Prognosis of children with mixed phenotype acute leukemia treated on the basis of consistent immunophenotypic criteria

Background

Mixed phenotype acute leukemia (MPAL) represents a diagnostic and therapeutic dilemma. The European Group for the Immunological Classification of Leukemias (EGIL) scoring system unambiguously defines MPAL expressing aberrant lineage markers. Discussions surrounding it have focused on scoring details, and information is limited regarding its biological, clinical and prognostic significance. The recent World Health Organization classification is simpler and could replace the EGIL scoring system after transformation into unambiguous guidelines.

Design and Methods

Simple immunophenotypic criteria were used to classify all cases of childhood acute leukemia in order to provide therapy directed against acute lymphoblastic leukemia or acute myeloid leukemia. Prognosis, genotype and immunoglobulin/T-cell receptor gene rearrangement status were analyzed.

Results

The incidences of MPAL were 28/582 and 4/107 for children treated with acute lymphoblastic leukemia and acute myeloid leukemia regimens, respectively. In immunophenotypic principal component analysis, MPAL treated as T-cell acute lymphoblastic leukemia clustered between cases of non-mixed T-cell acute lymphoblastic leukemia and acute myeloid leukemia, while other MPAL cases were included in the respective non-mixed B-cell progenitor acute lymphoblastic leukemia or acute myeloid leukemia clusters. Analogously, immunoglobulin/T-cell receptor gene rearrangements followed the expected pattern in patients treated as having acute myeloid leukemia (non-rearranged, 4/4) or as having B-cell progenitor acute lymphoblastic leukemia (rearranged, 20/20), but were missing in 3/5 analyzed cases of MPAL treated as having T-cell acute lymphobastic leukemia. In patients who received acute lymphoblastic leukemia treatment, the 5-year event-free survival of the MPAL cases was worse than that of the non-mixed cases (53±10% and 76±2% at 5 years, respectively, P=0.0075), with a more pronounced difference among B lineage cases. The small numbers of MPAL cases treated as T-cell acute lymphoblastic leukemia or as acute myeloid leukemia hampered separate statistics. We compared prognosis of all subsets with the prognosis of previously published cohorts.

Conclusions

Simple immunophenotypic criteria are useful for therapy decisions in MPAL. In B lineage leukemia, MPAL confers poorer prognosis. However, our data do not justify a preferential use of current acute myeloid leukemia-based therapy in MPAL.     

An update on classification,genetics, and clinical approach to mixed phenotype acute leukemia (MPAL)

Mixed phenotype acute leukemia (MPAL) is an uncommon diagnosis, representing only about 2–5% of acute leukemia cases. The blast cells of MPAL express multilineage immunophenotypic markers and may have a shared B/T/myeloid phenotype. Due to historical ambiguity in the diagnosis of MPAL, the genetics and clinical features of this disease remain poorly characterized. Based on the 2008 and 2016 World Health Organization classifications, myeloid lineage is best determined by presence of myeloperoxidase, while B and T lymphoid lineages are demonstrated by CD19 and cytoplasmic CD3 expression. MPAL typically carries a worse prognosis than either acute myeloid leukemia (AML) or acute lymphoid leukemia (ALL). Given the rarity of MPAL, there is a lack of prospective trial data to guide therapy; treatment generally relies on ALL-like regimens followed by consolidation chemotherapy or hematopoietic stem cell transplant (HSCT). Here, we review the updated classification, biology, clinical features, and treatment approach to MPAL.     

Diagnostic workup of acute leukemias of ambiguous lineage

Acute leukemias of ambiguous lineage (ALAL) comprise acute undifferentiated leukemias (AUL) and mixed-phenotype acute leukemias (MPAL). In the revised fourth edition of the World Health Organization (WHO) classification provided further refinements to the diagnostic criteria for ALAL. Molecular characterization of MPALs using comprehensive next-generation sequencing (NGS) has provided insights into their underlying biology and enabled a deeper understanding of ALAL classification. This review addresses the various components of pathologic assessment to establish a diagnosis of ALAL, and to further subclassify individual cases as AUL or MPAL, with an emphasis on the most up-to-date revisions to diagnostic criteria. In addition, key issues related to the detection of minimal residual disease (MRD) in ALALs and MPALs, and recently uncovered novel molecular diagnostic findings that may be helpful in better distinguishing various types of MPALs from each other, and from their “non-mixed” phenotypic correlates, are also discussed.     

Acute leukemias of ambiguous lineage in adults: molecular and clinical characterization

Acute leukemias of ambiguous lineage represent a heterogeneous group of rare, poorly characterized leukemias with adverse outcome. No larger studies have yet performed a combined approach of molecular and clinical characterization of acute undifferentiated leukemia (AUL) and biphenotypic acute leukemia (BAL) in adults. Here we describe 16 adults with AUL and 26 with BAL and performed mutational as well as expression studies of genes with prognostic impact in acute leukemia (BAALC, ERG, MN1, WT1, and IGFBP7). AUL showed overexpression of these genes compared to T-lymphoblastic leukemia (T-ALL), B-precursor ALL, and to acute myeloid leukemia (AML). Genotype alterations were not detectable in AUL. BAL samples were characterized by frequent WT1 mutations (18 %) and BCR-ABL translocations (30 %). ALL-based treatment protocols induced complete remissions in 40 % and AML-like therapies in 22 % of AUL/BAL patients. The outcome in both groups was very poor; a long-term survival was only observed in patients undergoing allogeneic stem cell transplantation (SCT). Our findings indicate that AUL and BAL share important molecular and high-risk features of both myeloid and lymphoid leukemias. BAL patients exhibited genetic alterations, which can be targeted therapeutically. Importantly, ALL therapy might be more effective than AML protocols and AUL/BAL patients should be considered for allogeneic SCT.     

Acute leukaemias of ambiguous lineage in children: characterization,prognosis and therapy recommendations

Acute leukaemias of ambiguous lineage (ALAL) represent a rare type of leukaemia, expressing both myeloid and lymphoid markers. This study retrospectively analyzed data from 92 children (biphenotypic n = 78, bilineal n = 6, lineage switch n = 8) with ALAL registered in the Berlin‐Frankfürt‐Münster (BFM) acute myeloid leukaemia (AML) and acute lymphoblastic leukaemia (ALL) studies between 1998 and 2006 (2·4% of all cases with acute leukaemia). Our cohort of ALAL patients was characterized by comparatively high median age (8·9 years), high median white blood cell count (14·9 × 10⁹/l), as well as frequent hyperleucocytosis (18·5%) and central nervous system involvement (24·1%). The most frequent cytogenetic abnormalities were ETV6/RUNX1 fusion (16%) and trisomy 8 (14·6%). Complete remission rate was significantly lower than in ALL‐BFM patients (91·8% vs. 99·1%, P < 0·001), but comparable to AML‐BFM patients (87·9%). Event‐free survival (EFS) and overall survival (OS) of ALAL patients were low, at 62 ± 5%. 5‐year probability of EFS was significantly worse than in ALL patients (80 ± 1%, P < 0·001), but better than for AML patients (49 ± 2%, P = 0·027). Our data suggest that an intensive therapy regimen including stem cell transplantation may be favourable for bilineal or lineage switch cases, whereas patients with ETV6/RUNX1 fusion, lymphoid morphology and patients with expression of cyCD22 and cyCD79a should be treated with an ALL‐directed therapy.     

Allogeneic hematopoietic stem cell transplantation for adult patients with mixed phenotype acute leukemia: results of a matched‐pair analysis

Adult patients with mixed phenotype acute leukemia (MPAL) have a poor prognosis, and the therapeutic role of allogeneic stem cell transplantation (allo‐SCT) for MPAL remains to be elucidated. Thus, we retrospectively assessed the efficacy of allo‐SCT for MPAL. Eighteen patients with MPAL were identified from the transplant outcome database of Kanto Study Group for Cell Therapy (KSGCT). We also selected 215 patients with acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) as control cohorts using an optimal matching method. The 5‐yr overall survival (OS) rate of patients with MPAL was 48.1%, and patients in remission at the time of transplant showed significantly better survival than those not in remission (5‐yr OS: 71.8% vs. 0%, P = 0.001). No significant differences were seen in OS when stratifying patients according to immunophenotype, cytogenetic abnormalities, or the type of induction therapy. The 5‐yr OS rate of patients with MPAL was not significantly different compared with AML control patients (48.1% vs. 48.1%; P = 0.855) or ALL control patients (48.1% vs. 37.8%; P = 0.426). These results suggested that allo‐SCT is an effective treatment for MPAL, especially early in the disease course, and innovative transplant approaches are warranted to improve the transplant outcome of patients with MPAL who are not in remission.     

Gene expression profiling for improved dissection of acute leukemia: a recently identified immature myeloid/T-lymphoid subgroup as an example

In this concise overview, we discuss recent findings concerning a distinct subgroup of acute myeloid/T-lymphoid leukemia. We describe how we identified these leukemias in multiple cohorts of acute myeloid leukemia (AML) using a combination of gene expression profiling and additional analytic approaches, and how we obtained insight in possible mechanisms leading to their phenotype.     

CD1-reactive leukemic cells in bone marrow: presence of Langerhans cell marker on leukemic monocytic cells.

Langerhans cells originate in bone marrow and probably belong to the monocyte-macrophage lineage. CD1 is a specific marker of Langerhans cells. By immunofluorescence and immunoelectron microscopy, CD1a antigen and myeloid markers (CD11, CD13, CD14, CD15, CD33, HLA-DR) were studied in 53 cases of acute myeloid leukemias (AML) and 3 acute lymphoblastic leukemias (ALL). The 11 ANLL without monocytic component were CD1a negative. 2/5 of acute myelomonocytic leukemias (AML4) and 9/37 of acute monocytic leukemias (AML5) were positive. All 3 ALL were negative. No correlation was found between CD1a and myeloid markers. CD1a+ AML did not differ from CD1a- AML with regard to cytogenetics or response to therapy. The CD1a positive cells may originate from an abnormal proliferation of CD1a positive cells which are present in bone marrow and which may differentiate into Langerhans cell precursors.     

Genomic analysis reveals few genetic alterations in pediatric acute myeloid leukemia

Pediatric de novo acute myeloid leukemia (AML) is an aggressive malignancy with current therapy resulting in cure rates of only 60%. To better understand the cause of the marked heterogeneity in therapeutic response and to identify new prognostic markers and therapeutic targets a comprehensive list of the genetic mutations that underlie the pathogenesis of AML is needed. To approach this goal, we examined diagnostic leukemic samples from a cohort of 111 children with de novo AML using single-nucleotide-polymorphism microarrays and candidate gene resequencing. Our data demonstrate that, in contrast to pediatric acute lymphoblastic leukemia (ALL), de novo AML is characterized by a very low burden of genomic alterations, with a mean of only 2.38 somatic copy-number alterations per leukemia, and less than 1 nonsynonymous point mutation per leukemia in the 25 genes analyzed. Even more surprising was the observation that 34% of the leukemias lacked any identifiable copy-number alterations, and 28% of the leukemias with recurrent translocations lacked any identifiable sequence or numerical abnormalities. The only exception to the presence of few mutations was acute megakaryocytic leukemias, with the majority of these leukemias being characterized by a high number of copy-number alterations but rare point mutations. Despite the low overall number of lesions across the patient cohort, novel recurring regions of genetic alteration were identified that harbor known, and potential new cancer genes. These data reflect a remarkably low burden of genomic alterations within pediatric de novo AML, which is in stark contrast to most other human malignancies.     

Children Diagnosed as Mixed-Phenotype Acute Leukemia Didn’t Benefit from the CCLG-2008 Protocol,Retrospective Analysis from Single Center

Mixed phenotype acute leukemia (MPAL) is a rare type of acute leukemia with a poor clinical outcome which lacks specific therapy. To evaluate the therapeutic efficiency of CCLG-2008 protocol used for acute lymphoblastic leukemia (ALL) in China on MPAL children who were initially diagnosed as ALL by morphology, we reviewed patients’ database diagnosed as ALL and MPAL according to WHO classification and compared their outcomes from July 2008 to June 2012. Total newly enrolled ALL in this study were 309 cases by morphology, in which ten cases were identified as MPAL mainly by immunophenotyping: B+ myeloid (3/10), T+ myeloid (2/10), B + T (4/10), trilineage (1/10). Two cases were classified as intermediate risk (IR) and 8 cases were high risk (HR) according to the CCLG-2008 criteria. Only one case of IR survived and others died due to primary resistance of chemotherapy and relapse. Compared with MPAL, ALL children in IR and HR had a longer survival (28.1 vs 9.5 months, p < 0.0001) and lower relapse (16.3 vs 85.7 %, p = 0.0002). In a summary, our result indicated that MPAL in children is a poor-risk disease which needs personalized therapy to improve outcome.

Electronic supplementary material

The online version of this article (doi:10.1007/s12288-014-0372-6) contains supplementary material, which is available to authorized users.     

Monoclonal antiglycophorin as a probe for erythroleukemias

A monoclonal antibody (LICR.LON.R10) specific for the major sialoglycoprotein of the erythroid cell membrane, glycophorin A (alpha), has been used to test the possibility that "cryptic" erythroleukemia may be diagnosed as acute lymphoblastic leukemia (ALL) or acute myeloblastic leukemia (AML). In addition to 27 overt erythroleukemias, 724 leukemias, including 329 ALL (103 in relapse), 205 AML, and 109 blast crises of Ph1-positive chronic myeloid leukemia, were analyzed. Twenty cases with a significant proportion of glycophorin-A-positive (gA+) cells were found; 8 of these (5 AML and 3 blast crises of chronic myeloid leukemia, CML) had an obvious erythroid component, but 12 others were diagnosed as AML (2), AMML (1), CML in myeloid blast crisis (4) or megakaryoblastic blast crisis (1), acute megakaryoblastic leukemia (2), or acute lymphoblastic leukemia (2). The latter two patients had no immunologic evidence supporting a diagnosis of ALL and were resistant to chemotherapy. We conclude that AML and ALL only very rarely express gA, and these are probably genuine "cryptic" erythroleukemias. Other gA+ leukemias (megakaryoblastic and CML blast crises) may arise from bi- or pluripotent stem cells and contain distinct and separable blast cell populations.     

Analysis of the cellular DNA and RNA content in acute leukemias by flow cytometry

Summary In the present study bone marrow samples from 573 patients with newly diagnosed acute myeloid (AML) and lymphoblastic or undifferentiated leukemias (ALL/AUL), were analysed for their cellular DNA und DNA/RNA content, respectively, by means of flow cytometry. From 237 patients with AML 35.4% revealed aneuploid DNA stemlines. While no relation of DNA aneuploidy with other pretherapeutic parameters, including FAB subtype, white blood cell count, lactate dehydrogenase, S-phase index and percentage of blasts in the bone marrow, was observed, cases with aneuploid DNA stemlines revealed a tendency towards longer remission duration. In ALL/AUL 21.8% of 280 patients expressed DNA aneuploidies, which were less frequently found in T-cell ALL (11.1%) as compared to common(C)-ALL (21.4%) or null-cell(null)-ALL (23.5%). DNA aneuploidy was not related with other clinically defined risk factors such as age, white blood cell count, and rapid achievement of remission. Patients with DNA indices <1.0, however, tended to have shorter remissions. A significant difference in RNA indices was observed between AML and ALL/AUL with median values of 14.4 and 10.1, respectively (P<0.05). These data indicate the usefulness of flow cytometric analyses of cellular DNA and RNA content for the characterization and classification of acute leukemias, complementing the identification of clinical risk factors, immuno-phenotyping and cytogenetics.Abbreviations AML acute myeloid leukemia - ALL acute lymphoblastic leukemia - AUL acute undifferentiated leukemia - T-ALL, C-ALL and Null-ALL T-cell, common and Null-cell ALL     

Acute myeloid leukemia associated with FGFR1 abnormalities

Hematologic malignancies associated with FGFR1 abnormalities present in heterogeneous forms, including myeloproliferative neoplasm, acute myeloid leukemia (AML), T- or B-lineage lymphoblastic leukemia/lymphoma, and even mixed phenotype acute leukemia. In the present report, we describe three new cases of AML associated with FGFR1 abnormalities: AML with minimal differentiation with 45,XY,-7,t(8;13)(p11.2;q12), acute myelomonocytic leukemia with eosinophilia with 48,XY,t(8;9)(p11.2;q33),+19,+21, and AML with minimal differentiation with 46,XX,add(8)(p11.2). FGFR1 abnormalities were confirmed by fluorescence in situ hybridization. We reviewed the records of 19 patients reported from Asian countries, and found that approximately 40 % of cases manifested as acute leukemia associated with myeloid lineage, and 47 % were not accompanied with eosinophilia. These findings highlight the need for detection of FGFR1 abnormalities, not only in myeloproliferative disorder, but also in AML patients even without eosinophilia. The prognosis for this group of neoplasms is poor, and there is no recognized effective targeted treatment. Two patients, including our case, who received allogeneic hematopoietic stem cell transplantation (allo-HSCT) survived. Accumulation of long-term follow-up data can help determine the allo-HSCT protocol or the need for new therapeutic trials to improve the survival rate of patients with FGFR1 abnormalities.     

The 2016 WHO classification of acute myeloid leukemia: What the practicing clinician needs to know

In 2016 a revision of the World Health Organization (WHO) classification of acute myeloid leukemia (AML) was introduced that included changes to several disease categories. The WHO approach results in disease categories that are defined by a combination of clinical, morphologic, immunophenotypic, and genetic features in an attempt to define clinically relevant, biologic entities. This review summarizes the WHO approach as well as the priority of specific features for disease classification. Changes to specific categories, including AML with myelodysplasia-related changes, AML with mutated NPM1, AML with biallelic mutations of CEBPA and erythroleukemia are summarized. The importance of additional gene mutations as well as germline predisposition in AML is also reviewed.     

Down Syndrome with Different Hematological Manifestations: A Short Series of 3 Cases with Review of Literature

Children with Down syndrome (DS) present with a spectrum of hematolymphoid malignancies ranging from transient myeloproliferative disorder (TMD) which regress spontaneously to frank acute leukemia of both myeloid and lymphoid lineage. Here we present a series of three cases with different manifestation in DS. Three cases of DS presented with TMD, acute myeloid leukemia (AML–M2) and acute megakaryoblastic leukemia (AMKL), respectively. This case series displays the spectrum of hematological manifestations in children with DS. Although TMD and AMKL are strongly associated with DS, other AML subtypes can also be seen in such patients.     

Diagnosing and treating mixed phenotype acute leukemia: a multicenter 10-year experience in México

Mixed phenotype acute leukemia (MPAL) in adults represents nearly 2 to 5 % of all acute leukemia cases. There are two large studies throughout the world and only case reports and small series have been reported in Latin America. This study retrospectively analyses the clinical characteristics and survival of 27 patients with MPAL evaluated in three medical institutions of Mexico. All cases meet World Health Organization 2008 criteria; 70.3 % of patients had B lymphoid/myeloid lineage MPAL. Induction chemotherapy protocols included 7?+?3 hyper-CVAD, high-density schedules, and pediatric-like regimens such as New York II and total XI. Complete remission was achieved in 23/27 patients (85.2 %). Only one patient died due to chemotherapy-induced aplasia during remission induction (5.2 %). In 68 % of cases, we were able to administer maintenance therapy as a regimen in lymphoblastic leukemia. At the time of analysis, 70.4 % of the patients in the entire cohort had died mainly as result of disease progression (73.6 %). Disease-free survival was 13 months (95 % CI, 9.6–16.3 months) and overall survival was 14.8 months (95 % CI 13.4–16.27). Survival rates are low and standardized therapy for the management of this type of leukemia is still lacking. This is the largest series reported in Mexico and to the best of our knowledge in Latin America.     

Myeloid lineage specificity and high sensitivity of monoclonal antibody GM 58/8 proves its usefulness as a diagnostic reagent in acute leukemia

Monoclonal antibody (MoAb) GM 58/8 was earlier reported to be directed against an antigen expressed by myeloid progenitors (CFU-GM), myeloid precursors, granulocytes, and monocytes. Immunophenotyping of 216 cases of acute leukemia [acute myeloblastic leukemia (AML) = 147 and acute lymphoblastic leukemia (ALL) = 69] and 18 cases of chronic granulocytic leukemia in blast crisis (CGLBC) with this antibody showed that GM 58/8 reacted with 92% of AML cases (M1-M5) and 100% of myeloblastic crisis in CGL cases. All cases of ALL, lymphoblastic crisis in CGL, erythroleukemia, and erythroblastic crisis in CGL were unreactive with GM 58/8. The antibody revealed the myeloid phenotype in an additional 15 cases of otherwise unclassifiable acute leukemia and six cases of CGLBC. Eleven cases of acute "mixed lineage" leukemia were also diagnosed with the help of GM 58/8. The high specificity (100%) and sensitivity (92%) of MoAb GM 58/8 for myeloblastic leukemia is unmatched by almost all previously described myeloid MoAb and proves its usefulness as a single diagnostic reagent for AML and myeloblastic crisis in CGL.     

CD133-2 (AC141) expression analysis in acute leukemia immunophenotyping in correlation to CD34 and P-glycoprotein

A total of 49 newly diagnosed patients with acute leukemia were studied in order to assess the diagnostic value of clone AC141 of CD133 antibody by flow cytometry. AC141 expression was further compared to CD34 and P-glycoprotein, immunophenotype, morphology and cytogenetic/molecular data. Flow cytometry allowed for the detection of AC141 expression in 42.8% of the patients. A strong correlation with myeloid lineage was observed. All AC141(+) acute myeloid leukemia (AML) cases were of immature morphology and a strong concordance with CD34 expression was found. However, discordant patterns were also observed. Besides, AC141 expression correlated with CD7 in the absence of mature markers (CD14, CD15 and CD64). Similarly to CD34, P-glycoprotein levels were also significantly higher in AC141(+) AML cases. No correlation was found with cytogenetic/molecular data of the patients. In conclusion, membrane expression of AC141, in combination with other antigens, might facilitate a more precise immunologic characterization of acute leukemias and may serve as an alternative to CD34 for purging purposes in selected patients.