. Sorafenib pharmacokinetics and plasma inhibitory activity were measured in a subset of patients. RESULTS: The maximum tolerated dose of sorafenib was 200 mg/m2 once daily; dose-limiting toxicities included rash (n = 2; 1 grade 3 and 1 grade 2), grade 2 hand-foot syndrome, and grade 3 fever. Pharmacokinetics/plasma inhibitory activity data demonstrated that measured plasma concentrations were sufficient to inhibit phosphorylated FLT3. Although outcomes were superior with sorafenib in cohorts 2 and 3, patients treated with sorafenib also underwent hematopoietic stem-cell transplant more frequently than the comparator population. Multivariable analysis that accounted for both hematopoietic stem-cell transplant and favorable co-occurring mutations confirmed sorafenib's benefit. Specifically, risk of an event was approximately two-fold higher in HAR FLT3/ITD+ patients who did not receive sorafenib (event-free survival from study entry: hazard ratio [HR] 2.37, 95% CI, 1.45 to 3.88, P < .001, disease-free survival from complete remission: HR 2.28, 95% CI, 1.08 to 4.82, P = .032, relapse risk from complete remission: HR 3.03, 95% CI 1.31 to 7.04, P = .010). CONCLUSION: Sorafenib can be safely added to conventional AML chemotherapy and may improve outcomes in pediatric HAR FLT3/ITD+ AML.

Cytarabine dose reduction in patients with low-risk acute myeloid leukemia: A report from the Children's Oncology Group.

BACKGROUND: The optimal number of chemotherapy courses for low-risk (LR) pediatric acute myeloid leukemia (AML) is not known. OBJECTIVE: To compare outcomes for four (21.6 g/m2 cytarabine) versus five (45.6 g/m2 cytarabine) chemotherapy courses for LR-AML using data from Children's Oncology Group (COG) AAML0531 and AAML1031. METHODS: We compared relapse risk (RR), disease-free survival (DFS), and overall survival (OS), and the differential impact in LR subgroups for patients receiving four versus five chemotherapy courses. Cox (OS and DFS) and risk (RR) regressions were used to estimate hazard ratios (HR) to compare outcomes. RESULTS: A total of 923 LR-AML patients were included; 21% received five courses. Overall, LR-AML patients who received four courses had higher RR (40.9% vs. 31.4%; HR = 1.40, 95% confidence interval [CI]: 1.06-1.85), and worse DFS (56.0% vs. 67.0%; HR = 1.45, 95% CI: 1.10-1.91). There was a similar decrement in OS though it was not statistically significant (77.0% vs. 83.5%; HR = 1.45, 95% CI: 0.97-2.17). Stratified analyses revealed the detrimental effects of cytarabine dose de-escalation to be most pronounced in the LR-AML subgroup with uninformative cytogenetic/molecular features who were minimal residual disease (MRD) negative after the first induction course (EOI1). The absolute decrease in DFS with four courses for patients with favorable cytogenetic/molecular features and positive MRD was similar to that observed for patients with uninformative cytogenetic/molecular features and negative MRD at EOI1, though not statistically significant. CONCLUSIONS: Our results support de-escalation of cytarabine exposure through the elimination of a fifth chemotherapy course only for LR-AML patients who have both favorable cytogenetic/molecular features and negative MRD after the first induction cycle.

Outcomes of intensification of induction chemotherapy for children with high-risk acute myeloid leukemia: A report from the Children's Oncology Group.

BACKGROUND: High-risk pediatric acute myeloid leukemia confers a poor prognosis, and alternative strategies are needed to improve outcomes. We hypothesized that intensifying induction on the AAML1031 clinical trial would improve outcomes compared to the predecessor trial AAML0531. METHODS: Patients on AAML0531 received cytarabine (1600 mg/m2 )/daunorubicin (150 mg/m2 )/etoposide (ADE) for induction II and patients on AAML1031 received mitoxantrone (48 mg/m2 )/cytarabine (8000 mg/m2 ) (MA). Stem cell transplant (SCT) conditioning included busulfan/cyclophosphamide on AAML0531, whereas AAML1031 used busulfan/fludarabine and liberalized donor eligibility. Patients were included in this analysis if they met high-risk criteria common to the two trials by cytogenics or poor disease response after induction I ADE. RESULTS: MA provided no benefit over ADE at: induction II response (complete response [CR]: 64% vs. 62%, p = .87; measurable residual disease [MRD]+: 57% vs. 46%, p = .34); or intensification I response (CR: 79% vs. 94%, p = .27; MRD+: 27% vs. 20%, p = 1.0). When considered with altered SCT approach, MA did not improve 5-year disease-free survival (24% ± 9% vs. 18% ± 15%, p = .63) or 5-year overall survival (35% ± 10% vs. 38% ± 18%, p = .66). MA was associated with slower neutrophil recovery (median 34 vs. 27 days, p = .007) and platelet recovery (median 29 vs. 24.5 days, p = .04) and longer hospital stay (32 vs. 28 days, p = .01) during induction II. CONCLUSION: Intensification of induction II did not improve treatment response or survival, but did increase toxicity and resource utilization. Alternative strategies are urgently needed to improve outcomes for pediatric patients with high-risk acute myeloid leukemia (trials registered at clinicaltrials.gov NCT01371981, NCT00372593).

CEBPA-bZip mutations are associated with favorable prognosis in de novo AML: a report from the Children's Oncology Group.

Biallelic CEBPA mutations are associated with favorable outcomes in acute myeloid leukemia (AML). We evaluated the clinical and biologic implications of CEBPA-basic leucine zipper (CEBPA-bZip) mutations in children and young adults with newly diagnosed AML. CEBPA-bZip mutation status was determined in 2958 patients with AML enrolled on Children's Oncology Group trials (NCT00003790, NCT0007174, NCT00372593, NCT01379181). Next-generation sequencing (NGS) was performed in 1863 patients (107 with CEBPA mutations) to characterize the co-occurring mutations. CEBPA mutational status was correlated with disease characteristics and clinical outcomes. CEBPA-bZip mutations were identified in 160 (5.4%) of 2958 patients, with 132 (82.5%) harboring a second CEBPA mutation (CEBPA-double-mutated [CEBPA-dm]) and 28 (17.5%) had a single CEBPA-bZip only mutation. The clinical and laboratory features of the 2 CEBPA cohorts were very similar. Patients with CEBPA-dm and CEBPA-bZip experienced identical event-free survival (EFS) of 64% and similar overall survival (OS) of 81% and 89%, respectively (P = .259); this compared favorably to EFS of 46% and OS of 61% in patients with CEBPA-wild-type (CEBPA-WT) (both P < .001). Transcriptome analysis demonstrated similar expression profiles for patients with CEBPA-bZip and CEBPA-dm. Comprehensive NGS of patients with CEBPA mutations identified co-occurring CSF3R mutations in 13.1% of patients and GATA2 mutations in 21.5% of patients. Patients with dual CEBPA and CSF3R mutations had an EFS of 17% vs 63% for patients with CEBPA-mutant or CSF3R-WT (P < .001) with a corresponding relapse rate (RR) of 83% vs 22%, respectively (P < .001); GATA2 co-occurrence did not have an impact on outcome. CEBPA-bZip domain mutations are associated with favorable clinical outcomes, regardless of monoallelic or biallelic status. Co-occurring CSF3R and CEBPA mutations are associated with a high RR that nullifies the favorable prognostic impact of CEBPA mutations.

Gemtuzumab Ozogamicin Improves Event-Free Survival and Reduces Relapse in Pediatric KMT2A-Rearranged AML: Results From the Phase III Children's Oncology Group Trial AAML0531.

PURPOSE: We investigated the impact of the CD33-targeted agent gemtuzumab ozogamicin (GO) on survival in pediatric patients with KMT2A-rearranged (KMT2A-r) acute myeloid leukemia (AML) enrolled in the Children's Oncology Group trial AAML0531 (NCT01407757). METHODS: Patients with KMT2A-r AML were identified and clinical characteristics described. Five-year overall survival (OS), event-free survival (EFS), disease-free survival (DFS), and relapse risk (RR) were determined overall and for higher-risk versus not high-risk translocation partners. GO's impact on response was determined and outcomes based on consolidation approach (hematopoietic stem cell transplant [HSCT] v chemotherapy) described. RESULTS: Two hundred fifteen (21%) of 1,022 patients enrolled had KMT2A-r AML. Five-year EFS and OS from study entry were 38% and 58%, respectively. EFS was superior with GO treatment (EFS 48% with GO v 29% without, P = .003), although OS was comparable (63% v 53%, P = .054). For patients with KMT2A-r AML who achieved complete remission, GO was associated with lower RR (40% GO v 66% patients who did not receive GO [No-GO], P = .001) and improved 5-year DFS (GO 57% v No-GO 33%, P = .002). GO benefit was observed in both higher-risk and not high-risk KMT2A-r subsets. For patients who underwent HSCT, prior GO exposure was associated with decreased relapse (5-year RR: 28% GO and HSCT v 73% No-GO and HSCT, P = .006). In multivariable analysis, GO was independently associated with improved EFS, improved DFS, and reduced RR. CONCLUSION: GO added to conventional chemotherapy improved outcomes for KMT2A-r AML; consolidation with HSCT may further enhance outcomes. Future clinical trials should study CD33-targeted agents in combination with HSCT for pediatric KMT2A-r AML.

Morphologic remission status is limited compared to ΔN flow cytometry: a Children's Oncology Group AAML0531 report.

Risk stratification for acute myeloid leukemia (AML) uses molecular and cytogenetic abnormalities identified at diagnosis. Response to therapy informs risk, and morphology continues to be used more frequently than flow cytometry. Herein, the largest cohort of pediatric patients prospectively assessed for measurable residual disease (MRD) by flow cytometry (N = 784) is reported. The "difference from normal" (ΔN) technique was applied: 31% of all patients tested positive (AML range, 0.02% to 91%) after the first course of treatment on Children's Oncology Group study AAML0531. Detection of MRD following initial chemotherapy proved the strongest predicator of overall survival (OS) in univariable and multivariable analyses, and was predictive of relapse risk, disease-free survival, and treatment-related mortality. Clearance of MRD after a second round of chemotherapy did not improve survival. The morphologic definition of persistent disease (>15% AML) failed 27% of the time; those identified as MRD- had superior outcomes. Similarly, for patients not achieving morphologic remission (>5% blasts), 36% of patients were MRD- and had favorable outcomes compared with those who were MRD+ (P < .001); hence an increase in myeloid progenitor cells can be favorable when ΔN classifies them as phenotypically normal. Furthermore, ΔN reclassified 20% of patients in morphologic remission as having detectable MRD with comparable poor outcomes. Retrospective analysis using the relapse phenotype as a template demonstrated that 96% of MRD- patients had <0.02% of the relapse immunophenotype in their end of induction 1 marrow. Thus, the detection of abnormal myeloid progenitor cells by ΔN is both specific and sensitive, with a high predictive signal identifiable early in treatment. This trial was registered at www.clinicaltrials.gov as #NCT00372593.

Deciphering the Significance of CD56 Expression in Pediatric Acute Myeloid Leukemia: A Report from the Children's Oncology Group.

BACKGROUND: In patients with acute myeloid leukemia (AML), CD56 expression has been associated with adverse clinical outcome. We reported on a phenotype associated with very poor prognosis (RAM) in children enrolled in the Children's Oncology Group trial AAML0531 (Brodersen et al. Leukemia 30 (2016) 2077-2080). RAM is also characterized in part by high-intensity expression of the CD56 antigen. Herein, we investigate underlying biological and clinical differences among CD56-positive AMLs for patients in AAML0531. METHODS: For 769 newly diagnosed pediatric patients with de novo AML enrolled in AAML0531, bone marrow specimens were submitted for flow cytometric analysis. For each patient, an immunophenotypic expression profile (IEP) was defined by mean fluorescent intensities of assayed surface antigens. Unsupervised hierarchical clustering analysis (HCA) was completed to group patients with similar immunophenotypes. Clusters were then evaluated for CD56 expression. Principal component analysis (PCA) was subsequently applied to determine whether CD56-positive patient groups were nonoverlapping. RESULTS: HCA of IEPs revealed three unique phenotypic clusters of patients with CD56-positive AML, and PCA showed that these three cohorts are distinct. Cohort 1 (N = 77) showed a prevalence of t(8;21) patients (72%), Cohort 2 (N = 52) a prevalence of 11q23 patients (69%), and Cohort 3 (RAM) (N = 16) a prevalence of patients with co-occurrence of the CBFA2T3-GLIS2 fusion transcript (63%). The 5-year event-free survival (EFS) for Cohorts 1, 2, and 3 were 69, 39, and 19%, respectively. CONCLUSIONS: When leukemia is considered by its multidimensional immunophenotype and not by the expression of a single antigen, correlations are seen between genotype and there are significant differences in patient outcomes. © 2019 International Clinical Cytometry Society.

Outcomes of Measurable Residual Disease in Pediatric Acute Myeloid Leukemia before and after Hematopoietic Stem Cell Transplant: Validation of Difference from Normal Flow Cytometry with Chimerism Studies and Wilms Tumor 1 Gene Expression.

We enrolled 150 patients in a prospective multicenter study of children with acute myeloid leukemia undergoing hematopoietic stem cell transplantation (HSCT) to compare the detection of measurable residual disease (MRD) by a "difference from normal" flow cytometry (ΔN) approach with assessment of Wilms tumor 1 (WT1) gene expression without access to the diagnostic specimen. Prospective analysis of the specimens using this approach showed that 23% of patients screened for HSCT had detectable residual disease by ΔN (.04% to 53%). Of those patients who proceeded to transplant as being in morphologic remission, 10 had detectable disease (.04% to 14%) by ΔN. The disease-free survival of this group was 10% (0 to 35%) compared with 55% (46% to 64%, P < .001) for those without disease. The ΔN assay was validated using the post-HSCT specimen by sorting abnormal or suspicious cells to confirm recipient or donor origin by chimerism studies. All 15 patients who had confirmation of tumor detection relapsed, whereas the 2 patients with suspicious phenotype cells lacking this confirmation did not. The phenotype of the relapse specimen was then used retrospectively to assess the pre-HSCT specimen, allowing identification of additional samples with low levels of MRD involvement that were previously undetected. Quantitative assessment of WT1 gene expression was not predictive of relapse or other outcomes in either pre- or post-transplant specimens. MRD detected by ΔN was highly specific, but did not identify most relapsing patients. The application of the assay was limited by poor quality among one-third of the specimens and lack of a diagnostic phenotype for comparison.

Phenotype in combination with genotype improves outcome prediction in acute myeloid leukemia: a report from Children's Oncology Group protocol AAML0531.

Diagnostic biomarkers can be used to determine relapse risk in acute myeloid leukemia, and certain genetic aberrancies have prognostic relevance. A diagnostic immunophenotypic expression profile, which quantifies the amounts of distinct gene products, not just their presence or absence, was established in order to improve outcome prediction for patients with acute myeloid leukemia. The immunophenotypic expression profile, which defines each patient's leukemia as a location in 15-dimensional space, was generated for 769 patients enrolled in the Children's Oncology Group AAML0531 protocol. Unsupervised hierarchical clustering grouped patients with similar immunophenotypic expression profiles into eleven patient cohorts, demonstrating high associations among phenotype, genotype, morphology, and outcome. Of 95 patients with inv(16), 79% segregated in Cluster A. Of 109 patients with t(8;21), 92% segregated in Clusters A and B. Of 152 patients with 11q23 alterations, 78% segregated in Clusters D, E, F, G, or H. For both inv(16) and 11q23 abnormalities, differential phenotypic expression identified patient groups with different survival characteristics (P<0.05). Clinical outcome analysis revealed that Cluster B (predominantly t(8;21)) was associated with favorable outcome (P<0.001) and Clusters E, G, H, and K were associated with adverse outcomes (P<0.05). Multivariable regression analysis revealed that Clusters E, G, H, and K were independently associated with worse survival (P range <0.001 to 0.008). The Children's Oncology Group AAML0531 trial: clinicaltrials.gov Identifier: 00372593.

Acute Myeloid Leukemia with RAM Immunophenotype: A Pediatric Case with Unusual Morphologic Features.

The RAM immunophenotype has been recently described as a subtype of acute myelogenous leukemia (AML) that is characterized clinically by extremely poor prognosis. We present a case of AML with RAM immunophenotype in a 5-year-old patient that resulted in poor outcome despite early hematopoietic cell transplant. We describe the unusual morphologic features that, along with the distinct immunophenotype, may provide initial diagnostic clues and further justify the classification of this AML variant as a rather distinct subtype.

Immunophenotypic analysis of erythroid dysplasia in myelodysplastic syndromes. A report from the IMDSFlow working group.

Current recommendations for diagnosing myelodysplastic syndromes endorse flow cytometry as an informative tool. Most flow cytometry protocols focus on the analysis of progenitor cells and the evaluation of the maturing myelomonocytic lineage. However, one of the most frequently observed features of myelodysplastic syndromes is anemia, which may be associated with dyserythropoiesis. Therefore, analysis of changes in flow cytometry features of nucleated erythroid cells may complement current flow cytometry tools. The multicenter study within the IMDSFlow Working Group, reported herein, focused on defining flow cytometry parameters that enable discrimination of dyserythropoiesis associated with myelodysplastic syndromes from non-clonal cytopenias. Data from a learning cohort were compared between myelodysplasia and controls, and results were validated in a separate cohort. The learning cohort comprised 245 myelodysplasia cases, 290 pathological, and 142 normal controls; the validation cohort comprised 129 myelodysplasia cases, 153 pathological, and 49 normal controls. Multivariate logistic regression analysis performed in the learning cohort revealed that analysis of expression of CD36 and CD71 (expressed as coefficient of variation), in combination with CD71 fluorescence intensity and the percentage of CD117+ erythroid progenitors provided the best discrimination between myelodysplastic syndromes and non-clonal cytopenias (specificity 90%; 95% confidence interval: 84-94%). The high specificity of this marker set was confirmed in the validation cohort (92%; 95% confidence interval: 86-97%). This erythroid flow cytometry marker combination may improve the evaluation of cytopenic cases with suspected myelodysplasia, particularly when combined with flow cytometry assessment of the myelomonocytic lineage.

Clone-specific MYD88 L265P and CXCR4 mutation status can provide clinical utility in suspected Waldenström macroglobulinemia/lymphoplasmacytic lymphoma.

MYD88 L265P, a diagnostic marker for lymphoplasmacytic lymphoma (LPL)/Waldenström macroglobulinemia (WM) can also be detected in other hematopoietic malignancies. We demonstrate a novel approach to increase the specificity of this marker for WM/LPL diagnosis by combining flow cytometric cell sorting with molecular analysis. Clonal B-lymphocyte and co-occurring clonal plasma cell populations of low-grade B-cell lymphomas were sorted by flow cytometry and analyzed for immunoglobulin gene rearrangements (PCR), and for MYD88 and CXCR4 mutations. Identical clonal origin was confirmed by PCR for 21 LPL/WM cases and MYD88 L265P was detected in both B-cell and plasma cell fractions. 9/20 other B-cell lymphomas with identical light chain restriction on B-cells and plasma cells were genotypically identical by PCR and MYD88 L265P was detected in both cell fractions in 7/9 whereas in 11/20 specimens with different clonal origin, MYD88 L265P was absent (5/11), or only found in B-lymphocytes (4/11), or plasma cells (2/11). CXCR4 mutations were detected in 17/39 cases, but missed in 63% of these without cell sorting. Confirming MYD88L265P in both B-cells and plasma cell fractions can provide a novel and powerful discriminator to distinguish LPL/WM from phenotypically similar disorders. Furthermore, this approach significantly increases CXCR4 detection sensitivity.

Detection of clonal evolution in hematopoietic malignancies by combining comparative genomic hybridization and single nucleotide polymorphism arrays.

BACKGROUND: Array comparative genomic hybridization (aCGH) has become a powerful tool for analyzing hematopoietic neoplasms and identifying genome-wide copy number changes in a single assay. aCGH also has superior resolution compared with fluorescence in situ hybridization (FISH) or conventional cytogenetics. Integration of single nucleotide polymorphism (SNP) probes with microarray analysis allows additional identification of acquired uniparental disomy, a copy neutral aberration with known potential to contribute to tumor pathogenesis. However, a limitation of microarray analysis has been the inability to detect clonal heterogeneity in a sample. METHODS: This study comprised 16 samples (acute myeloid leukemia, myelodysplastic syndrome, chronic lymphocytic leukemia, plasma cell neoplasm) with complex cytogenetic features and evidence of clonal evolution. We used an integrated manual peak reassignment approach combining analysis of aCGH and SNP microarray data for characterization of subclonal abnormalities. We compared array findings with results obtained from conventional cytogenetic and FISH studies. RESULTS: Clonal heterogeneity was detected in 13 of 16 samples by microarray on the basis of log2 values. Use of the manual peak reassignment analysis approach improved resolution of the sample's clonal composition and genetic heterogeneity in 10 of 13 (77%) patients. Moreover, in 3 patients, clonal disease progression was revealed by array analysis that was not evident by cytogenetic or FISH studies. CONCLUSIONS: Genetic abnormalities originating from separate clonal subpopulations can be identified and further characterized by combining aCGH and SNP hybridization results from 1 integrated microarray chip by use of the manual peak reassignment technique. Its clinical utility in comparison to conventional cytogenetic or FISH studies is demonstrated.