Leukemia Of Ambiguous Lineage Research Articles

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.

Successful Treatment of Life-Threatening Cerebral Bleeding Associated with Disseminated Intravascular Coagulation Using Recombinant Human Soluble Thrombomodulin in a Patient with Mixed Phenotype Acute Leukemia with t (9; 22) (q34; q11.2); <i>Bcr-Abl1</i>

Recently, mixed phenotype acute leukemia (MPAL) with t (9; 22) (q34; q11.2); bcr-abl1 was described as one kind of acute leukemia of ambiguous lineage in the 2008 World Health Organization Classification of Tumors of Hematopoietic and Lymphoid Tissues. However, treatment strategy remains difficult for this uncommon MPAL. In addition, this type of MPAL is at high risk of tumor lysis syndrome (TLS) because of high chemo-sensitivity. Here, we report a MPAL with t (9; 22) (q34; q11.2); bcr-abl1 case that suffered from life-threatening cerebral bleeding associated with disseminated intravascular coagulation (DIC) with TLS after bcr-abl positive acute lymphoblastic leukemia (ALL) type induction therapy who was successfully treated with recombinant human thrombomodulin (rhTM). This case reached complete remission without additive cerebral bleeding. In conclusion, bcr-abl positive ALL type induction therapy was effective for MPAL with t (9; 22) (q34; q11.2); bcr-abl1 and rhTM was effective against DIC with TLS.

Allo-HSCT for acute leukemia of ambiguous lineage in adults: the comparison between standard conditioning and intensified conditioning regimens

Knowledge concerning the clinical and biological characteristics of acute leukemia of ambiguous lineage (ALAL) is limited so that there has been a lack of uniformity in treatment. In this report, we retrospectively investigated the effect of intensified conditioning on adult ALAL undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT). A total of 59 patients with ALAL (male in 37 cases and female in 22 cases) were consecutively enrolled in the data analyses. Twenty-four patients received the standard conditioning (total body irradiation (TBI) + cyclophosphamide (CY) or busulfan + CY protocol) and 35 received the intensified conditioning (TBI + CY + etoposide or fludarabine + cytarabine plus TBI + CY + etoposide protocol). Five-year transplant-related mortality was 17.6 ± 9.6% and 25.5 ± 8.0%, the 5-year overall survival (OS) post-transplantation was 23.8 ± 8.9% and 64.0 ± 8.4%, disease-free survival was 16.7 ± 7.6% and 55.8 ± 9.4%, the 5-year cumulative incidence of relapse was 80.8 ± 8.5% and 28.8 ± 9.9%, respectively, in the standard and the intensified group (P = 0.380, P = 0.029, P = 0.005, and P < 0.001). Both univariate and multivariate analysis indicated that the intensified conditioning regimen and acute graft-versus-host disease were favorable factors to reduce the relapse. The younger patients, patients with CR at the time of transplantation, and the intensified conditioning regimen were favorable factors to elevate the survival. In conclusion, intensified conditioning regimens followed by allo-HSCT might improve long-term survival and decrease relapse of leukemia in adult ALAL compared to the standard conditioning regimens.

Microrna Profiling Classifies Acute Leukemias of Ambiguous Lineage As Either Acute Myeloid Leukemia or Acute Lymphoid Leukemia

Abstract 1443 IntroductionClassification of acute leukemia (AL) is based on commitment of the leukemic cells to either the myeloid or the lymphoid lineage. However, a small percentage of AL cases lacks immunophenotypical lineage commitment or displays features of both hematopoietic lineages. These leukemias of ambiguous lineage represent a heterogeneous category of AL that cannot be classified as either myeloid AL (AML) or lymphoid AL (ALL). This presents a major hurdle for choice of treatment, because it is unsettled whether these patients benefit from AML or ALL treatment regimens or a mixture of both. Better diagnostic tools to define the lineage of origin for these AL would be instrumental to direct therapy decision making.MicroRNAs are small single stranded RNA molecules which regulate gene expression by promoting degradation of mRNAs or repressing their translation. MicroRNA expression profiles have been shown to accurately discriminate AL of the lymphoid lineage from AL of the myeloid lineage. Here, we investigated microRNA expression profiles of leukemias of ambiguous lineage to classify them as either AML or ALL. MethodsMicroRNA expression profiles of nine patients with leukemia of ambiguous lineage and eleven patients with AML, B-ALL or T-ALL were analyzed using microarray analysis. MicroRNAs differentially expressed between the myeloid and lymphoid lineage were selected using Linear Model for Microarray Analysis (LIMMA) and used for unsupervised clustering of the ambiguous lineage leukemias with the AML, B-ALL and T-ALL samples. The top five most discriminating microRNAs were selected for qRT-PCR validation in eight additional ambiguous lineage cases, five AML or ALL cases and two control cell lines. ResultsUnsupervised clustering analysis of the AML, B-ALL and T-ALL samples resulted in a clear separation between the myeloid and lymphoid lineages. Top differentially expressed microRNAs were miR-199b, miR-27a/b, miR-223, miR-23a, miR-221 and miR-150. When comparing leukemias of ambiguous lineage with AML, B-ALL and T-ALL using LIMMA, no clear differences were found, indicating that leukemias of ambiguous lineage are not a separate entity. Moreover, unsupervised clustering of all AL samples using the top 10 percent of most variable microRNAs resulted in clustering of leukemias of ambiguous lineage with either AML, B-ALL or T-ALL and showed comparable expression profiles. Expression analysis by qRT-PCR of the five most discriminative microRNAs on the additional samples, including leukemias of ambiguous lineage, was able to accurately assign these leukemias to the lineage of origin. ConclusionMicroRNA expression profiling of leukemias of ambiguous lineage indicated the presence of a myeloid or lymphoid lineage-specific genotype despite an indistinct immunophenotype. Analyzing microRNA expression at diagnosis to classify acute leukemias of ambiguous lineage as either AML or ALL might be instrumental to therapeutic decision making. Disclosures:No relevant conflicts of interest to declare.

Myeloid Sarcoma of the Orbit and Ocular Adnexae

Abstract 1463 Background:Myeloid sarcoma of the orbit and ocular adnexae (OMS) is a rare extramedullary manifestation of acute myeloid leukemia (AML), myelodysplastic syndromes (MDS), and myeloproliferative neoplasms (MPNs). Currently there are no standard treatment guidelines for OMS. Methods:On a retrospective review of the medical records, we identified 10 patients (pts) with OMS evaluated at our institution. We explored the pathologic, conventional cytogenetic and molecular cytogenetic features of these pts and analyzed their clinical features, treatments, and outcomes. Results:Among 10 acute leukemia pts with OMS, 6 (60%) were male; the median age of OMS onset was 49 yrs (range, 19 to 79). Morphological review of bone marrow (BM) aspirate smears and core biopsy specimens revealed treatment-related AML (t-AML) (n=4), AML with t(8;21) (q22;q22) (n=2), AML without maturation (n=1), AML with mutated NPM1 (n=1), CML-blast phase (n=1), and myeloperoxidase (MPO)-positive acute leukemia of ambiguous lineage (n=1). Monocytic or myelomonocytic differentiation was noted in 5 pts (50%). Conventional and/or FISH analyses performed on bone marrow in 10 pts demonstrated: +8 (n=3), t(8;21)(q22;q22) (n=2), −7 (n=2), del(7q) (n=2), t(1;3)(q21;q21) (n=1), t(2;11)(q14;q24) (n=1), t(10;11)(p13;q21) (n=1), diploid (n=2), and BCR/ABL1 rearrangement (n=1). Molecular genetic analysis, available in 8 cases, demonstrated NPM1 mutation (n=1) and NRAS mutation (n=1).Ocular symptoms were the first sign of leukemia in 4 pts, 2 with concurrent BM disease. Of the 2 presenting with isolated OMS without BM disease, one developed BM disease 321 days after OMS, while the other remained without BM disease. Six pts developed OMS after the initial leukemia diagnosis: during disease progression before treatment initiation (n=1), during AML treatment of resistant disease (n=1), at relapse with concurrent BM relapse (n=2), or as the site of isolated relapse without concurrent BM involvement (n=2).Clinical manifestations of OMS included: conjunctivitis, iris infiltration, lacrimal gland lesion, episcleritis, orbital mass, periorbital erythema or swelling, ocular secretions, ocular pain, visual problems, and/or retinal infiltrates. The diagnosis of OMS was confirmed by histologic and immunophenotypic analyses of biopsy specimens in the 8 pts from whom tissue could be obtained safely. Of the 2 pts who were not candidates for biopsy, 1 had a conjunctival lesion that involved the lacrimal gland and biopsy-confirmed skin and mediastinal disease. The second pt had intermittent bilateral blurred vision, with fundoscopically-visualized leukemic retinal infiltrates, intraretinal hemorrhages, and cotton wool spots. In both pts the ocular symptoms and lesions resolved with chemotherapy.Treatment modalities included chemotherapy alone (n=3, including 2 who received intrathecal chemotherapy), chemotherapy and surgery (n=5), chemotherapy with focal radiation (XRT) (n=1), and XRT with surgery (n=1). Three pts underwent allogeneic stem cell transplantation (SCT) after OMS diagnosis.In addition to OMS, 7 pts developed extramedullary disease in at least one other site, either concurrently with OMS or at other times during the disease course, including: CSF (n=1), breast (n=1), skin (n=3), lymph node (n=1), lung/mediastinum (n=2), and bone (n=1).Two pts are in CR, 3 and 4 years after OMS diagnosis. Six died either of progressive leukemia or treatment complications. The median time from OMS diagnosis to death was 278 days (range, 39–779 days). Of the 6 pts who died, the OMS either improved or resolved with local or systemic treatment. Two pts have been lost to follow-up, 1 in CR 2 years after OMS diagnosis, and 1 with breast and BM relapse 10 months after OMS diagnosis. Conclusion:OMS may be the initial sign of AML, with or without concurrent BM disease. OMS may also present as an isolated manifestation of relapse, without BM relapse. Extramedullary disease often develops in other sites during the disease course in AML pts with OMS. While OMS symptoms are non-specific, leukemic ocular involvement should be considered and a complete ocular exam is warranted. While t(8,21) is known to occur with extramedullary myeloid sarcoma and OMS, we also identified trisomy 8 in 30% of pts in our series. Disclosures:No relevant conflicts of interest to declare.

Analysis of Infection-Related Mortality in Allogeneic Hematopoietic Stem Cell Transplantation Recipients with Refractory/Relapse Acute Leukemia

Abstract 4513 BackgroundInfection-related mortality (IRM) is a major cause of transplant-related mortality (TRM) in recipients of allogeneic hematopoietic stem cell transplant (allo-HSCT). However, the data about IRM in refractory/relapse acute leukemia patients undergoing allo-HSCT was scarce. We conducted a retrospective study to describe the incidence, causes and risk factors for IRM in recipients with refractory/relapse acute leukemia at a single center. MethodsClinic datas of 127 cases received transplant for refractory/relapse acute leukemia between January 1997 and August 2011 were reviewed. There were 67 cases died at the end of follow-up. In this 67 cases, 27 patients were female and 40 were male; the media age was 31 years (range, 11–61).Uderlying disease was acute myeloid leukemia (AML) in 37 cases, acute lymphoblastic leukemia (ALL) in 21 and acute leukemia of ambiguous lineage in 9. Status of underlying diseases was CR in 19 cases, PR in 15 and NR in 33. There were 25 cases with residual infection diseases before HSCT, including 21 in stable state and 4 in active state with effective control. A total of 59 cases received the conditioning regimen which is 4.5Gy TBI for 2 days and CTX 60mg·kg−1·d−1 for 2 days and 8 patients received busulfan 3.2 mg·kg−1·d−1 for 4 days and CTX 60mg·kg−1·d−1 for 2 days. Patients with PR and NR status of underlying diseases also received fludarabine 50mg·d−1 for 5 days and cytarabine 2 g·d−1 for 5 days as the conditioning regimen. Forty-two cases received related-donor transplant and 25 received unrelated-donor transplant; 30 cases had HLA typing (HLA-A, B and DR) matched-family donor, 12 cases has mismatched-family donors and 25 cases had unrelated-donors. The most common source of stem cells for HSCT was the peripheral blood (n=49), followed by bone marrow plus peripheral blood (n=10) and bone marrow (n=8). Cyclosporine A (CsA) with methotrexate (MTX) (on days +1, +3 and +6) was administered for GVHD prophylaxis in patients undergoing HLA-matched sibling donor transplantation. CsA only or CsA with MTX (on days +1 and +3) was administered for GVHD prophylaxis in patients not received CR before transplant. Patients receiving an unrelated donor or HLA-mismatched sibling donor transplant received CsA + MTX and human antithymocyte globulin (ATG 6–7.5 mg/kg) as GVHD prophylaxis. Logistic regression was used for the identification of risk factors of IRM. ResultsWithin a median 23 months follow-up for patients alive, 67 cases died including 36 died of leukemia relapse and 31 died of TRM. The incidence of 5-years overall survival was 35.2% whereas disease-free survival was 30.8%. Of the 127 cases, 36 cases occurred IRM and the 2-year cumulative incidence of IRM was 36.6%. Fungal infections were responsible for a major proportion of the cases of IRM followed by virus and bacteria infections. Multivariate analyses showed II∼IV°aGVHD (OR=3.017, 95%CI 1.007–9.041, P=0.049) and IFI (OR=3.223, 95%CI 1.103–9.421, P=0.032) were the risk factors of IRM. ConclusionsAs a common cause of transplant-related mortality, IRM occurred more frequently in refractory/relapse acute leukemia than in the standard-risk patients. Reduction of IRM will require effective prophylaxis and treatment of severe GVHD. Disclosures:No relevant conflicts of interest to declare.

Flow Cytometry Thresholds of Myeloperoxidase Detection to Discriminate Between Acute Lymphoblastic or Myeloblastic Leukemia

AbstractAbstract 1450▪▪This icon denotes a clinically relevant abstract Background:The WHO 2008 classification emphasizes the role of myeloperoxidase (MPO) detection as the only requirement for assigning myeloid lineage to a blast population, notably for the diagnosis of acute leukemia of ambiguous lineage. WHO highlights flow cytometry (FCM) as the preferred method for MPO detection and EGIL proposed a 10% cut-off for FCM but 3% for cytochemistry. Here we performed a reevaluation of the MPO positivity threshold by FCM comparing as background reference isotype controls or the autofluorescence of residual normal lymphocytes. Methods:A multicenter retrospective trial was set-up which compared retrospectively 128 acute lymphoblastic leukemias (ALL) and 75 acute myeloid leukemias without maturation (AML1), defined as MPO negative or positive by benzidine-based cytochemistry. Blasts were considered MPO positive by flow cytometry when their mean fluorescence intensity exceeded that of blast cells incubated with an isotype control or that of residual lymphocytes in the MPO-stained sample. In order to homogenize the interpretation of MPO staining, five various cut-offs were assessed for both negative controls, respectively 2%, 1%, 0.75%, 0.5% and 0.25%. Besides, as Ratio Fluorescence Intensity (RFI) may be a useful parameter to analyze staining for markers with unimodal distribution, we evaluated the RFI of blast cells MPO fluorescence relative to both controls, comparing mean and median intensities. Results:The harmonized method that was developed to interpret MPO staining by FCM between 4 French centers can be applied regardless of antibodies, permeabilization reagents or instruments used. For both negative controls, the 1% cut-off provided the best discrimination by ROC curve, and was used to assess the percentage of stained blasts in the 203 cases. The EGIL 10% threshold of stained blasts to discriminate between ALL and AML, using the isotype control to assess positivity, provided 100% sensitivity and 85.4% specificity, the optimal threshold being 13% (sensitivity 95.1%, specificity 91.7%). Residual normal lymphocytes proved to be an advantageous alternative reference, a threshold of 28% yielding improved 97.4% sensitivity and 96.1% specificity (Figure). The correlation between both methods was excellent, yet the percentage of positive blasts was higher using lymphocytes as control compared to isotype control. Using the RFI method, the isotype control appeared as the more relevant negative control to discriminate ALL against AML1 with a threshold of 3.42. However, with 90% sensivity and 95,83% specificity, the RFI method was less performing than percentages in this study.Finally, we assessed the relevance of this analysis method and positivity thresholds on 18 AMLs with minimal differentiation (AML0), MPO-negative by definition in cytochemistry, yet liable to be positive in FCM. Interestingly, with these new appropriate thresholds, MPO staining was positive for 10 of 18 AML0 when using lymphocytes as negative controls and only 3 of 17 cases when using an isotype control. Conclusion:i) MPO detection by flow cytometry can be interpreted indifferently of the negative control used if an appropriate threshold is applied; ii) Our analysis method of MPO expression is relevant for ALL and AML discrimination and can be useful for AML0 or mixed phenotype acute leukemia assessment, especially using residual lymphocytes as reference, since the latter bypass the higher non-specific binding of isotype controls on blast cells. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Acute leukemias of ambiguous lineage

The 2008 edition of the WHO Classification of Tumors of Haematopoietic and Lymphoid Tissues recognizes a special category called "leukemias of ambiguous lineage." The vast majority of these rare leukemias are classified as mixed phenotype acute leukemia (MPAL), although acute undifferentiated leukemias and natural killer lymphoblastic leukemias are also included. The major immunophenotypic markers used by the WHO 2008 to determine the lineage for these proliferations are myeloperoxidase, CD19, and cytoplasmic CD3. However, extensive immunophenotyping is necessary to confirm that the cells indeed belong to 2 different lineages or coexpress differentiation antigens of more than 1 lineage. Specific subsets of MPAL are defined by chromosomal anomalies such as the t(9;22) Philadelphia chromosome BCR-ABL1 or involvement of the MLL gene on chromosome 11q23. Other MPAL are divided into B/myeloid NOS, T/myeloid NOS, B/T NOS, and B/T/myeloid NOS. MPAL are usually of dire prognosis, respond variably to chemotherapy of acute lymphoblastic or acute myeloblastic type, and benefit most from rapid allogeneic hematopoietic stem cell transplantation.

Acute Leukemias of Ambiguous Lineage: The Roswell Park Experience

Abstract 2546Acute leukemias of ambiguous lineage (ALAL) are defined by the lack of clear evidence of differentiation along a single lineage and include mixed-phenotype acute leukemias (MPAL) and acute undifferentiated leukemia (AUL). These rare acute leukemias (AL) pose diagnostic and therapeutic challenges. Criteria for ALAL/MPAL diagnosis were proposed by the European Group for the Immunological Characterization of Leukemias (EGIL) and incorporated into the 2001 World Health Organization (WHO) classification of myeloid neoplasms. The most recent 2008 WHO classification proposed a much simplified diagnostic approach to MPAL/ALAL that requires clinical validation.We retrospectively analyzed the pathologic and clinical features of 29 cases of acute leukemia diagnosed and treated at our institution between 1995–2010. All AL patients with any of the following key words identified in the pathology and flow cytometry databases were reviewed: T/myeloid, B/myeloid, undifferentiated, mixed phenotype, anomalous expression, unclassified, biphenotypic, bilineal, biclonal. Pathology, flow cytometry, and cytogenetics reports were reviewed by 2 hematopathologists and reclassified according to EGIL and 2008 WHO criteria. Treatment regimens were reviewed and overall survival assessed for each patient.There were 29 patients,19 adults and 10 children, with a median age of 39 years (range 2–87). Eighteen were male and 11 female. Using EGIL, 10/29 (34%) were classified as ALAL as compared to 2008 WHO where only 6/29 (21%) fulfilled the new criteria. Based on either EGIL and/or 2008 WHO classification scheme,13 out of 29 cases (45%) were classified as ALAL. The remaining 16 cases were non-ALAL, most concordant in both systems: B-cell lymphoblastic leukemia (B-ALL) (n=11, 38%) and T ALL (n=3, 10%) and discrepant in only 2 cases (7%), both classified as acute myeloid leukemia (AML) by EGIL and B-ALL by 2008 WHO. The most common cytogenetic change was t(9;22), present in 6 (33%) of non-ALAL and 2/13 (15%) ALAL. A single 11q23 translocation case was seen in the non-ALAL group. We compared ALAL (n=13) vs. non-ALAL (n=16) clinical characteristics and treatment outcome and found no significant difference in age at leukemia diagnosis, presenting white blood cell count, hemoglobin, and platelet count, or marrow blast percentage between ALAL and non-ALAL patients. Significantly more ALAL patients were treated with acute myeloid leukemia (AML) based regimens (70%) than non-ALAL patients (12.5%) (p= 0.0016), since 14 of 16 non-ALAL cases were ALL with aberrant myeloid marker expression at re-review. Patients classified as ALAL (n=13) demonstrated a trend to worse overall survival than non-ALAL patients (median 12.25 vs. 20 months, p=0.08) (Figure 1) and potentially worse overall survival following AML than ALL therapy (median 14 vs 17 months, p=0.38). The ALAL patients with the longest survival following AML induction chemotherapy (i.e. 70.6, 27.1, and 17 months) were those who underwent subsequent allogeneic stem cell transplantation. This was not true in the non-ALAL patients and ALAL patients treated with ALL regimens where long-term survival was achieved following chemotherapy in the absence of transplantation. Among all 29 patients identified here, administration of ALL-based chemotherapy independent of EGIL/WHO classification was significantly associated with improved overall survival (p=0.015) (Figure 2). [Display omitted] [Display omitted] In summary, this single institution retrospective review shows that 2008 WHO classification simplifies the diagnosis and limits the number of ALAL/MPAL compared to EGIL score system but this rare entity remains a diagnostic challenge. Over half of these 29 cases preliminarily diagnosed as acute leukemia with dual or uncertain lineage were re-classified as ALL. Although limited by small patient numbers, our preliminary findings suggest that patients diagnosed with ALAL using either EGIL or WHO criteria fare worse than non-ALAL patients and achieve better outcomes (in the absence of allogeneic stem cell transplantation) following ALL-based chemotherapy. Given the overall difficulty in assigning ALAL vs. non-ALAL diagnoses based on EGIL vs. WHO criteria, our results support the upfront treatment of AL of unclear lineage with ALL-based chemotherapy regimens. Disclosures:No relevant conflicts of interest to declare.

Outcome of Patients with Refractory Acute Leukemia Treated with a Third Induction Chemotherapy Regimen

Abstract 4305 BackgroundTreatment options are limited for refractory acute leukemia (AL) patients. Few data are available on the potential utility vs futility of a third course of induction chemotherapy. We have reviewed the characteristics and outcome of 35 patients treated with a third intensive induction course for refractory AL at our institution and other centers affiliated to the Quebec Leukemia Cell Bank (BCLQ). Methods and patientsAdult AL patients who never achieved complete remission (CR) or who relapsed within 6 months after 2 consecutive induction courses and who received a third line intensive induction course between 2000 and 2010 were included in this study. Clinical, laboratory and treatment data were collected from hospital and/or BCLQ files. Outcomes were measured after the third induction course. Primary outcomes were achievement of CR/CRi, overall survival (OS) at 12 months and event-free survival (EFS), defined as the percentage of patients alive and leukemia free at 12 months. Secondary outcomes included median OS, median duration of remission for those obtaining CR and treatment related mortality (TRM) associated with the third induction course. OS was estimated using the Kaplan-Meier method and survival curves were compared using the Log-rank test. Alive patients were censored at their last follow-up. ResultsThirty five patients met the inclusion criteria. The median age was 43 years old (20–62). Thirty one patients (89%) were diagnosed with AML and 4 patients (11%) with ALL or acute leukemia of ambiguous lineage. The median white blood cell count at diagnosis was 23 × 109/L (0.7–223). For AML patients, cytogenetics was favourable in 1 patient (3%), intermediate in 17 (55%) and adverse in 13 (42%). None of the 6 patients with normal karyotype for whom molecular characterisation was performed had a favourable genotype defined as NPM1 mutated without FLT3-ITD mutation. The patient with favourable karyotype had a variant t(8;21)(q22;q22) translocation without KIT mutation. Eighteen patients (51%) were primarily refractory to prior therapy and 17 (49%) had relapsed within 6 months of CR. The third line induction regimen consisted of cyclophosphamide and etoposide in 11 patients (31%), high dose cytarabine-based regimen in 10 (29%), mitoxantrone and etoposide in 7 (20%), fludarabine, cytarabine and idarubicin in 5 (14%) and other regimen in 2(6%). Patients with sustained remission after their third induction course were followed for a minimum of 12 months except for one patient alive and leukemia free at 6 months. Patients who were not in sustained remission have all been followed until death or transition to palliative care.CR after the third induction course was achieved in 6 patients (17%). At 12 months, OS was 11% and EFS was 6%. After the third induction, median OS was 5 months and median duration of remission was 6 months. Among 20 patients treated in our institution, the third induction TRM was 15% and median duration of hospitalisation, excluding patients who died in aplasia, was 38 days (20–70). Median OS was higher in the intermediate than in the adverse cytogenetics group (8 vs 2 mo, p=0.0008, figure 1) and in patients who achieved CR after third induction vs no CR (12 vs 4 mo, p=0.03). There was no difference in median OS between primary refractory and relapsed patients (8 vs 4 months, p=0.52).Allogeneic stem cell transplantation (SCT) was performed in 10 patients (29%) of whom 4 (40%) were in CR before transplantation. The only two long term survivors were in the allogeneic SCT group. Their percentages of blasts at transplant were 3 and 69 %. ConclusionOnly two refractory patients are long term survivors and they share several features: non adverse cytogenetics, young age (20 and 23) and they underwent allogeneic SCT. Adverse cytogenetics impinges negatively on outcome, even in this highly refractory group of subjects.The prognosis of AL patients with persistent disease after 2 induction courses is extremely poor and represents a major unmet therapeutic need. Refractory patients with adverse cytogenetics were unsalvageable in this series. The best approach for patients not in CR after 2 inductions probably remains allogeneic SCT, when possible. We consider that in patients who cannot be offered an allogeneic SCT, administration of a third course of conventional chemotherapy is practically useless. These patients should be considered candidates for investigational treatments. Disclosures:Bergeron: Merck: Research Funding; Roche: Honoraria; Celgene: Research Funding. [Display omitted]

Allogeneic hematopoietic stem cell transplantation for acute leukemias of ambiguous lineage in adults: a comparison between the conditioning of different intensities

To evaluate the efficacy of allogeneic hematopoietic stem cell transplantation (allo-HSCT) in the conditioning of different intensities for acute leukemias of ambiguous lineage (ALAL). A total of 38 ALAL patients were treated with two conditioning of different intensities in our hospital from March 2002 to August 2010. The standard conditioning included TBI + Cy or Bu + Cy, intensified conditioning included Fludarabine + Ara-C + TBI + Cy. Cyclosporine A (CsA) and methotrexate (MTX) were administered in patients with human leukocyte antigen-matched sibling donor. And CsA, MTX plus antihuman thymocyte globulin and/or mycophenolate were used in all patients with HLA-mismatched related donor and unrelated donors transplants for graft-versus-host disease (GVHD) prophylaxis. COX regression was used to evaluate the prognostic factors of ALAL. Among 38 ALAL patients, 19 received the standard conditioning while another 19 the intensified conditioning. All patients achieved hematopoietic reconstitution. The 5-year overall survival (OS) and the disease-free survival (DFS) were 35.5% and 25.7% respectively. The 5-year OS rates were 20.2% and 48.1% (P = 0.233) and DFS 6.5% and 43.1% (P = 0.031) in the standard and intensified conditioning groups respectively. The 5-year cumulative relapsing incidence was 58.9% in all patients and 87.6% vs 30.4% in the standard and intensified conditioning groups respectively (P = 0.003). Through a COX regression model for univariate analysis, the intensified conditioning and chronic GVHD were protective factors for DFS (P = 0.001, 0.031). The intensified conditioning in ALAL patients undergoing allo-HSCT may improve the long-term patient survival and decrease the relapse of leukemia. The graft versus leukemic effect has some efficacy in ALAL patients undergoing allo-HSCT.

Acute leukemia of ambiguous lineage with monosomy 7 and Philadelphia chromosome

Acute leukemia of ambiguous lineage with monosomy 7 and Philadelphia chromosome

Gemtuzumab Ozogamicin In Refractory Childhood Acute Myeloid Leukemia.

AbstractAbstract 1075 Introduction:Gemtuzumab ozogamicin, a CD33 linked calicheamicin, has been used in children with refractory AML since some years. In a phase I/II study, the treatment feasibility and moderate toxicity of the compound have been demonstrated (Zwaan et al. British Journal of Haematology, 2010).Here we report the treatment results of 62 children and adolescents with refractory AML after at least two intensive regimens (n=62; male n=40; female n=22) and/or allogeneic stem cell transplant (n=9). Twenty-eight children received GO-monotherapy (7.5mg/m2 day 1 and day 15; group A)*, in 34 children GO 3mg/m2 was combined with cytarabine (500mg/m2/d 96h continuous infusion; group B).The median age at initial diagnosis was 10 years (range 0.2 to 21 years). Nine children had secondary AML following a previous malignant disease and in 4 cases an acute leukemia of ambigious lineage (ALAL) had been diagnosed.The morphological subtypes were AML FAB M0 n=4; M1/M2 n=17 M3 n =1; M4/M5 n=25; M4Eo n=1; M6/M7 n=9, undifferentiated (AUL)/acute leukemia of ambiguous lineage (ALAL) n=5.In cytogenetics, most patients had a normal karyotype (n=16; including 7 with FLT3-ITD) followed by MLL-rearrangements (n=14), numerical aberrations (n=7; including FLT3-ITD n=2), others aberrations (n=10; FLT3-ITD n=1), complex karyotype (n=3) and BCR/ABL positive AML (n=1). Favorable translocations [t(8;21), inv 16, t(15;17)] were diagnosed in 4 patients. In 7 patients no cytogenetic results were available. Results:Five children suffered acute anaphylactic reaction during or short after infusion (CTC grade III n=3, grade IV n=2). Severe, long lasting myelosuppression has been observed in almost all patients. While the frequency of severe infection during myelosuppression was moderate, side effects such as mucositis have rarely been reported. A vena occlusive-disease (VOD)-like syndrome was observed in 5 children after alloSCT, in two cases with fatal outcome.An overall response (CR, CRp, PR) has been documented in 32 children (group A n=10/28, 36%; group B n=22/34, 65%). A higher response rate has been observed in children with AML FAB M1/2 with auer rods (n=18 response n=12, 67%) and AML with favorable aberrations (n=3/4). Six out of 10 children with FLT3-ITD positive AML responded to GO whereas patients with AUL/ALAL (n=9; response n=1, 11%) and AML FAB M6/7 (n=10; response n=3, 30%) showed a poorer response.Thirty-five patients received allogeneic SCT following GO treatment, 30 of the responders and 5 patients without GO response. In total 13 children are alive. Out of the responders 11 children or adolescents are still in complete remission (follow-up 2.7 years, 0.5 to 8.2 yrs.). Again children with AML FAB M1/2 and/or favorable cytogenetics had the best prognosis (7 /19 children alive, 37%). By contrast, none of the children with AML FAB M6/7, AUL or ALAL survived.Out of the 30 patients who did not respond to GO, following alloSCT 2 children are alive, however, the follow-up of 0.5 years (0.3 to 0.9 yrs) is very short. In conclusion,in this particular poor group of heavily pretreated children and adolescents with multiple relapsed, refractory AML, GO seems to improve the prognosis in some children and enables further treatment e.g. alloSCT. Based on the specific needs of children it is of importance that clinical trials continue in childhood AML to identify the appropriate patient group and schedule of GO combined with chemotherapy.* Seven patients out of this group were eligible for the International AML 2001/02 study “Salvage of refractory childhood AML with Mylotarg, Zwaan et al. BJH, 2010. Disclosures:No relevant conflicts of interest to declare.

Molecular Characterization of Acute Undifferentiated and Biphenotypic Leukemia

AbstractAbstract 1692 Background:Acute leukemias of ambiguous lineage account for only 3–5% of all cases of acute leukemia and represent a heterogeneous group of rare, poorly characterized leukemia with adverse outcome. The aim of this study was to further molecularly characterize this rare subtype to gain insights into the pathogenesis of acute undifferentiated leukemia (AUL) and biphenotypic acute leukemia (BAL). These molecular insights may then lead to targeted treatment strategies for specific molecular subgroups. Design and Methods:The European Group for Immunologic Classification of Leukemia (EGIL) defines BAL as a single blast population with co-expression of myeloid- and lymphoid-associated antigens according to a scoring system. Cases without expression of lineage-specific antigens are designated as AUL. Based on the EGIL scoring system, we identified 11 and 20 adults with newly diagnosed AUL and BAL, respectively. In all samples, flow cytometry was performed using a panel of monoclonal antibodies for precursor cells (CD34, TdT), lymphoid-associated antigens, including surface and cytoplasmic antigens (CD79a, CD22, IgM, CD19, CD20, CD10, CD24, CD3, TCR, CD2, CD5, CD8, CD7, CD1a), and myeloid-associated antigens including myeloperoxidase, CD13, CD33, CD65s, CD117, CD14, CD15, CD64. In order to further molecularly characterize AUL and BAL, samples were studied for expression and genotype alterations of candidate genes with prognostic impact or with relevance in the pathogenesis of acute leukemias. The mRNA expression levels of BAALC, IGFBP7, MN1, and WT1 were determined by quantitative real-time PCR. In addition, samples were studied for WT1 mutations in exons 7 and 9, for FLT3 mutations [internal tandem duplications (ITD) and mutations in the tyrosine kinase domain (TKD835)], as well as for BCR-ABL (breakpoints: e1/a2; b3/a2; b2/a2) and MLL-AF4 rearrangements. Results were than compared to data generated from acute myeloid leukemia (AML; n = 75), acute T-lymphoblastic leukemia (T-ALL; n = 242) and B-precursor ALL (n = 368) patients. Results:AUL patients were characterized by a specific gene expression pattern. In particular, in AUL over-expression was shown for IGFBP7 (P = 0.01) and MN1 (P = 0.04) compared to BAL; for BAALC (P = 0.001), IGFBP7 (P = 0.0001) and MN1 (P = 0.02) compared to T-ALL; for BAALC (P = 0.02) and IGFBP7 (P = 0.001) compared to AML; as well as for IGFBP7 (P = 0.0001) compared to B-precursor ALL. Genotype alterations (WT1, FLT3, BCR-ABL, MLL-AF4) were not detectable in AUL samples.The majority of BAL cases expressed either T-lineage plus myeloid antigens (n = 9; 45%) or B-lineage plus myeloid markers (n = 9; 45%), while co-expression of T/B lymphoid antigens was only seen in 2 samples of BAL. Cell surface expression of CD34 (≥ 20%) was most frequently found in BAL (17 of 20 cases; 85%). Gene expression studies revealed over-expression of BAALC and IGFBP7 in BAL compared to T-ALL, AML and B-precursor ALL, but less pronounced as shown for AUL. Moreover, BAL samples were characterized by frequent WT1 mutations in exon 7 (sufficient DNA was available in 14 samples of which 4 had WT1 mutations; 29%). In contrast, WT1 mutations were only found in 8% of T-ALL and have previously been reported in about 10% of AML patients with normal cytogenetics (Paschka et al, 2008). In concordance with published data, the BCR-ABL fusion gene was detected in 32% of BAL samples (19 samples studied of which 9 were positive for BCR-ABL). MLL-AF4 translocations and FLT3 mutations were not detectable in this BAL cohort. Conclusion:Here we present a comprehensive molecular study investigating mRNA expression and genotype alterations in the poorly characterized subgroup of acute leukemias with ambiguous or lacking lineage commitment. We show that AUL and BAL patients share important prognostic features of both myeloid and lymphoid leukemias. Over-expression of stem cell associated genes (BAALC and IGFBP7) and genes associated with an adverse prognosis (BAALC and MN1) was most prominent in AUL. As lineage-directed therapies are likely ineffective for these high-risk patients and the fact that AUL lack known specific molecular targets, further studies are warranted to unravel molecular aberrations, which might allow the development of more specific therapies. BAL patients however, were characterized by genetic aberrations including BCR-ABL rearrangements and WT1 mutations, which may imply targeted therapy options. Disclosures:No relevant conflicts of interest to declare.

Acute leukemias of ambiguous lineage: diagnostic consequences of the WHO2008 classification

Acute leukemias of ambiguous lineage: diagnostic consequences of the WHO2008 classification

Mixed phenotype acute leukemia with t(11;19)(q23;p13.3)/ MLL‐MLLT1(ENL), B/T‐lymphoid type: A first case report

The majority of cases of acute leukemia belong to a specific lineage origin, either lymphoid or myeloid, and therefore are classified as acute lymphoblastic leukemia (ALL) or acute myelogenous leukemia (AML), based on morphologic features and cytochemical and immunophenotypic profile of the blast cells. A minority of acute leukemias however, show no clear evidence of differentiation along a single lineage. These are now classified under acute leukemias of ambiguous lineage by the most recent WHO classification and account for <4% of all cases of acute leukemia [1]. They include leukemias with no lineage specific antigens (acute undifferentiated leukemias) and those with blasts that express antigens of more than one lineage to such degree that it is not possible to assign the leukemia to any one particular lineage with certainty (mixed phenotype acute leukemias). The latter can either be leukemias with two distinct populations of blasts, each expressing antigens of a different lineage (historically referred to as "bilineal" leukemias) or a single blast population expressing antigens of multiple lineages (historically referred to as "biphenotypic" acute leukemias) [2]. Acute leukemias of ambiguous lineage may harbor a variety of genetic lesions. Those with t(9;22)(q34;q11) or translocations associated with mixed lineage leukemias (MLL) gene, i.e., t(11;V)(q23;V), occur frequently enough and are associated with distinct features, that are considered as separate entities according to the recent WHO classification. Co-expression of myeloid and B-lymphoid antigens is most common in mixed phenotype acute leukemia (MPAL), followed by co-expression of myeloid and T-lymphoid antigens, accounting for 66-70% and 23-24% of MLLs, respectively. Coexpression of B- and T-lineage associated antigens or antigens of all three lineages is exceedingly rare, accounting for <5% of MLLs [3,4]. The requirements for assigning more than one lineage to a single blast population has been established by current WHO classification [1].

Blastic Plasmacytoid Dendritic Cell Neoplasm: Report of Three Cases.

Abstract 4703The World Health Organization (WHO) recently published a revised, updated edition of the WHO Classification of Tumours of the Haematopoietic and Lymphoid Tissues, including new criteria for the recognition of some previously described neoplasms as well as clarification and refinement of the defining criteria for others. It also adds entities – some defined mainly by genetic and immunophenotypic features – that have only recently been characterized. Particularly, the diagnosis and classification of acute leukemias of ambiguous lineage is debated; among these: “blastic NK-cell lymphoma” and “agranular CD4+/CD56+ hematodermic neoplasms”. Both of them are now known to be, in virtually all cases, a tumor derived from precursors of a specialized subset of dendritic cells, plasmacytoid dendritic cells, and so are myeloid-related neoplasms defined as blastic plasmacytoid dendritic cell neoplasm (BPDCN). This is a clinically aggressive neoplasm that is usually characterized at onset by solitary or multiple skin lesions, often with associated regional lymphadenopathy, and frequently by involvement of the PB and BM. Leukemic cells show submembranous cytoplasmic vacuoles and pseudopodia-like extensions of agranular cytoplasm. The blasts in such cases do not express myeloperoxidase or nonspecific esterase, and are characterized by the expression of CD4, CD43, CD56, CD123, BDCA-2/CD303, TCL1, and CLA; CD7 and CD33 are not uncommonly expressed as well, and TdT is expressed in about 30% of cases. There is no expression of CD34 or CD117. Here we report three cases with clinical data, cytological and immunophenotypic findings strongly suggesting the diagnosis of BPDCN. Case 1An 80 year-old-man was admitted to our institution on December 2006. He referred the occurrence of skin lesions since January 2005, when a diagnosis of extranodal B-cell non-Hodgkin lymphoma was made and treatment with conventional chemotherapy was performed, but without achieving any response. At our evaluation he presented leukocytosis (144 × 109/L) associated with purplish, firm nodules on the trunk, arms and face. Peripheral blood and bone marrow aspirate showed the presence of blast cells with a lymphoid appearance, granular periodic acid-Schiff (PAS) positivity and a high expression of CD33, CD4, and CD56. He was treated with AML-like therapy, but died of disease progression. Case 2A 79-year old woman was admitted in December 2006 with a 2-month history of anemia, splenomegaly, and weight loss of 10 kg in the last year. Laboratory tests were as follows: Hb, 41 g/L; leukocytes, 2.5 × 109/L (with 10% of blast cells); platelets, 43 × 109/L. No lymphadenopathy or skin lesions were present. Bone marrow examination revealed 41% of small to medium-sized blast cells without Auer rods or granula and negative reactivity to myeloperoxidase, esterase and PAS. She was treated with an AML-like protocol; she achieved partial response, but died after three months, of disease progression. Case 3A 69-year-old man was admitted to our Institution for cytopenia in June 2009. He referred the occurrence of brownish-purple firm nodules on the trunk since April 2009. At our evaluation he presented pancytopenia; bone marrow aspiration was performed and revealed infiltration by 65% of blasts with reticulated chromatin, evident nucleoli, a vacuolated cytoplasm and pseudopodia-like expansions. The blasts were negative for myeloperoxidase, monocyte esterase and PAS staining. Skin biopsy revealed a dermal infiltration by the same blastic-cell BM population. He underwent AML-like therapy and, although the skin lesions disappeared, 30% blastic bone marrow infiltration persisted.Morphological revision of these cases, selected for their peculiar immunophenotype reported in the following Table, revealed the same cytological features and cytochemical reactivity in cases 2 and 3; case 1 had a lymphoblastic-like morphology and showed PAS positivity, but the lack of cCD3 was not consistent with the diagnosis of ALL. All the cases were FLT3-ITD+. We suggest that a correct modern panel of MoAb with a careful morphological examination could help to pose the diagnosis of BPDCN, which typically affects older patients and is characterized by poor prognosis.CaseCD4CD56HLA-DR7.1cMPOcCD3cCD79aCD34CD117CD33CD3031++++-----+nt2++++-----++3++++-----++ Disclosures:No relevant conflicts of interest to declare.

Acute lymphoplasmacytoid dendritic cell (DC2) leukemia: Results from the Hellenic Dendritic Cell Leukemia Study Group

We present a cohort of 22 patients with type 2 dendritic cell (DC2) acute leukemia (or blastic plasmacytoid dendritic cell neoplasm-BPDCN, as it has been recently named), diagnosed in Greece over the past 12-year period, according to the main clinical and immunophenotypic features of this entity. Four additional cases are discussed, classified as leukemia of ambiguous lineage (LAL), because of the simultaneous detection of a CD56 negative DC2 population and of a second myeloid precursor cell population. The morphological features and cytogenetic findings of the typical BPDCN cases were similar to those previously described. Acute lymphoblastic leukemia-type chemotherapeutic regimens were more efficient in controlling the disease. Immunophenotyping of typical BPDCN cases revealed CD4 +, CD56 +, HLA-DR + and CD123 bright neoplastic cells, in the absence of major B-, T- and myeloid-associated markers, while the phenotype of the four cases characterized as LAL highlights the risk of misdiagnosis. Based on our experience, we propose a flow cytometric algorithmic approach for the distinction of typical BPDCN from certain types of acute myeloid leukemia, but also for the identification of acute myeloid leukemia, admixed with CD56 negative DC2 cells, which could be misdiagnosed as BPDCN.

Clinical characteristics and outcome of children with biphenotypic acute leukemia

Knowledge concerning the clinical and biological presentation, as well as the outcome of treatment, of biphenotypic acute leukemia in children is limited. This retrospective review analyzes the clinical features and outcome of children with biphenotypic acute leukemia diagnosed and treated over an 8-year period. According to the EGIL scoring system 24 (3.7%) of 633 patients with acute leukemia were classified as having biphenotypic acute leukemia. The diagnostic work-up and results were reviewed specifically for this study in the light of the newly published WHO criteria for the diagnosis of leukemia of ambiguous lineage. Based on these criteria, 11 (1.7%) patients were categorized according to the new nomenclature as having mixed phenotype acute leukemia. The majority of the patients (58.3%) had a B-lymphoid/myeloid phenotype, followed by the T-lymphoid/myeloid phenotype. The most frequent chromosomal abnormality involved the 14q32 locus. Patients received therapy based on a treatment regimen for acute lymphocytic leukemia regimen, which included myeloid-effective agents. At a median follow up of 4 years (range, 6 month - 7 years) the overall survival rate was 75.7% and the event-free survival rate was 73.5%. The survival of those patients who underwent hematopoietic stem cell transplantation in first complete remission was not different from that of the patients who were treated with chemotherapy alone (overall survival: 70.1% versus 81.1%, respectively, p=0.39; event-free survival: 70.1% versus 76.2%, respectively, p=0.75). The outcome of the 11 patients who were retrospectively classified as having mixed phenotype acute leukemia according to the new WHO criteria was excellent, with no relapses or deaths occurring among these patients. An acute lymphocytic leukemia type of induction therapy, using agents that are active against lymphoid and myeloid leukemias, appears to be more effective in achieving and maintaining complete remissions regardless of whether the patients are classified according to EGIL criteria or the new WHO criteria. Hematopoietic stem cell transplantation may not be necessary for all patients in first complete remission.

Definitive Criteria for Ambiguous Lineage Leukemias

Biphenotypic leukemias are acute leukemias of ambiguous lineage in which there is proliferation of more than one distinct cell type. In this case, both myeloid and lymphoid phenotypes are present in the blast population. As opposed to leukemias in which one specific myeloid or lymphoid population is found, these biphenotypic leukemias present a unique diagnostic challenge in these patients. Treatment is determined by identification of the predominant lineage. This study explores the successful use of the European international scoring system to define biphenotypic acute leukemias. Of nine cases of leukemias of mixed phenotype, determined by flow cytometric analysis, three were scored as biphenotypic by traditional 2001 WHO standards, whereas six were of ambiguous lineage but did not meet the scoring criteria for mixed phenotype. However, on re‐evaluation using the 2008 WHO guidelines, three of these cases were reclassified as biphenotypic based on the increased significance of myeloperoxidase (MPO) and combined CD14/CD64 positivity for myeloid lineage. Considering the worse prognosis of biphenotypic leukemia than AML or ALL, early identification of mixed lineage is critical for appropriate treatment.