Cases Of B-lineage Acute Lymphoblastic Leukemia Research Articles

Droplet Digital PCR Improves IG-/TR-based MRD Risk Definition in Childhood B-cell Precursor Acute Lymphoblastic Leukemia.

Minimal residual disease (MRD) is the most powerful prognostic factor in pediatric acute lymphoblastic leukemia (ALL). Real-time quantitative polymerase chain reaction (RQ-PCR) represents the gold standard for molecular MRD assessment and risk-based stratification of front-line treatment. In the protocols of the Italian Association of Pediatric Hematology and Oncology (AIEOP) and the Berlin-Frankfurth-Munschen (BFM) group AIEOP-BFM ALL2009 and ALL2017, B-lineage ALL patients with high RQ-PCR-MRD at day+33 and positive at day+78 are defined slow early responders (SERs). Based on results of the AIEOP-BFM ALL2000 study, these patients are treated as high-risk also when positive MRD signal at day +78 is below the lower limit of quantification of RQ-PCR (“positive not-quantifiable,” POS-NQ). To assess whether droplet digital polymerase chain reaction (ddPCR) could improve patients’ risk definition, we analyzed MRD in 209 pediatric B-lineage ALL cases classified by RQ-PCR as POS-NQ and/or negative (NEG) at days +33 and/or +78 in the AIEOP-BFM ALL2000 trial. ddPCR MRD analysis was performed on 45 samples collected at day +78 from SER patients, who had RQ-PCR MRD ≥ 5.0 × 10–4 at day+33 and POS-NQ at day+78 and were treated as medium risk (MR). The analysis identified 13 of 45 positive quantifiable cases. Most relapses occurred in this patients’ subgroup, while ddPCR NEG or ddPCR-POS-NQ patients had a significantly better outcome (P < 0.001). Overall, in 112 MR cases and 52 standard-risk patients, MRD negativity and POS-NQ were confirmed by the ddPCR analysis except for a minority of cases, for whom no differences in outcome were registered. These data indicate that ddPCR is more accurate than RQ-PCR in the measurement of MRD, particularly in late follow-up time points, and may thus allow improving patients’ stratification in ALL protocols.

Normal Absolute Monocyte Count at the Time of Relapse is Associated with Improved Survival After First Salvage Therapy in Adult Patients with Early Relapsed B-Lineage Acute Lymphoblastic Leukemia.

BackgroundPeripheral monocytes, a key cell type for innate immunity, have been shown to be associated with survival in various types of hematological malignancies. However, no previous studies regarding the prognostic impact of peripheral absolute monocyte count (AMC) in early relapsed B-lineage acute lymphoblastic leukemia (B-ALL) have been reported.MethodsForty-nine cases of early relapsed adult B-ALL were reviewed. The upper (0.80 × 109/L) and lower limits (0.12 × 109/L) of the normal value for AMC were used as cut-off points. Kaplan–Meier curves and Log rank test were used for comparison of overall survival (OS). The univariate and multivariate Cox proportional hazards models were used for investigating the factors associated with OS.ResultsMore than half (59.2%) of all patients showed a normal AMC (0.12–0.80 × 109/L). The median follow-up was 5.3 months from the start of first salvage therapy. Univariate analysis revealed that normal AMC (versus low/high AMC) at the time of relapse was a prognostic factor for improved OS (P = 0.021). On multivariate analysis, normal AMC (versus low/high AMC) at the time of relapse remained an independent prognostic factor for improved OS (hazard ratio = 0.43, P = 0.030).ConclusionAMC at the time of relapse, which can be easily derived from routine clinical laboratory testing of complete blood count, might be used as a prognostic marker for survival outcomes in adult patients with early relapsed B-ALL.

Flow cytometric false myeloperoxidase-positive childhood B-lineage acute lymphoblastic leukemia.

Flow cytometric intracellular myeloperoxidase (MPO) staining of leukemic blasts is a useful tool in diagnosis of leukemia subtype. Interpretation of high MPO-positivity can be a diagnostic challenge in B-lineage acute lymphoblastic leukemia (B-ALL). While very few such cases have been reported, high MPO positive B-ALL cases without additional myeloid antigen positivity are suspect and require further investigation. Three pediatric cases of B-ALL with strong MPO staining (clone 8E6; Invitrogen) at diagnosis and three others with negative MPO staining were studied by flow cytometry and immunohistochemistry. In-vitro drug cytotoxicity, oxidative stress generation, and immunophenotyping using other MPO clones were performed to further investigate MPO presence. Expectedly, normal myeloid cells in all six samples were positive and mature lymphocytes negative for MPO staining. However, MPO monoclonal antibody (mAb) obtained from clones other than Invitrogen and other myeloid-specific mAbs gave negative results suggesting false positivity of the initial MPO staining. Immunohistochemistry for MPO was also negative on all six cases tested. Furthermore, in-vitro vincristine cytotoxicity was greater in leukemic cells from MPO false-positive cases compared with MPO-negative B-ALL samples, demonstrating indirect lack of MPO activity. Moreover, drug treatment did not lead to generation of reactive oxidative species, also reflective of lack of significant MPO presence. The cause of false-positive MPO staining remains unknown in these three cases; a cross reactivity could be the culprit. Caution should be given to similar phenomena and detailed investigation may contribute to the understanding of altered protein expression in such outlier cases. © 2017 International Clinical Cytometry Society.

Flow cytometric detection of minimal residual disease in B-lineage acute lymphoblastic leukemia by using “MRD lite” panel

BackgroundNormal B lymphoid precursors that express CD19, CD10, and/or CD34 are highly sensitive to corticosteroids and after two weeks of remission-induction therapy, they form less than 0.01% of the bone marrow population. More than 0.01% of such cells indicate minimal residual disease (MRD). MRD “lite” panel uses only three antibodies, namely CD19, CD10, and CD34 for MRD detection in cases of B-lineage acute lymphoblastic leukemia (B-ALL) expressing CD19, CD10, and/or CD34 by flow cytometry. MethodsFifteen cases of B-ALL were studied for MRD at Day 19 of remission-induction therapy by employing a simplified MRD detection protocol using a 3-color fluorochrome conjugated antibody panel (CD19, CD10, and CD34) on bone marrow aspirate samples. ResultsAll cases at diagnosis expressed CD19, CD10, and CD34. Of fifteen patients, five (33.33%) were MRD negative with less than 0.01% of mononuclear cells and remaining ten cases (66.66%) were MRD positive, with the level of 0.01% to less than 0.1% cells. ConclusionThe MRD assay used in this study is a simplified method for detecting MRD at Day 19 of remission-induction therapy for B-lineage ALL. This MRD assay is an effective and useful methodology in cases of B-ALL expressing CD19, CD10, and/or CD34 by flow cytometry.

Rapid Identification of BCR/ABL1 -like Acute Lymphoblastic Leukemia (ALL) Cases By Quantitative Real Time-PCR (Q-RT-PCR). Generation and Validation of a Predictive Statistical Model

Introduction: Among B-lineage ALL, BCR/ABL1-like cases have clearly emerged as a subgroup with peculiar features. By gene expression profiling (GEP), they have a transcriptional signature similar to that of BCR/ABL1+ cases. Moreover, these cases often have IKZF1 deletions and CRLF2 deregulation, and harbor several lesions involving tyrosine kinases (TKs) that may be patient specific. The incidence varies with age (from 10% of B-lineage ALL in children, to 27% in adolescents/young adults, with a decrease in adults) and they are usually characterized by an inferior outcome, though risk-directed therapy based on minimal residual disease levels has improved prognosis in childhood. Even if GEP promptly recognizes these patients, this technique is expensive, not readily available and not applicable to all centers.Aims: To generate a simple and reliable tool, based on a Q-RT-PCR approach, aimed at the rapid identification of BCR/ABL1-like cases, and to build a statistical predictive model. Finally, to correlate the results obtained with the clinico-biologic features and outcome.Methods and patients: Through a meta-analysis of previously published GEP data focused on BCR/ABL1-like cases, and on our in house results, 9 commonly overexpressed genes were selected and evaluated by Q-RT-PCR; CRLF2 was also included, because of its association with this signature (total=10 genes). Q-RT-PCR was performed using the ABI PRISM 7300 Sequence Detection System and FastStart Universal SYBR Green Master (Rox). Overall, 149 B-lineage ALL cases, negative for known major rearrangements, i.e. BCR/ABL1, ETV6/RUNX1, E2A/PBX1 and ALL1 (B-NEG ALL), were tested. Median age was 28.8 years (range 1-78), 59 were females and 90 males. To generate a predictive model for BCR/ABL1-like identification, one round of cross-validation was performed; the full dataset was divided into two panels (discovery and screening). The discovery panel included 52 cases previously evaluated for GEP analysis and comprised 26 BCR/ABL1-like and 26 B-NEG ALL samples, while the screening panel (n=97) was based on 81 cases including 16 B-NEG ALL samples harboring JAK2, IL7R or CRLF2 mutations, tested as internal control, given their association with a BCR/ABL1-like signature.Results: The statistical model was built on the discovery panel and was validated, in terms of sensitivity and specificity, on the screening panel. Q-RT-PCR values of the 10 genes were grouped in the discovery panel according to principal component analysis to reduce multicollinearity and a logistic regression model was used to examine the association among the first three principal components (accounting for more than 80% of the total variance) and BCR/ABL1-like cases. Finally, a score was computed and validated in the screening panel. The model had a sensitivity and specificity of 93.8% and 93.8%, respectively, with a positive predictive value of 75%. Within the screening panel, 10/81 (12.3%) and 6/16 JAK2, IL7R or CRLF2 mutated cases (37.5%) were predicted as BCR/ABL1-like (total=16.5%). GEP was performed on 14/16 BCR/ABL1-like samples and confirmed the similarity with BCR/ABL1+ ALL cases by unsupervised and supervised analysis. Molecular screening, comprising the screening of JAK1/2, CRLF2, IL7R (JAK/STAT pathway) mutations and IKZF1 deletions, confirmed a significant enrichment of the JAK/STAT pathway (48% vs 15%, p=0.001) and of IKZF1 deletions (82 vs 46%, p=0.006) in BCR-ABL1-like cases as opposed to the other B-NEG ALL cases. Finally, BCR-ABL1 -like patients showed a significantly higher white blood cells (WBC) count (46x109/l vs 12.8x109/l, p=0.003) and poorer prognosis compared to the other B-NEG ALL patients, in terms of complete remission achievement (70.3 vs 87%, p=0.07), overall survival (41.1% vs 59.1%, p=0.06) and disease-free survival (DFS) (31.1 % vs 46%, p=0.008).Conclusions: We describe a Q-RT-PCR approach and a statistical model capable of rapidly identifying BCR-ABL1-like cases. We confirm a significant association with JAK/STAT mutations, IKZF1 deletions and poorer outcome. The prompt recognition of BCR-ABL1-like ALL at presentation can: i) identify cases in which additional lesion/s involving a TK - that might sustain the BCR/ABL1-like signature - should be further investigated; ii) drive therapeutic decisions, since these patients might benefit by the addition of targeted therapies, particularly TK inhibitors. DisclosuresFoà:Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Cytogenetic Profile of De Novo B lineage Acute Lymphoblastic Leukemia: Determination of Frequency, Distribution Pattern and Identification of Rare and Novel Chromosomal Aberrations in Indian Patients.

Chromosomal aberrations identified in acute lymphoblastic leukemia (ALL) have an important role in disease diagnosis, prognosis and management. Information on karyotype and associated clinical parameters are essential to physicians for planning cancer control interventions in different geographical regions. In this study, we present the overall frequency and distribution patterns of chromosomal aberrations in both children and adult de novo B lineage ALL Indian patients using conventional cytogenetics, interphase FISH and multiplex RT-PCR. Among the 215 subjects, cytogenetic results were achieved in 172 (80%) patients; normal karyotype represented 37.2% and abnormal 62.8% with a distribution as follows: 15.3% hypodiploidy; 10.3% hyperdiploidy; 15.8% t(9;22); 9.8% t(1;19); 3.7% t(12;21); 2.8% t(4;11); 2.8% complex karyotypes. Apart from these, we observed several novel, rare and common chromosomal rearrangements. Also, FISH studies using LSI extra-signal dual-color probes revealed additional structural or numerical changes. These results demonstrate cytogenetic heterogeneity of ALL and confirm that the incidence of chromosomal abnormalities varies considerably. To the best of our knowledge, this is one of the largest reported series of cytogenetic investigations in Indian B-lineage ALL cases. In addition, ongoing cytogenetic studies are warranted in larger groups of B-lineage ALL cases to identify newly acquired chromosomal abnormalities that may contribute to disease diagnosis and management.

NSE/αNAE positivity in B-lineage acute lymphoblastic leukemia: revisiting a potential cytochemical diagnostic pitfall

Cytochemical staining for leukemia typing is declining in hematology laboratories, but the use of flow cytometry may not be possible in some settings. Aberrant cytochemical nonspecific esterase/α-naphthyl acetate esterase (NSE/αNAE) positive B-lymphoblasts can cause confusion with monoblasts, a potentially dangerous pitfall. This unusual cytochemical NSE/αNAE positivity had been associated with relatively poorer outcome of acute lymphoblastic leukemia (ALL) in the era prior to the advent of routine multicolor flow cytometric immunophenotyping. We reviewed morphological, cytochemical and flow-cytometric data from five cases of B-lineage ALL that showed NSE/αNAE positivity and were diagnosed definitively using multi-parametric flow cytometric immunophenotypic analysis. Diffuse or dot-like (localized) strong cytochemical NSE/αNAE activity was detected in all cases and all showed one or more features of high risk disease. The number of NSE/αNAE positive blasts in the marrow varied from 10 to 75%. The morphological differential diagnoses included T-lymphoid lineage ALL and acute monoblastic leukemia (AML-M5). Flow cytometric data revealed B-lineage antigens and the absence of monocytic or other myeloid markers resolved the diagnosis. These cases underscore the importance of immunophenotyping in all cases of suspected ALL regardless of the cytochemical findings. Although the numbers are small, the association with high risk disease observed in all five of our cases may corroborate the previously reported poor prognostic value of such aberrant cytochemical staining.

Clinical importance of myeloid antigen expression in Moroccan patients with adult B-lineage acute lymphoblastic leukemia.

The prognostic significance of myeloid antigen (MyAg) expression in acute lymphoblastic leukemias (ALL), especially in adult patients, is still controversial. In the present report, frequency and clinical significance of MyAg (CD13 and/or CD33) in blast cells were assessed in 80 consecutive adult (≥18 years) patients with B-lineage acute lymphoblastic leukemia (B-ALL), representing 66.7% of 120 patients diagnosed as having ALL during the study period. Immunophenotyping was used to classify leukemic cells as Bor Tlymphoblasts and to identify the aberrant expression of myeloid-associated antigens. MyAg expression was documented in 52.5% of the 80 B-ALL cases analyzed. CD13 was the most commonly antigen expressed (36.3%) followed by CD33 (28.8%). No significant associations were found between the expression of MyAg and the presence of known adverse prognostic features (eg: age>30 years, male gender, high WBC count and Philadelphia positivity). Also, we failed to observe any statistically significant difference between MyAg-positive and MyAg-negative patients in terms of achievement of complete remission and overall survival at 3 years. This study demonstrates that the presence of MyAg on lymphoblastic cells lacks prognostic value In Moroccan patients with adult B-ALL.

Detection of Common Chimeric Fusion Transcripts in Acute Lymphoblastic Leukemia (ALL) Patients Using Multiplex Reverse Transcriptase Polymerase Chain Reaction Assay: A North Indian Tertiary Care Centre Experience

Abstract 4234 Introduction:Chromosomal abnormalities constitute an important parameter to identify prognostically relevant subgroups in acute lymphoblastic leukemia (ALL). Many western studies report the incidence of common chimeric fusion transcripts as 30–35%, however the data regarding the Indian patients is very scarce. Aims and Objectives:The aim of the present study was to detect presence of common chimeric fusion transcripts of t(12;21), t(9;22), t(1;19) and t(4;11) in adult and pediatric ALL cases using the Multiplex RT-PCR assay. Materials and Methods:This prospective study included 100 consecutive ALL cases diagnosed during last one year; 54 patients were <12 years of age. Diagnosis of ALL was made on the basis of bone marrow (BM) examination and flowcytometric immunophenotypic (FCM-IP) analysis. Approximately 2 ml of peripheral blood (PB) sample or 0.5 ml of BM sample was collected in EDTA. RNA extraction and cDNA synthesis was performed using commercial kits, according to standard protocols. RT-PCR assay was carried out using primers specific to the fusion transcripts of TEL-AML1 t(12;21); BCR-ABL t(9;22); E2A-PBX t(1;19) and MLL-AF4 t(4;11). The results of RT-PCR and FCM-IP were blinded and compared later. Results:Out of the 100 cases enrolled in the study, 54% were pediatric and 46% adult cases. Among the pediatric ALL cases, 49 were diagnosed as B-lineage ALL, 3 T-lineage ALL and 2 Biphenotypic ALL. Whereas among the adult ALL cases, 30 were diagnosed as B-lineage ALL, 12 T-lineage ALL and 4 Biphenotypic ALL. A total of 26% showed positivity for various fusion transcripts: 13% each pediatric and adult cases being positive. Of the 12 positive pediatric B-lineage ALL cases, 8 were positive for TEL-AML1 transcripts, 1 for BCR-ABL transcripts, 1 for E2A-PBX transcripts and 2 for MLL-AF4 transcripts. Out of the 11 positive adult cases, 7 were positive for BCR-ABL transcripts, 2 were positive for TEL-AML1 transcripts and 1 each for E2A-PBX transcripts and MLL-AF4 transcripts. BCR-ABL transcripts were detected in 1 out of 2 pediatric biphenotypic ALL and 2 out of 4 adult biphenotypic ALL cases. None of the pediatric cases was positive for the MLL-AF4 transcripts. None of the 4 transcripts were detected in any T-lineage ALL. Conclusion:Results of our study are comparable to those of other western and Asian studies, with TEL-AML1 transcripts being the most common fusion transcripts seen in pediatric B-ALL cases and BCR-ABL in adult cases. However two Indian studies had reported much lower incidence of TEL-AML1 and MLL-AF4 transcript in B-ALL cases. Identification of good prognostic groups and rationalization of therapy assumes great importance in our set ups as many patients are economically under-privileged. Relationship of these transcripts to the prognosis of our ALL patients will be evaluated in future. Disclosures:No relevant conflicts of interest to declare.

Expression of Ikaros Isoform 6 in Chinese Children With Acute Lymphoblastic Leukemia

Ikaros is a zinc-finger transcription factor that plays an important role in the differentiation and proliferation of lymphocytes. Dominant-negative Ikaros isoform 6 (Ik6), one of its common subtypes, is overexpressed in leukemia patients and is associated with unfavorable prognosis in childhood B-cell progenitor acute lymphoblastic leukemia (ALL). This study was to identify specific isoforms, especially Ik6, in Chinese pediatric patients with ALL. The mRNA expression of Ikaros was detected in 88 children with previously untreated ALL by nested reverse transcription-polymerase chain reaction (RT-PCR). Sequencing of the PCR products was performed to identify specific isoforms. The expression of fusion genes was determined by using multiplex RT-PCR. The functional isoforms Ik1, Ik2/3, and dominant negative isoforms Ik4, 6, 8, 9, 10 identified by nested RT-PCR were further confirmed by sequence analysis. In the 88 cases, the Ik6 was found to be overexpressed in 8 of 70 cases of B-lineage ALL and in 1 of 18 cases of T-lineage ALL patients. Among Ik6 B-lineage ALL patients, 3 had expression of BCR/ABL fusion gene and 1 had HOX11 expression. Ik6 overexpression was independent of age, white blood cell count at diagnosis, risk group, and expression of the fusion genes currently measured in China except BCR/ABL (P<0.01). And it was strongly associated with elevated levels of minimal residual disease at day 28 (P<0.01). Ik6 can be included as a high-risk factor at diagnosis. In developing countries with limited resources, it can be economically detected by nested RT-PCR.

Flow cytometric analysis of de novo acute lymphoblastic leukemia in childhood: report from the Japanese Pediatric Leukemia/Lymphoma Study Group

Although the antigen expression patterns of childhood acute lymphoblastic leukemia (ALL) are well known, little attention has been given to standardizing the diagnostic and classification criteria. We retrospectively analyzed the flow cytometric data from a large study of antigen expression in 1,774 children with newly diagnosed ALL in JPLSG. T- and B-lineage ALL accounted for 13 and 87% of childhood ALL cases, respectively. Cytoplasmic CD3 and CD7 antigens were positive in all T-ALL cases. More than 80% of T-ALL cases expressed CD2, CD5 and TdT. In B-lineage ALL, the frequencies of early pre-B, pre-B, transitional pre-B and B-ALL were 81, 15.5, 0.6 and 2.9%, respectively. More than 90% of early pre-B ALL cases expressed CD19, CD79a, CD22, CD10 and TdT. CD34 was expressed in three-fourths of early pre-B ALL cases. The frequencies of TdT and CD34 expression were lower in pre-B ALL than in early pre-B ALL. B-ALL showed less frequent expression of CD22, CD10, CD34 and TdT than other B-lineage ALL cases. Expression of CD13 and CD33, aberrant myeloid antigens, was significantly more frequently associated with B-lineage ALL than with T-ALL. Based on this retrospective study of antigen expression in 1,774 de novo childhood ALL cases in JPLSG, we propose standardized clinical guidelines for the immunophenotypic criteria for diagnosis and classification of pediatric ALL.

Abstract 4356: Cytotoxicity of the anti-CD22 immunotoxin HA22 against pediatric acute lymphoblastic leukemia (ALL)

Abstract Cure rates for children with acute lymphoblastic leukemia (ALL) have steadily improved, but a proportion of patients still relapse despite intensive and prolonged chemotherapy. New therapies are needed to overcome drug resistance and reduce non-specific toxicities of chemotherapy. CD22 is a B-lineage differentiation antigen expressed on most B-lineage ALL blasts. We have developed recombinant anti-CD22 immunotoxins composed of the Fv portion of the anti-CD22 mAb RFB4 linked to a 38kDa truncated protein derived from Pseudomonas exotoxin A (PE38). These produce cell death through a unique and highly specific mechanism of action, entailing binding to cell surface CD22, rapid internalization, intracellular processing, and inhibition of protein synthesis through ADP-ribosylation of elongation factor-2. In phase I and II trials of patients with hairy cell leukemia the first generation agent, BL22, produced complete responses (CR) of 61% and 47% respectively. However, activity was modest in children with ALL (Blood 2007;110:262a). A modified agent, HA22, was derived by complementarity-determining region mutagenesis resulting in a significantly higher binding affinity for the CD22 antigen. We cultured pre-B ALL cells from newly diagnosed (n = 13) and relapsed children (n= 22), as well as human ALL cells derived from xenografts in immunodeficient mice (n = 4) and the cell line KOPN-8 on telomerase-immortalized human bone marrow mesenchymal cells. On day 2 of culture, HA22 was added at concentrations ranging from 0 to 500ng/ml and the cells were incubated for an additional 72 hours. Dexamethasone 10umol/L was used as a positive control. The number of viable CD19+ cells at the end of the cultures was determined by flow cytometry. Viability was assessed using gates set for light-scatter properties and Annexin-PE/7-AAD staining on Days 2 and 5. The percentage of CD19+ viable cells relative to the control was determined and the 50% inhibitory concentration (IC50) calculated. Mesenchymal-supported cell cultures maintained ALL cell viability (mean 77%, median 52%, range 6% - 280% viable cells at 96 hours). ALL samples varied in their sensitivity to HA22. IC50s ranged from 1-80 ng/mL in 29 of 40 samples. In 11 samples 50% killing was not achieved. Resistance to cytotoxicity from dexamethasone was also observed, which correlated with resistance to HA22 for samples from newly diagnosed patients (r = 0.55, p = 0.053). Peripheral blood blasts appeared to be more sensitive to HA22 in comparison to bone marrow derived blasts (p = 0.008). Blasts with higher CD22 site densities showed increased cytotoxicity to HA22 (r = −0.34, p = 0.16). These results indicate that most cases of childhood B-lineage ALL are sensitive to HA22 at concentrations well below clinically achievable blood levels. Clinical testing of HA22 in children with drug-resistant ALL has been initiated. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4356.

CD22 Expression in Pediatric B-Lineage Acute Lymphoblastic Leukemia.

Abstract 4119While the majority of pediatric patients with newly diagnosed B-lineage acute lymphoblastic leukemia (ALL) are cured with standard chemotherapy regimens, treatment is associated with multiple toxicities, and ALL remains the most frequent cause of cancer mortality in childhood. CD22, a B-lineage surface glycoprotein involved in B cell signaling and adhesion, is expressed in most cases of B-lineage ALL. We are conducting clinical trials of anti-CD22 immunotoxins [RFB4(dsFv)-PE38] for pediatric ALL. To assess eligibility for such targeted therapy, CD22 expression by ALL cells was studied in peripheral blood and/or bone marrow aspirate samples from 50 patients with relapsed ALL. The level of CD22 expression by ALL cells was quantitated by measuring mean anti-CD22 antibody binding per ALL cell (ABC) under saturating conditions using flow cytometry and the BD Biosciences QuantiBRITE system for fluorescence quantitation. Patients ranged in age from 3 to 22 years (median 10 years) and included 27 males and 23 females. CD22 expression was detected in all samples, and the vast majority of cases demonstrated expression of CD22 in 100% of leukemic blasts. CD22 antigen density in ALL cells varied widely among patients at baseline (range 451 - 14,519; mean 4276; median 3824; standard deviation 2976; see graph). CD22-directed immunotoxin therapy was initiated in 29 of the 50 patients, 19 of whom had samples quantitated for CD22 expression levels both before and after immunotoxin therapy. Most patients exhibited limited variation in the mean number of anti-CD22 molecules bound per ALL cell when comparing multiple specimens. In conclusion, CD22 expression varies widely in pediatric B-lineage ALL and persists despite repeated exposure to CD22-directed therapy.(MedImmune, LLC, sponsored the clinical studies of anti-CD22 immunotoxin CAT-8015.) [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Translocation (14;14)(q11;q32) with simultaneous involvement of the IGH and CEBPE genes in B-lineage acute lymphoblastic leukemia

Translocation (14;14)(q11;q32) is one of the recurrent chromosome aberrations in ataxia-teleangiectasia (AT) and T-cell malignancies. In patients with the t(14;14), the TCL1 and TCRα/δ genes were found to be involved at the molecular level. However, t(14;14)(q11;q32) is an exceedingly rare phenomenon in B-lineage acute lymphoblastic leukemia (B-ALL). To date, it has been reported in only 5 B-ALL cases. Here, we report another B-ALL case with t(14;14)(q11;q32) in a 39-year-old female. The immunophenotype of the blasts showed positivity for CD79a, CD10, CD19, and HLA-DR. Chromosome analysis of the bone marrow (BM) cells at presentation showed the karyotype 47,XX,+4,t(14;14)(q11;q32). Fluorescence in situ hybridization (FISH) demonstrated trisomy 4 and the simultaneous involvement of the IGH gene at 14q32 and the CEBPE gene at 14q11, which differs from the genes involved in T-cell leukemias. After chemotherapy, the patient achieved complete remission (CR). Later, she received allogeneic peripheral blood stem cell transplantation. After CR, the karyotype of the BM cells was normal. She was disease-free at a 6-month follow-up. We suggest that t(14;14)(q11;q32) involving the IGH and CEBPE genes in B-ALL is rare, but it is a recurrent abnormality that could identify a new subgroup of B-ALL.

ZAP-70 expression in acute lymphoblastic leukemia: association with the E2A/PBX1 rearrangement and the pre-B stage of differentiation and prognostic implications

We evaluated the expression of 2 members of the Syk family, ZAP-70 and Syk, in acute lymphoblastic leukemia (ALL) samples, using data derived from a series of 33 T-ALL and 95 B-lineage adult ALL patients analyzed by oligonucleotide arrays. Of the B-lineage ALL cases, 37 were BCR/ABL+, 10 were ALL1/AF4+, 5 were E2A/PBX1+, and 43 carried no known molecular abnormality. ZAP-70 was highly expressed in T-ALL. A high ZAP-70 expression was also found in a proportion of B-lineage ALL, the highest levels being associated with the E2A/PBX1+ group and the lowest with ALL1/AF4+ cases (P < .001). A higher ZAP-70 expression was also observed in the pre-B group (P < .001). Remarkably, Syk expression was always preserved, suggesting that ZAP-70 expression is not substitutive of Syk. At the protein level, ZAP-70 was evaluated on 39 newly diagnosed ALL patients (25 adults, 14 children) and was detected in 23 cases (59%). ZAP-70 expression was consistently found in Ig mu+ cases. Evaluation of long-term outcome in cases without molecular abnormalities showed that the higher levels of ZAP-70 were coupled to a higher relapse rate. In ALL, ZAP-70 expression is associated with the E2A/PBX1 rearrangement and pre-B stage and may have a prognostic role and be a candidate molecule for targeted therapies.

Genome-Wide Expression Profiling and Minimal Residual Disease in Childhood Acute Lymphoblastic Leukemia.

The assessment of early treatment response based on minimal residual disease (MRD) detection is a powerful prognostic indicator in childhood acute lymphoblastic leukemia (ALL). To identify genes whose expression is associated with poorer early response and to define gene expression signatures predictive of MRD findings, we correlated gene expression profiles of diagnostic bone marrow blasts in 236 children with ALL enrolled in St. Jude Total Therapy XIIIA-XV protocols with MRD results obtained at days 19 and 46 of remission induction treatment. The dataset consisted of 46 T-lineage ALL and 190 B-lineage ALL; the latter included 10 BCR-ABL, 11 E2A-PBX1, 12 MLL rearranged, 49 TEL-AML1, 46 hyperdiploid >50 chromosomes (HD>50) karyotype, 3 BCR-ABL plus HD>50, and 59 other cases. RNA expression profiles were obtained using Affymetrix U133A gene chips; MRD was assessed by a flow cytometric assay that allows the identification of one leukemic cell among 10,000 normal bone marrow cells or greater and is applicable to approximately 95% of patients. We used a general linear model to eliminate the possible confounding influence of genetic subtypes known to be associated with treatment response. Then, we applied a t-test with the P value threshold of 0.006, determined by the profile information criterion for large-scale multiple tests. By this criterion, 279 probe sets were associated with MRD at day 19 (estimated false-discovery rate [FDR] 0.42) and 606 probe sets with MRD at day 46 (estimated FDR 0.17); 41 probe sets were associated with MRD at both time points. The expression of CASP8A2 (FLASH, CED-4), which encodes a key mediator of apoptosis and participates in glucocorticoid signaling, was significantly lower in cases with MRD at both time points. In a cluster analysis using the probe sets associated with MRD, the capacity to predict results of the MRD assay was limited. For example, only 69% of MRD-negative and 81% of MRD-positive results at day 19 were correctly classified. Similar results were obtained using the day 46 data. We also determined whether MRD status could be predicted by an unsupervised cluster analysis of all 236 cases with 17,269 probe sets (after removing transcripts not expressed in any of the samples). Although there was a strong association of cluster formation with lineage and genetic subtypes, there was no significant association with MRD status at days 19 or 46. Moreover, there was no significant association with MRD status in analyses limited to a series of 66 ‘standard-risk' B-lineage ALL cases (excluding those with BCR-ABL, TEL-AML1, MLL, hypodiploid <45 chromosomes or HD>50), or to cases of each individual genetic subtype. In conclusion, leukemic cells at diagnosis express genes that are associated with MRD. Although gene expression profiles can accurately identify leukemia cell lineage and genotype, they cannot accurately predict MRD status, probably owing to the multifactorial nature of treatment response, which is influenced not only by cellular drug resistance but also by clinical and pharmacologic variables of the host.

Post-induction residual disease in translocation t(12;21)-positive childhood ALL.

t(12;21)(p1 3;q22), the most frequent chromosomal translocation found in childhood acute lymphoblastic leukemia (ALL), occurs in approximately 25% of B-lineage ALL cases and has been claimed to carry a good prognosis. As part of the Nordic Society of Pediatric Hematology and Oncology (NOPHO) ALL-MRD 95 study, which includes children from Iceland, Norway, and Denmark diagnose d with ALL, patients were screened for the presence of t(12; 21) by reverse transcriptase-polymerase chain reaction (RT-PCR) at diagnosis, and their residual disease was quantified after 4 weeks of induction therapy (prednisolone, vincristine, doxorubicin, i.t. methotrexate) by a competitive, clone-specific, semi-nested PCR analysis. Among 96 children diagnosed with ALL, and quantified for post induction residual disease, 32 were t(12;21)-positive. The median residual disease was similar for B-precursor ALL patients with and without t(12;21) (0.009 vs. 0.03%, P = 0.12). Al though patients with t(12;21)-positive ALL have been claimed to have a good outcome, these data indicate that this does not reflect a high sensitivity to prednisolone, vincristine, and doxorubicin given during induction therapy.

Genetics of childhood acute lymphoblastic leukemia

Genetic alterations that underlie the pathogenesis of individual leukemia subtypes have important prognostic implications in acute lymphoblastic leukemia (ALL). Heretofore, specific genetic abnormalities with prognostic relevance were identified mainly by cytogenetic analysis or reverse-transcriptase polymerase-chain-reaction, and were largely limited to B-lineage ALL cases. Moreover, there is heterogeneity among specific genetic subtypes of ALL and treatment has over-riding impact on the prognostic information conveyed by the genetic abnormalities. In Philadelphia chromosome-positive (Ph+) ALL, an age of 1 to 9 years and a low initial leukocyte count confer a favorable prognosis. In each category of 11q23/MLL abnormality, infants less than 1 year of age have a worse treatment outcome than do older patients. Allogeneic transplantation with HLA-matched related donor improved outcome for patients with Ph+ ALL but not those with the t(4;11) andMLL-AF4 fusion. Using oligonucleotide microarrays, T-cell ALL can now be classified into four major subtypes with prognostic significance:MLL-ENL andHOX11 clusters with favorable prognosis andTAL1 andLYL1 clusters with inferior outcome. In our recent study of 327 ALL cases, distinct expression profiles could not only accurately identify each of the known prognostically important leukemia subtypes, but also individual cases that would eventually relapse or develop therapy-related acute myeloid leukemia. We have also identified a novel ALL subgroup with unique expression profile. Examination of the genes comprising the different expression signatures provided important insights into the biologic differences between the leukemia subgroups. The importance of host pharmacogenetics has also been recognized. Thus, genetic polymorphisms of certain drug-metabolizing enzymes, drug transporters or drug receptors have been linked with treatment efficacy and toxicity, as well as host susceptibility to the development of de novo leukemia or therapy-related second cancers. The best-characterized genes (e.g. thiopurine methyltransferase, cytochrome P450, glutathione transferase, methylenetetrahydrofolate reductase and NAD(P)H: quinone oxidoreductase) will be discussed. Ultimately, patients will be treated with individualized therapy based on leukemic cell gene expression profile and host pharmacogenetics.

Interleukin-4 variant (BAY 36-1677) selectively induces apoptosis in acute lymphoblastic leukemia cells

AbstractInterleukin 4 (IL-4) suppresses the growth of acute lymphoblastic leukemia (ALL) cells, but its clinical usefulness is limited by proinflammatory activity due mainly to the interaction of cytokine with endothelial cells and fibroblasts. Stroma-supported cultures of leukemic lymphoblasts were used to test the antileukemic activity of an IL-4 variant, BAY 36-1677, in which the mutations Arg 121 to Glu and Thr 13 to Asp ensure high affinity for IL-4Rα/IL-2Rγ receptors expressed by lymphoid cells, without activation of the IL-4Rα/IL-13Rα receptors mainly expressed by other cells. BAY 36-1677 (25 ng/mL) was cytotoxic in 14 of 16 cases of B-lineage ALL; the median reduction in cell recovery after 7 days of culture was 85% (range, 17%-95%) compared to results of parallel cultures not exposed to the cytokine. Twelve of the 14 sensitive cases had t(9;22) or 11q23 abnormalities; 3 were obtained at relapse. BAY 36-1677 induced apoptosis in leukemic lymphoblasts but did not substantially affect the growth of normal CD34+ cells, thus conferring a growth advantage to normal hematopoietic cells over leukemic lymphoblasts in vitro. BAY 36-1677 had antileukemic activity equal or superior to that produced by native IL-4, but it lacked any effects on the growth of endothelial cells and fibroblasts. The molecular manipulation of IL-4 to abrogate its proinflammatory activity has generated a novel and therapeutically promising cytokine for the treatment of high-risk ALL.

Flow Cytometric Diagnosis of the Cell Lineage and Developmental Stage of Acute Lymphoblastic Leukemia by Novel Monoclonal Antibodies Specific to Human Pre–B-Cell Receptor

Three novel monoclonal antibodies (MoAbs) have been established that recognize distinct epitopes of a human pre–B-cell receptor (pre-BCR) composed of a μ heavy (μH) chain and a λ5/VpreB surrogate light (SL) chain. HSL11 reacts with λ5 whereas HSL96 reacts with VpreB. Intriguingly, HSL2 does not bind to each component of the pre-BCR but does bind to the completely assembled pre-BCR complex. Flow cytometric analyses with cytoplasmic staining of a panel of human cell lines showed that HSL11 and HSL96 specifically stained cell lines derived from the pro–B and pre–B-cell stages of B-cell development. In contrast, HSL2 stained exclusively cell lines derived from the pre–B-cell stage. These results prompted us to explore the possibility of clinical application of these MoAbs for the determination of the cell lineage and developmental stage of acute lymphoblastic leukemia (ALL). Whereas none of mature B-lineage ALLs (B-ALLs), T-lineage ALLs (T-ALLs), and acute myeloid leukemias analyzed were stained in the cytoplasm with these three MoAbs, the vast majority of non–B- and non–T-ALLs (53 out of 56 cases) were found positive for either λ5, Vpre-B, or both in their cytoplasm. Among these 53 cytoplasmic SL chain-positive ALLs, 19 cases were also positive for cytoplasmic μH chain, indicative of pre–B-cell origin. Interestingly, 6 out of these 19 pre–B-ALL cases were found negative for cytoplasmic staining with HSL2. From these results, we propose a novel classification of B-ALL in which five subtypes are defined on the basis of the differential expression of SL chain, μH chain, pre-BCR, and light chain along the B-cell development.