Rapid identification of BCR/ABL1‐like acute lymphoblastic leukaemia patients using a predictive statistical model based on quantitative real time‐polymerase chain reaction: clinical,prognostic and therapeutic implications

BCR/ABL1‐like acute lymphoblastic leukaemia (ALL) is a subgroup of B‐lineage acute lymphoblastic leukaemia that occurs within cases without recurrent molecular rearrangements. Gene expression profiling (GEP) can identify these cases but it is expensive and not widely available. Using GEP, we identified 10 genes specifically overexpressed by BCR/ABL1‐like ALL cases and used their expression values – assessed by quantitative real time‐polymerase chain reaction (Q‐RT‐PCR) in 26 BCR/ABL1‐like and 26 non‐BCR/ABL1‐like cases to build a statistical “BCR/ABL1‐like predictor”, for the identification of BCR/ABL1‐like cases. By screening 142 B‐lineage ALL patients with the “BCR/ABL1‐like predictor”, we identified 28/142 BCR/ABL1‐like patients (19·7%). Overall, BCR/ABL1‐like cases were enriched in JAK/STAT mutations (P < 0·001), IKZF1 deletions (P < 0·001) and rearrangements involving cytokine receptors and tyrosine kinases (P = 0·001), thus corroborating the validity of the prediction. Clinically, the BCR/ABL1‐like cases identified by the BCR/ABL1‐like predictor achieved a lower rate of complete remission (P = 0·014) and a worse event‐free survival (P = 0·0009) compared to non‐BCR/ABL1‐like ALL. Consistently, primary cells from BCR/ABL1‐like cases responded in vitro to ponatinib. We propose a simple tool based on Q‐RT‐PCR and a statistical model that is capable of easily, quickly and reliably identifying BCR/ABL1‐like ALL cases at diagnosis.     

Involvement of primary mesenchymal precursors and hematopoietic bone marrow cells from chronic myeloid leukemia patients by BCR‐ABL1 fusion gene

For decades now, it is well established that chronic myeloid leukemia (CML) is a hematopoietic stem cell (HPC) disorder. However, it remains to be determined whether BCR‐ABL1 gene rearrangement occurs in a HPC or at an earlier stem cell and whether the degree of involvement of hematopoiesis by the BCR‐ABL1 fusion gene relates to the response to therapy. Here, we have investigated by interphase fluorescence in situ hybridization (iFISH) the distribution of BCR‐ABL1 fusion gene in FACS‐sorted bone marrow (BM) populations of mesenchymal precursor cells (MPC) and other hematopoietic cell populations from 18 newly diagnosed CML patients. Overall, our results showed systematic involvement at relatively high percentages of BM maturing neutrophils (97% ± 15%), basophils (95% ± 12%), eosinophils (90% ± 8%), CD34⁺ precursors cells (90% ± 7%), monocytes (84% ± 30%), nucleated red blood cells (87% ± 24%), and mast cells (77% ± 33%). By contrast, MPC (30% ± 34%), B‐cells (15% ± 27%), T‐lymphocytes (50% ± 26%), and NK‐cells (35% ± 34%) were involved at lower percentages. In 8/18 CML patients, ≥2 tumor BCR‐ABL1⁺ subclones were detected by iFISH. Of note, all tumor cell subclones were systematically detected in CD34⁺ cells, whereas MPC were only involved by the ancestral tumor cell subclone. In summary, here we confirm the presence at diagnosis of the BCR‐ABL1 fusion gene in MPC, CD34⁺ precursors, and other different BM hematopoietic myeloid cell lineages from CML patients, including also in a significant fraction of cases, a smaller percentage of T, B, and NK lymphocytes. Interestingly, involvement of MPC was restricted to the ancestral BCR‐ABL1⁺ subclone. Am. J. Hematol. 89:288–294, 2014. © 2013 Wiley Periodicals, Inc.     

Laboratory testing in BCR‐ABL1‐like (Philadelphia‐like) B‐lymphoblastic leukemia/lymphoma

BCR‐ABL1‐like B‐lymphoblastic leukemia/lymphoma (BCR‐ABL1‐like B‐ALL), also known as Philadelphia‐like (Ph‐like) ALL, is a neoplasm of B‐lineage lymphoblasts characterized by a pattern of gene expression similar to that of B‐ALL with the BCR‐ABL1 translocation but lacking the BCR‐ABL1 fusion protein. The diagnosis of BCR‐ABL1‐like B‐ALL is associated with a high rate of relapse and poor clinical outcomes. In recognition of the difficulty in screening these leukemias for diagnostic workup, the 2016 update/revision to the World Health Organization (WHO) 2008 edition included BCR‐ABL1‐like B‐ALL as a provisional entity. This review addresses the various clinical considerations and methodologies currently used in the pathologic diagnosis, subclassification, and molecular characterization of cases of BCR‐ABL1‐like B‐ALL, with particular attention paid to emerging methods useful in identification of molecular lesions potentially amenable to targeted therapy.     

Very early onset of an acute myeloid leukemia in an adult patient with B‐cell lymphoblastic leukemia

We report on a case of a 30‐year‐old male with acute B‐lymphoblastic leukemia (B‐ALL) with immunophenotype CD19⁺, CD22⁺, CD20⁺, CD10⁺, with aberrant expression of CD13 and CD117, and IgH gene rearrangements. Three months after treatment with GMALL‐2003 and Ida/FLAG protocols bone marrow showed predominance of blasts with myeloid morphology and phenotype MPO⁺, CD13⁺, CD33⁺, CD64⁺, CD15⁺, CD56⁺, EVI‐1 gene overexpression and lack of IgH rearrangements. The case is the first report of a very early emergence of myeloid leukemia during the induction treatment for B‐ALL in an adult patient. Different pathogenetic mechanisms are discussed – clonal evolution or selection, lineage switch or development of a de novo or therapy‐induced leukemia.     

BCR‐ABL1‐like B‐lymphoblastic leukemia/lymphoma: Review of the entity and detection methodologies

BCR‐ABL1‐like B‐lymphoblastic leukemia/lymphoma (BCR‐ABL1‐like ALL or Ph‐like ALL) is a neoplastic proliferation of lymphoblasts that has a gene expression profile similar to that of B‐ALL with t(9;22)(q34.1;q11.2) BCR‐ABL1, but lacks that gene fusion. It is associated with poor prognosis and is seen in 10%‐20% of pediatric cases and 20%‐30% of adult cases of ALL. It is included as a provisional entity in the revised 4th edition of the WHO Classification. A variety of different genetic abnormalities are identified in this entity, but they all converge on pathways that are potentially responsive to the addition of targeted therapy to conventional chemotherapy. Thus, it is important to screen for BCR‐ABL1‐like ALL, particularly in adults and pediatric patients with high‐risk clinical features. Here, we provide a brief overview of the genetic profile and clinical features of BCR‐ABL1‐like ALL and review laboratory methodologies for routine identification of this genetically heterogeneous entity.     

Acute myeloid and T-cell acute lymphoblastic leukaemia with aberrant antigen expression exhibit similar TCRδ gene rearrangements

TCRδ gene recombination patterns were analysed by Southern blot, polymerase chain reaction and nucleotide sequencing in acute myeloid leukaemias with coexpression of lymphoid antigens (Ly⁺ AML, n = 10) as well as in early T-cell acute lymphoblastic leukaemias with (My⁺ T-ALL, n = 10) and without coexpression of myeloid antigens (My^? T-ALL, n = 9). These 29 acute leukaemias exhibiting TCRδ gene rearrangements were selected from 66 Ly⁺ AML, 14 My⁺ T- ALL and 12 My^? T-ALL cases. Similar recombination patterns, namely Dδ2Jδ1 and Vδ1Jδ1 gene rearrangements, were observed in Ly⁺ AML and My⁺ T-ALL. In contrast to Vδ2Dδ3 rearrangements in B-cell precursor ALL, these rearrangements require activation of a T-cell-specific TCRδ enhancer. Comparison of My⁺ T-ALL and Ly⁺ AML with My^? T-ALL exhibited a higher incidence of incomplete Dδ2Jδ1 rearrangements in My⁺ T-ALL and Ly⁺ AML. Since a Dδ2Jδ1 rearrangement is an early event in TCRδ recombination, these leukaemias seem to be arrested at an earlier stage of differentiation. Similar patterns of TCRδ rearrangements in My⁺ T-ALL and Ly⁺ AML suggest existence of a common myeloid/T-lymphoid progenitor cell. Although weak or missing expression of terminal deoxynucleotidyl transferase (TδT) was found in 7/10 Ly⁺ AML cases, no difference was observed in numbers of N-nucleotides inserted in junctional regions when comparing with 3/10 cases exhibiting TdT expression. Since TdT activity is necessary for N-nucleotide addition, this finding suggests down-regulation of TδT expression after rearrangement took place in these Ly⁺ AML cases.     

Myeloid neoplasms with concurrent BCR‐ABL1 and CBFB rearrangements: A series of 10 cases of a clinically aggressive neoplasm

Chronic myeloid leukemia (CML) is defined by the presence of t(9;22)(q34;q11.2)/BCR‐ABL1. Additional chromosomal abnormalities confer an adverse prognosis and are particularly common in the blast phase of CML (CML‐BP). CBFB rearrangement, particularly CBFB‐MYH11 fusion resulting from inv(16)(p13.1q22) or t(16;16)(p13.1;q22), is an acute myeloid leukemia (AML)‐defining alteration that is associated with a favorable outcome. The co‐occurrence of BCR‐ABL1 and CBFB rearrangement is extremely rare, and the significance of this finding remains unclear. We identified 10 patients with myeloid neoplasms harboring BCR‐ABL1 and CBFB rearrangement. The study group included six men and four women with a median age of 51 years (range, 20‐71 years). The sequence of molecular alterations could be determined in nine cases: BCR‐ABL1 preceded CBFB rearrangement in seven, CBFB rearrangement preceded BCR‐ABL1 in one, and both alterations were discovered simultaneously in one patient. BCR‐ABL1 encoded for p210 kD in all cases in which BCR‐ABL1 preceded CBFB rearrangement; a p190 kD was identified in the other three cases. Two patients were treated with the FLAG‐IDA regimen (fludarabine, cytarabine, idarubicin, and G‐CSF) and tyrosine kinase inhibitors (TKI); seven with other cytarabine‐based regimens and TKIs, and one with ponatinib alone. At last follow up (median, 16 months; range 2‐85), 7 of 10 patients had died. The co‐existence of BCR‐ABL1 and CBFB rearrangement is associated with poor outcome and a clinical course similar to that of CML‐BP, and unlike de novo AML with CBFB rearrangement, suggesting that high‐intensity chemotherapy with TKI should be considered in these patients.     

High reliability and sensitivity of the BCR/ABL1 D-FISH test for the detection of BCR/ABL rearrangements

The BCR/ABL1 fusion gene is mainly caused by the t(9; 22)(q34; q11.2) translocation, which results in the Philadelphia (Ph) chromosome. The Ph chromosome is the typical hallmark in chronic myeloid leukemia (CML), but can also be present in acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). The BCR/ABL1 rearrangement is an important tumor classification marker and a useful prognostic factor allowing an adequate therapy management. Ph chromosome detection by conventional cytogenetics (CC) can be hampered by low quantity and quality of metaphases from tumor cells. Furthermore, BCR/ABL1 rearrangements may be hidden due to cryptic rearrangements or complex aberrations. Therefore, molecular cytogenetic methods turned out to be useful tools for the detection of BCR/ABL1 rearrangements. We performed fluorescent in situ hybridization (FISH) with the recently developed BCR/ABL1 D-FISH probe (QBIOgene, Illkirch, F) on cultured bone marrow and peripheral blood cells of 71 patients with CML, ALL, AML, and myeloproliferative disorder (MPD). FISH results and the results of banding methods were directly compared. Based on the analyses of >200 nuclei per patient, D-FISH correlated closely with CC and allowed an accurate quantification of BCR/ABL1 rearrangements even in a low percentage of aberrant cells. No false-positive or false-negative results were obtained. Furthermore, the D-FISH probe detected three cryptic and one complex BCR/ABL1 rearrangement, which were not visible by CC. We conclude that D-FISH reliably detects standard Ph chromosomes as well as its variant translocations and accurately quantifies BCR/ABL1 rearrangements prior and during cancer treatment as well as in the phase of remission, in daily routine tumor cytogenetic diagnostics.     

Immunophenotypic analysis of T‐acute lymphoblastic leukemia. A CD5‐based ETP‐ALL perspective of non‐ETP T‐ALL

T‐cell antigens [CD5,CD1a,CD8] define early T‐cell precursor acute lymphoblastic leukemia (ETP‐ALL). To understand immature T‐ALL of which ETP‐ALL is part, we used these antigens to subcategorize non‐ETP T‐ALL for examining expression of myeloid/stem cell antigens (M/S) and clinical features. Using CD5 (+/?) to start categorization, we studied 69 routinely immunophenotyped patients with T‐ALL. CD5^? was a homogenous (CD8,CD1a)^? M/S⁺ ETP‐ALL group (n = 9). CD5⁺ cases were (CD8,CD1a)^? pre‐T‐ALL (n = 22) or (CD8,CD1a)⁺ (n = 38) thymic/cortical T‐ALL; M/S⁺ 20/22 (90.91%) in former and 22/38 (57.89%) in latter (P = 0.007). ETP‐ and pre‐T‐ALL together (CD1a^?,CD5^?/+ immature T‐ALL group) were nearly always M/S⁺ (29/31; 93.55%). In multivariate analysis, only ETP‐ALL predicted poor overall survival (P = 0.02). We conclude (i) CD5 negativity in T‐ALL almost always means ETP‐ALL. CD1a and CD8 negativity, as much as CD5, marks immaturity in T‐ALL, and the CD5^+/?/CD1a^?/CD8^? immature T‐ALL group needs further study to understand the biology of the T‐ALL–myeloid interface. (ii) ETP‐ALL patients may be pre‐T‐ALL if CD2⁺; CD2⁺, conversely, CD5^?/CD1a^?/CD8^? pre‐T ALL patients are ETP‐ALL. (iii) Immunophenotypic workup of T‐ALL must not omit CD1a, CD5, CD8 and CD2, and positivity of antigens should preferably be defined as recommended for ETP‐ALL, so that this entity can be better evaluated in future studies of immature T‐ALL, a group to which ETP‐ALL belongs. (iv) ETP‐ALL has poor prognosis.     

Inhibition of the ABL Kinase Activity Blocks the Proliferation of BCR/ABLLeukemic Cells and Induces Apoptosis

ABSTRACT: The BCR/ABL fusion protein transforms myeloid stem cells. Both chronic myelogenous leukemias (CML) and a subset of acute lymphoblastic leukemias (ALL) are associated with the expression of BCR/ABL proteins. This knowledge has not yet been translated into any specific tool to control ABL driven neoplastic cells growth. CGP57148B is an ATP-competitive inhibitor of the ABL protein kinase; it has been shown to inhibit the kinase activity of ABL bothin vitroandin vivoand to inhibit the growth of v-abl and bcr/abl transfectants, as well as thein vitroformation of bone marrow (BM)-derived colonies in the presence of growth factors in some CML patients. These studies were performed to investigate the activity of CGP57148B on the spontaneous proliferation of both fresh and cultured, leukemic and normal, BCR/ABL positive and negative cells, and to study its mechanism of action. Six cell lines derived from BCR/ABL+ leukemias (K562, BV173, KCL22, KU812, MC3, LAMA84), thirteen BCR/ABL negative lines, both neoplastic (KG1, SU-DHL-1, U937, Daudi, NB4, NB4.306) and derived from normal cells (PHA blasts, LAK, fibroblasts, LCL, renal epithelial cells, endothelial cells, CD34⁺cells), and 14 fresh leukemic samples were tested using a tritiated thymidine uptake assay. Thein vivophosphorylation of the BCR/ABL protein was evaluated by western blot, while apoptosis was detected by the annexin V/propidium binding test. The induction of differentiation was assayed by immunofluorescence using multiple antibodies.All six BCR/ABL⁺lines showed a dose dependent inhibition of their spontaneous proliferative rate, which was not accompanied by differentiation. The treatment caused, within minutes, dephosphorylation of the BCR/ABL protein, followed in 16-24 hours by a decrease in cycling cells and induction of apoptosis. No significant inhibition of DNA synthesis was observed in any BCR/ABL negative normal or neoplastic line at concentrations ≤3 μM, with the exception of fibroblasts and CD34 cells. Proliferation inhibition was observed also when using fresh samples obtained from two Ph+ ALL and 12 consecutive CML patients. Induction of apoptosis was observed in these samples too.The activity of CGP57148B can be monitored inex vivoisolated or cultured cells using a simple and reproducible assay, without the need for exogenously added growth factors. This molecule possibly exerts its effects through the inhibition of the kinase activity of BCR/ABL and the subsequent initiation of apoptosis, without inducing cell differentiation. Some normal cells are also affected.These data support the use of CGP57148B in initial clinical studies; possible toxic effects on BM and fibroblast-derived cells will have to be closely monitored. Thein vivomonitoring of patients will have to be focused on the induction of apoptosis in leukemic cells.     

BCR‐ABL1 molecular remission after 90Y‐epratuzumab tetraxetan radioimmunotherapy in CD22+ Ph+ B‐ALL: proof of principle

Although targeted therapies are used increasingly in hematologic malignancies, we are unaware of any prior studies of radioimmunotherapy (RAIT) in B‐acute lymphoblastic leukemia (ALL), even though this radiosensitive tumor expresses CD22, potentially a good target for this approach. Here, we report a patient with Philadelphia chromosome‐positive B‐ALL in third relapse who received RAIT with ⁹⁰yttrium (⁹⁰Y)‐labeled anti‐CD22 epratuzumab tetraxetan. Seven weeks after initiating therapy, the patient achieved a BCR‐ABL1 molecular remission documented by RT‐qPCR, which is now continuing at 6 months while awaiting an allogeneic hematopoietic stem cell transplant. ⁹⁰Y‐Epratuzumab tetraxetan may be a promising therapeutic option for CD22⁺ B‐ALL patients.     

Poor responses to tyrosine kinase inhibitors in a child with precursor B‐cell acute lymphoblastic leukemia with SNX2‐ABL1 chimeric transcript

In addition to BCR, various rare fusion partners for the ABL1 gene have been reported in leukemia. We have identified the fusion gene SNX2‐ABL1 in a pediatric case of acute lymphoblastic leukemia (ALL), which has only once previously been reported in an adult patient. Cytogenetic analysis detected this fusion gene arising from a t(5;9)(q22;q34) translocation. ALL cells carrying a SNX2‐ABL1 fusion exhibited a BCR‐ABL1+ ALL‐like gene expression profile. The patient poorly responded to dasatinib but partially responded to imatinib. Treatment using tyrosine kinase inhibitors requires further investigation to optimize the genotype‐based treatment stratification for patients with SNX2‐ABL1 fusion.     

Philadelphia chromosome-positive acute lymphoblastic leukemia with monosomy 7 successfully treated with intermediate- and high-dose ara-C

A 52-year-old man was admitted for treatment of acute lymphoblastic leukemia (ALL). The bone marrow was hypercellular with 67.2% blasts, which were negative for peroxidase, and expressed CD13, CD33, CD34, CD10 and CD7. Cytogenetic and molecular studies revealed t(9;22) and -7(Ph/-7) with major BCR/ABL rearrangement. The patient was treated with the L-AdVP regimen, but failed to achieve complete remission (CR). He then received two courses of chemotherapy consisting of intermediate- and high-dose cytarabine (ara-C), resulting in CR. This case suggests that Ph/-7 ALL with major BCR/ABL gene rearrangement showing coexpression of myeloid antigens may be sensitive to intermediate- and high-dose ara-C.     

Patient perceptions of second transplants in myeloma: impact on recruitment in the British Society of Blood and Marrow Transplantation/UK Myeloma Forum Myeloma X Relapse (Intensive) Trial

The BCR/ABL1 fusion gene, usually carried by the Philadelphia chromosome (Ph) resulting from t(9;22)(q34;q11) or variants, is pathognomonic for chronic myeloid leukaemia (CML). It is also occasionally found in acute lymphoblastic leukaemia (ALL) mostly in adults and rarely in de novo acute myeloid leukaemia (AML). Array Comparative Genomic Hybridization (aCGH) was used to study six Ph(+)AML, three bi‐lineage and four Ph(+)ALL searching for specific genomic profiles. Surprisingly, loss of the IKZF1 and/or CDKN2A genes, the hallmark of Ph(+)ALL, were recurrent findings in Ph(+)AML and accompanied cryptic deletions within the immunoglobulin and T cell receptor genes. The latter two losses have been shown to be part of ‘hot spot’ genome imbalances associated with BCR/ABL1 positive pre‐B lymphoid phenotype in CML and Ph(+)ALL. We applied Significance Analysis of Microarrays (SAM) to data from the ‘hot spot’ regions to the Ph(+)AML and a further 40 BCR/ABL1(+) samples looking for differentiating features. After exclusion of the most dominant markers, SAM identified aberrations unique to de novo Ph(+)AML that involved relevant genes. While the biological and clinical significance of this specific genome signature remains to be uncovered, the unique loss within the immunoglobulin genes provides a simple test to enable the differentiation of clinically similar de novo Ph(+) AML and myeloid blast crisis of CML.     

Inhibition of calcineurin combined with dasatinib has direct and indirect anti‐leukemia effects against BCR‐ABL1+ leukemia

Treatment of BCR‐ABL1⁺ leukemia has been revolutionized with the development of tyrosine kinase inhibitors. However, patients with BCR‐ABL1⁺ acute lymphoblastic leukemia and subsets of patients with chronic myeloid leukemia are at high risk of relapse despite kinase inhibition therapy, necessitating novel treatment strategies. We previously reported synthetic lethality in BCR‐ABL1⁺ leukemia cells by blocking both calcineurin/NFAT signaling and BCR‐ABL1, independent of drug efflux inhibition by cyclosporine. Here, using RNA‐interference we confirm that calcineurin inhibition sensitizes BCR‐ABL1⁺ cells to tyrosine kinase inhibition in vitro. However, when we performed pharmacokinetic and pharmacodynamic studies of dasatinib and cyclosporine in mice, we found that co‐administration of cyclosporine increases peak concentrations and the area under the curve of dasatinib, which contributes to the enhanced disease control. We also report the clinical experience of two subjects in whom we observed more hematopoietic toxicity than expected while enrolled in a Phase Ib trial designed to assess the safety and tolerability of adding cyclosporine to dasatinib in humans. Thus, the anti‐leukemia benefit of co‐administration of cyclosporine and dasatinib is mechanistically pleiotropic, but may not be tolerable, at least as administered in this trial. These data highlight some of the challenges associated with combining targeted agents to treat leukemia. Am. J. Hematol. 89:896–903, 2014. © 2014 Wiley Periodicals, Inc.     

Current paradigms in the management of Philadelphia chromosome positive acute lymphoblastic leukemia in adults

Philadelphia chromosome‐positive (Ph‐positive) acute lymphoblastic leukemia (ALL) is a biologically, clinically, and genetically distinct subtype of precursor‐B ALL. The Ph chromosome, results from a reciprocal translocation of the ABL1 kinase gene on chromosome 9 to the breakpoint cluster region (BCR) gene on chromosome 22. Depending on the translocation breakpoint, typically a p210 BCR‐ABL1 or a p190 BCR‐ABL onc protein are generated; both are constitutively active tyrosine kinases that play a central role to alter signaling pathways of cell proliferation, survival, and self‐renewal, leading to leukemogenesis. In Ph‐positive ALL, the p190‐BCR‐ABL (minor [m]‐bcr) subtype is more frequent than the p210‐BCR‐ABL (major [M]‐bcr) subtype, commonly found in chronic myeloid leukemia. The Philadelphia chromosome is the most frequent recurrent cytogenetic abnormality in elderly patients with ALL. Its incidence increases with age, reaching ～50% in patients with ALL aged 60 years and over. Patients traditionally had a very poor outcome with chemotherapy, particularly if they do not undergo allogeneic hematopoietic cell transplantation (allo‐HCT) in first complete remission (CR1). With the availability of multiple tyrosine kinase inhibitors (TKI), the therapeutic armamentarium is expanding quickly. However, there is no consensus on how to best treat Ph‐positive ALL. With modern therapy, improved outcomes have led to the emergence of a number of controversies, including the need for intensive chemotherapy, the ideal TKI, and whether all eligible patients should receive an allo‐HSCT, and if so, what type. Here, we discuss these controversies in light of the available literature.     

Antigen receptor gene rearrangements reflect on the heterogeneity of adult Acute Lymphoblastic Leukaemia (ALL) with implications of cell‐origin of ALL subgroups – a UKALLXII study

Cytogenetic and molecular investigations of Acute Lymphoblastic Leukaemia (ALL) have identified the existence of distinct clinical subgroups. Molecular monitoring of clonal Immunoglobulin and T cell receptor (IG/TR) gene rearrangements has become an important tool in stratification of therapy of ALL. In order to determine whether certain features of the patient‐specific rearrangements could hold further prognostic clues or provide information on the cell of origin of ALL, a comprehensive analysis of structural and biological features (V gene usage, coding frame and mutational status and complementarity‐determining region ‐III length) of 473 IG/TR rearrangements identified in 229 adults with ALL was carried out. Distinct variable‐gene usage profiles were identified between ALL subgroups, particularly for patients positive for BCR‐ABL1 compared to MLL‐AFF1 positive leukaemias; suggesting that the former is derived from a more mature B progenitor. Interestingly, occurrence of TRGV1‐TRGV8 was prognostic for better event‐free survival (31% at 4 years with vs. 0% at 4 years without, P = 0·05). The heterogeneity in clinical outcome is suggested by the basic molecular processes of antigen receptor gene rearrangements as shown in this work.     

Prognostic relevance of cytometric quantitative assessment in patients with myelodysplastic syndromes

Objectives: Morphology and cytogenetics are currently used to define prognosis in myelodysplastic syndromes (MDS). However, these parameters have some limits. Flow cytometry has been recently included in the diagnostic panel for MDS, and its prognostic significance is under evaluation. Methods: Marrow aspirates from 424 MDS patients were analyzed by flow cytometry to evaluate the impact of bone marrow cell immunophenotype on overall survival (OS) and leukemia‐free survival (LFS). The immature compartment of myeloblasts was analyzed by the quantitative expression of CD34 (<3% vs. ≥3%), CD117, and CD11b^?/CD66b^? (<5% vs. ≥5%); myeloid maturation was analyzed by the expression of CD11b⁺/CD66b⁺⁺ (<15% vs. ≥15%) and CD11b⁺/CD66b⁺ (<25% vs. ≥25%). Results:  In univariate analysis, the expression of immaturity markers (CD34⁺, CD117⁺, and CD11b^?/CD66b^?) was associated with shorter LFS and OS (P < 0.0001); higher expression of differentiation markers (CD11b⁺/CD66b⁺⁺ and CD11b⁺/CD66b⁺) was associated with longer LFS (P < 0.0001 and P = 0.0002, respectively) and OS (P < 0.0001). In multivariate analysis, expression of CD34⁺ (P = 0.007), CD117⁺ (P = 0.013), and CD11b⁺/CD66b⁺⁺ (P = 0.023) retained independent prognostic value for OS, while only the expression of CD34⁺ was a prognostic factor for LFS (P = 0.0003). Two different risk groups were defined according to the presence of 0–1 or ≥2 of these factors with significant different LFS and OS (P < 0.0001). This score showed prognostic value in predicting survival even in subanalysis according to IPSS and WHO subgroups. Conclusions: Flow cytometric analysis in MDS may provide meaningful prognostic information. Blast percentage expressed as CD117⁺ or CD34⁺ cells and the quantitative assessment of myeloid maturation showed prognostic value for survival.     

Diagnostic application of monoclonal antibody KB90 (CD11c) in acute myeloid leukaemia

Summary The expression of membrane CD11c by leukaemic blast cells was examined (indirect immunorosetting) in 75 cases of acute leukaemia (myeloid, n=60; lymphoid, n=15) and evaluated as a potential marker for the diagnostic discrimination between monocytic (AMML-M4 and AMoL-M5) and non-monocytic (M1, M2 and M3) AML subtypes. Preliminary studies of normal bone marrow cells indicated that CD11c expression was not restricted to cells of monocytic lineage but was also present, with apparent lower density, on significant proportions of mature and immature granulocytes. Examination of acute myeloid leukaemia (AML) subtypes revealed that the non-monocytic leukaemias (n=33) were CD11c^–, defined as <30% positive cells, whereas all but one of the AMML-M4 (n=13) and AMoL-M5 (n=14) cases were CD11c⁺. All 15 cases of lymphoblastic leukaemia (ALL) showed <5% CD11c⁺ blasts. Membrane CD11c expression was also compared to the more widely used markers of monocytic differentiation; cytoplasmic alpha-naphthyl acetate esterase (ANAE) and membrane CD14 expression. This analysis showed that all 13 AMML-M4 leukaemias studied, including seven cases that were CD14^– and eight that were ANAE^–, were CD11c⁺. In addition, the AMoL-M5 cases (all of which were ANAE⁺) could be phenotypically subdivided into CD11c⁺ CD14⁺ (n=9), CD11c⁺ CD14^– (n=4) and CD11c^– CD14^– (n=1) subgroups. The study also confirmed that the discriminitive ability and sensitivity of the immunorosetting procedure for the detection of membrane CD11c compared favourably to immunofluorescent staining intensities as measured by flow cytometry.