Acute Leukemia Of Ambiguous Lineage Research Articles

Immunophenotype, microRNA expression and cytogenetic characterization of acute leukemias of ambiguous lineage

Acute leukemias of ambiguous lineage (ALAL) form a group of leukemias presenting the features of both acute lymphoid leukemia (ALL) and acute myeloid leukemia (AML). Based on Immunologic Classification of Leukemia (EGIL), these leukemias are recognized as biphenotype acute leukemia (BAL). According to WHO classification and based on immunophenotypic characteristics, ALAL is further categorized to acute undetermined leukemia (AUL), mixed phenotype acute leukemia (MPAL), and other ambiguous lineage leukemias. Immunophenotype and cytogenetic aspects are important in recognition of these leukemias; however, there is limited data about these leukemias. This has resulted in mistreatment of patients. Thanks to the recent advances and introducing the role of microRNAs in regulation of gene expression, a new avenue has been opened in the field of classification and pathogenesis of ambiguous lineage leukemias. MiRNA-based classification can help define myeloid or lymphoid origin of ambiguous lineage leukemias. Taking immunophenotype, cytogenetic, and miRNA profiles into account collectively, a better recognition of the disease is achieved, which results in accurate diagnosis and effective treatment of patients. In this review, we have discussed ambiguous lineage leukemias with regard to microRNA expression, immunophenotype, and cytogenetic aspects.

Mixed phenotype acute leukemia: B/T-cell type-case report and review of literature

Mixed phenotypic acute leukemia (MPAL), classified under acute leukemia of ambiguous lineage, pose diagnostic dilemma due to multiple lineage-specific antigen expression. World Health Organization has laid down strict criteria for assigning >1 lineage to a single blast population. Most reported cases of biphenotypic acute leukemia belong to myeloid/B-cell lineage or myeloid/T-cell lineage. However, MPAL with B-/T-cell phenotype is very rare. A 5-year-old girl was admitted with fever, petechial rash, pallor, generalized lymphadenopathy, and hepatosplenomegaly. Complete blood counts revealed leukocytosis, anemia, and thrombocytopenia. Bone Marrow Aspiration showed 97% blasts which were negative for myeloperoxidase, and nonspecific esterase. Immunophenotyping showed a single compact population of blasts which expressed CD19, CD79a, CD22, cytoplasmic CD3, and CD7. Thus, flow cytometric immunophenotyping helped to establish a final diagnosis of MPAL: B/T-cell type.

Extensive Molecular Analysis Strongly Improves the Distinction Between AML and ALL in Adult Acute Leukemias of Ambiguous Lineage

BackgroundIn 1995 the European Group for the Immunological Classification of Leukemias (EGIL) presented guidelines to classify bilineage and biphenotypic acute leukemias (BAL). Subsequently, the WHO in 2008 defined mixed phenotype acute leukemias (MPAL), as leukemias coexpressing antigens of both lymphoid and myeloid lineage, and leukemias with distinct blast populations of more than one lineage. However, a classifying diagnosis towards myeloid or lymphoid leukemia based on an extensive molecular-biological characterization could provide much better distinction, which could be useful for selecting AML or ALL like treatment approaches. We investigated whether extensive molecular analysis might improve a diagnosis of BAL/MPAL into predominant AML or ALL.Patients and methodsFor this study 25 adults, diagnosed between 2000 and 2009 with BAL/MPAL were identified. Flowcytometry and cytogenetics were performed at diagnosis. In addition, molecular analysis was performed to identify BCR-ABL, MLL, AML1-ETO, CBF-MYH11, SIL-TAL1, SET-NUP, NUP-ABL fusion transcripts, FLT3-ITD, CEBPA, NPM1, NOTCH1 mutations and IKZF1, CDKN2A, CDKN2B deletions. Furthermore, multiplex PCR assays to detect immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangements were performed. Gene expression profiles (GEP) were generated, and results were compared to an AML/ALL classifier profile. A classifying diagnosis of either ALL or AML was made if at least two of the 5 assays applied, were highly suggestive for a specific lineage without explicit contradicting results of the remaining assays.ResultsTwenty-four of the 25 BAL/MPAL cases could be diagnosed into predominant AML or ALL by extensive molecular analysis (Table 1). In 11 cases the WHO2008 criteria classified BAL cases as AML or ALL, and extensive molecular analysis also indicated the same lineage. Adding standard cytogenetic and molecular analysis improved the WHO classification in another 4 patients. Remarkably, extensive molecular analysis clearly showed preference for either AML or ALL in all of the MPAL cases. As a result, we were able to improve a diagnosis in all MPAL cases. Only 1 of the 25 patients remained unclassified. Statistical analysis did not show significant correlation between the different assays. Twelve patients received AML-based chemotherapy, including 3 patients ultimately classified as ALL. Moreover, among 12 patients who received ALL type therapy, 1 was retrospectively classified as AML. In general, outcome appeared very poor with a 3 year overall survival of 32% (Kaplan-Meier estimate, 95%CI: 14%-51%).ConclusionsThis study suggests that a differential diagnosis of AML versus ALL is partly improved by applying the WHO2008 criteria instead of the EGIL classification. However, extensive molecular analysis, including GEP and Ig/TCR, more strongly enabled the identification of the lineage of origin, ultimately resulting in a classifying diagnosis in 24 out of 25 patients. So far no assay appeared as golden standard but different complementary techniques showed additive value. Collectively these results strongly argue for advanced, centralized diagnostic procedures in patients with acute leukemia of ambiguous lineage, which may subsequently enable the development of specific therapeutic approaches in these poor-risk patients.Table 1Patient characteristicsAge (year)EGILWHO 2008Molecular aberrationsGEP probabilityIg/TCRClassificationAMLALL51TMMPAL*0,0001,000POSALL52TMMPALFLT3-ITD0,9700,030NEGAML20BMMPALMLL-AF4 CEBPA0,0001,000POSALL18BMMPALIKZF1 CDKN2A-2B0,0001,000POSALL49BMMPALBCR-ABL IKZF1NDNDPOSALL57BMMPALCDKN2A0,0001,000POSALL40BMMPALCDKN2A-2BNDNDPOSALL23TMPALCDKN2ANDNDPOSALL24BMPAL*0,0001,000POSALL65BMPALBCR-ABL IKZF1 CDKN2ANDNDPOSALL58BMAML*1,0000,000NEGAML52BMAMLNPM1NDNDNEGAML59BMAMLFLT3-ITD NPM1NDNDNEGAML41BMAMLFLT3-ITD CEBPA NPM1NDNDPOSAML69BMAMLMLLNDNDNEGAML18BMAMLCBF-MYH11NDNDPOSAML71BM#FLT3-ITD CDKN2A-2B0,7090,291NEGAML67BM#*0,0030,997POSALL15BMALLIKZF1 CDKN2A-2BNDNDPOSALL23BM#*0,9980,002POS?32BTM#*0,9380,062POSAML27BTMALLIKZF10,0010,999POSALL69BTMALLCDKN2A-2B0,0980,902NEGALL16BTMALLCDKN2A-2BNDNDPOSALL54TMALLNOTCH10,1480,852NEGALLAbbreviations: ND, not determined.* No abnormalities# Unclear classification according to WHO2008 DisclosuresNo relevant conflicts of interest to declare.

Langerhans cell histiocytosis in acute leukemias of ambiguous or myeloid lineage in adult patients: support for a possible clonal relationship

Four patients presented with acute leukemia of ambiguous or myeloid lineage in association with Langerhans cell histiocytosis and provide evidence suggesting a common origin of the two neoplasms. One patient had a non-constitutional trisomy 21 in both the leukemic blasts and the Langerhans cells indicative of a clonal relationship. A second case expressed CD2, CD13, and CD117 on both the Langerhans cells and the blasts suggesting a possible clonal relationship. All four cases exhibited geographic intermingling of the Langerhans cell histiocytosis and acute leukemia and shared unique features including extramedullary leukemia involving lymph nodes in all cases with Langerhans cell histiocytosis only present in sites involved by acute leukemia. T-cell antigen expression was present in all cases with one meeting criteria for mixed phenotype acute leukemia, T/myeloid, not otherwise specified. These findings support the concept that coexistent Langerhans cell histiocytosis and acute leukemia is clonally related in some cases. Furthermore, these cases of acute myeloid or acute leukemia of ambiguous lineage with Langerhans cell histiocytosis share some unique features suggesting a common underlying neoplastic hematopoietic stem cell.

Acute leukemia of ambiguous lineage with trisomy 4 as the sole cytogenetic abnormality: A case report and literature review

We describe a patient with acute leukemia of ambiguous lineage who had trisomy 4 as the sole cytogenetic abnormality. Clinical, pathological, immunophenotypic and molecular features are presented and compared with the previous 4 published cases. Over expression of c-kit, which is localized to chromosome 4, was documented on the leukemic blasts. Prognosis seems to be poor. Treatment with acute lymphoblastic leukemia like regimens seems to be superior compared to acute myeloid leukemia like regimens and allogeneic stem cell transplant is recommended after achieving remission.

Comparison of clinical efficacy between HLA-mismatched related and HLA-matched unrelated donor hematopoietic stem cell transplantation for hematopoietic malignancies

ObjectiveTo compare the clinical efficacy of HLA-mismatched related donor (MRD) and HLA-matched unrelated donor (MUD) hematopoietic stem cell transplantation (HSCT) for hematopoietic malignancies, 175 patients with hematopoietic malignancies undergoing allogeneic HSCT (allo-HSCT) (83 from MRD and 92 from MUD) from October 2002 to December 2012 were enrolled in this retrospective study. MethodsOf the 175 patients, 67 were female, and 108 were male. The median age was 27.0 (range 11-53) years. Primary diseases included acute lymphoblastic leukemia (ALL, n=56), acute myelogenous leukemia (AML, n=46), acute leukemias of ambiguous lineage (ALAL, n=13), chronic myeloid leukemia (CML, n=47), myelodysplastic syndrome (MDS, n=7), lymphoma (n=5) and aplastic anemia (AA, n=1). Fifty-seven patients were in the status of no remission (NR), and 118 were complete remission (CR) at the time of transplantation. Genomic molecular typing was used for HLA-A, -B and -DRB in most donors and recipients before May 2008, and then it was used for HLA-A, -B, -DRB, -DRQ and Cw. Hematopoietic engraftment, graft versus host disease (GVHD), relapse, overall survival (OS) and disease-free survival (DFS) were compared between MRD and MUD group. ResultsEighty-three patients received MRD transplantation, including sibling (n=47), parents (n=29), children (n=5) and others (n=2), and 93 received MUD transplantation. Of HLA 6/6 typing, 18 were one allele, 19 two alleles, 1 three alleles-mismatched; of HLA 10/10 typing, 4 were one allele, 17 two alleles, 9 three alleles, 7 four alleles, and 8 five alleles- mismatched in MRD group. Forty-one patients were HLA 6/6 and 51 we HLA 10/10-matched in MUD group. There was no significant difference between MRD and MUD group regarding age, gender, disease type and disease status pre-transplantation (all P>0.05). Except for 4 and 3 patients who died of regimen-related toxicity (RRT), respectively, in MRD and MUD group, other patients obtained hematopoietic engraftment.The median time to neutrophil and platelet engraftment was 11 (range, 9 to 22) days and 13 (range, 10 to 40) days in MRD group, and it was 12 (range, 10 to 29) days and 14 (range, 10 to 60) days in MUD group (P>0.05, P>0.05). The incidence of I-IV0acute GVHD (aGVHD) was 68.7% and 48.9% in MRD and MUD group (P<0.01), but there was no significant difference in the incidence of III-IV0 aGVHD between the two groups (14.5% vs. 8.7%, P>0.05). The incidence of chronic GVHD (cGVHD) was 22.9% and 41.3% in MRD and MUD group (P<0.05), but there was no significant difference in the incidence of extensive cGVHD between the two groups (7.2% vs. 12.0%, P>0.05).The mortality of GVHD was 14.5% and 18.6% in MRD and MUD group (P>0.05).With a median follow up of 51 months (range 1 day-130 months) post-transplantation, the 10- year OS and DFS were 47.2%±6.1% and 42.9%±6.1% in MRD group, compared with 55.1%±5.5% and 45.8%±5.4% in MUD group(P=0.236, P=0.686, respectively). The 10-year cumulative incidence of relapse was 24.8%±6.0% and 39.1%±6.1% in MRD and MUD group (P=0.230). ConclusionsAlthough the incidence of cGVHD was higher in MRD group, there was no significant difference in the incidence of I-IV0aGVHD, III-IV0 aGVHD and extensive cGVHD between MRD and MUD group. Hematopoietic engraftment, cumulative relapse rate, OS and DFS also did not differ significantly between the two groups. These results suggest that MRD is equivalent to MUD in efficacy and safety. Without HLA-matched related donors, MRD is superior to MUD because donor source is unlimited and choice of treatment could be made promptly according to disease status. Disclosures:Liu:It was supported by Science and Technology Program of Guangzhou of China (11A72121174).: Research Funding; It was supported by National Public Health Grand Research Foundation (Grant No. 201202017), National Natural Science Foundation of China (Grant No.81000231, No.81270647).: Research Funding; It was supported by 863 Program (No. 2011AA020105).: Research Funding.

Novel r(2)(p25q31) Cytogenetic Abnormality in a Pediatric Patient With Acute Leukemia of Ambiguous Lineage

ABSTRACT We describe a case of acute leukemia of ambiguous lineage with a novel cytogenetic abnormality. A 1-year-old boy presented with abnormal complete blood count findings, and was found to have blasts and mild dysgranulopoiesis. The blasts showed immunophenotypic evidence of myeloid and T-lineage differentiation. Subsequent cytogenetic analysis showed r(2)(p25q31) as the sole stem line cytogenetic defect with clonal evolution. While cytogenetic abnormalities can have a critical role in the classification and prognostication of acute lymphoblastic and acute myeloid leukemia, the significance of cytogenetic abnormalities in acute leukemia of ambiguous lineage remains unclear. This finding has not been reported previously to the best of our knowledge.

Two Cases of Near-Tetraploidy in Acute Leukemias of Ambiguous Lineage

Acute leukemia of ambiguous lineage (ALAL) is a rare subtype of acute leukemias that does not show any clear evidence of differentiation along a single lineage [1].

MicroRNA Profiling Can Classify Acute Leukemias of Ambiguous Lineage as Either Acute Myeloid Leukemia or Acute Lymphoid Leukemia

Classification of acute leukemia is based on the commitment of leukemic cells to the myeloid or the lymphoid lineage. However, a small percentage of acute leukemia cases lack straightforward immunophenotypical lineage commitment. These leukemias of ambiguous lineage represent a heterogeneous category of acute leukemia that cannot be classified as either acute myeloid leukemia (AML) or acute lymphoid leukemia (ALL). The lack of clear classification of acute leukemias of ambiguous lineage as either AML or ALL is a hurdle in treatment choice for these patients. Here, we compared the microRNA (miRNA) expression profiles of 17 cases with acute leukemia of ambiguous lineage and 16 cases of AML, B-cell acute lymphoid leukemia (B-ALL), and T-cell acute lymphoid leukemia (T-ALL). We show that leukemias of ambiguous lineage do not segregate as a separate entity but exhibit miRNA expression profiles similar to AML, B-ALL, or T-ALL. We show that by using only 5 of the most lineage-discriminative miRNAs, we are able to define acute leukemia of ambiguous lineage as either AML or ALL. Our results indicate the presence of a myeloid or lymphoid lineage-specific genotype, as reflected by miRNA expression, in these acute leukemias despite their ambiguous immunophenotype. miRNA-based classification of acute leukemia of ambiguous lineage might be of additional value in therapeutic decision making.

Abstract LB-246: MicroRNA profiling can classify acute leukemias of ambiguous lineage as either acute myeloid leukemia or acute lymphoid leukemia.

Abstract Classification 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 display features of both hematopoietic lineages and lacks immunophenotypical lineage commitment. These leukemias of ambiguous lineage represent a heterogeneous category of AL that cannot be classified as either myeloid AL (AML) or lymphoid AL (ALL). The lack of a clear classification of acute leukemias of ambiguous lineage as either AML or ALL is a hurdle in treatment choice for these patients. MicroRNAs are small single stranded RNA molecules which regulate gene expression by promoting degradation of mRNAs or repressing their translation. MicroRNA expression profiles have shown to be able to accurately discriminate AL of the lymphoid lineage from those of the myeloid lineage. Here, we compared the microRNA expression profiles of acute leukemias of ambiguous lineage with those of B-ALL, T-ALL and AML. MicroRNA expression profiles of nine patients with leukemia of ambiguous lineage and eleven patients with AML, B-ALL or T-ALL were analyzed. Unsupervised clustering analysis of the AML, B-ALL and T-ALL samples resulted in a clear separation between cases of the myeloid and lymphoid lineages. The top differentially expressed microRNAs between AML and ALL were miR-199b, miR-27a/b, miR-223, miR-23a, miR-221 and miR-150. Subsequently, we compared the general miRNA expression profiles of AL of ambiguous lineage with those of the AML, B-ALL and T-ALL cases thereby showing that these leukemias of ambiguous lineage do not segregate as a separate entity. Moreover, unsupervised clustering of all the 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. Furthermor, qRT-PCR expression analysis of the five most discriminative microRNAs between ALL and AML was able to define AL of ambiguous lineage as either AML or ALL. Thus, our results indicate the presence of a lymphoid or myeloid lineage specific microRNA expression profile in AL cases of ambiguous lineage, despite their mixed immunophenotype. At diagnosis, the classification of AL of ambiguous lineage by microRNA expression analysis might be of additional value in therapeutic decision making. Citation Format: Dave C. de Leeuw, Willemijn van den Ancker, Fedor Denkers, Rene X. Menezes, Theresia M. Westers, Gert J. Ossenkoppele, Arjan A. van de Loosdrecht, Linda Smit. MicroRNA profiling can classify acute leukemias of ambiguous lineage as either acute myeloid leukemia or acute lymphoid leukemia. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-246. doi:10.1158/1538-7445.AM2013-LB-246

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); &amp;lt;i&amp;gt;Bcr-Abl1&amp;lt;/i&amp;gt;

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.

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]

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.