Detection of acute leukemia cells with mixed lineage leukemia (MLL) gene rearrangements by flow cytometry using monoclonal antibody 7.1

Abstract Abstract 2477 Background: MLL gene rearrangements are found in more than 70% of the cases of infant leukemia, both acute lymphoblastic leukemia (ALL) or acute myeloid leukemia (AML), but are less frequent in leukemia from older children. MLL translocations are also found in approximately 10% of adult AML and in a small proportion of patients with therapy-related leukemia. Independently of their association with other high-risk features at presentation, MLL rearrangements are in most cases predictive of poor clinical outcome. In this study, we report the clinical characterization and frequency and type of MLL rearrangements present in a consecutive series of 45 patients that were diagnosed with acute leukemia in the Portuguese Oncology Institute, Porto, Portugal, over the last 13 years (1998–2011). Patients and Methods: Conventional cytogenetic, fluorescence in situ hybridization (FISH), and molecular genetic studies (RT-PCR and LDI-PCR) were used to characterize the type and frequency of MLL rearrangements in a consecutive series of 45 Portuguese patients with MLL-related leukemia treated in a single institution between 1998 and 2011. Additionally, a detailed patient clinical characterization was also performed and statistical analysis using the Kaplan-Meier method as used to evaluate patient survival. Results: In 43 patients (96% of the cases) we could identify the fusion partner, the most common being the MLLT3, AFF1, MLLT1, MLLT10, ELL, and MLLT4 genes, accounting for 88% of all cases. In the group of patients with acute lymphoblastic leukemia and an identified MLL fusion partner, 47% showed the presence of an MLL-AFF1 fusion, as a result of a t(4;11). In the remaining cases, a MLL-MLLT3 (27%), a MLL-MLLT1 (20%), or MLL-MLLT4 (7%) rearrangement was found. The most frequent rearrangement found in patients with acute myeloblastic leukemia was the MLL-MLLT3 fusion (42%), followed by MLL-MLLT10 (23%), MLL-MLLT1 (8%), MLL-ELL (8%), MLL-MLLT4 (4%), and MLL-MLLT11 (4%). In three patients, fusions involving MLL and a septin family gene (SEPT2, SEPT6, and SEPT9), were identified. The most frequently identified chromosomal rearrangements were reciprocal translocations, but insertions and deletions, some cryptic, were also observed. In our series, patients with MLL rearrangements were shown to have a poor prognosis, regardless of leukemia subtype and treatment protocol. However, patients that received a bone marrow transplant had a better survival than patients that received chemotherapy alone. Interestingly, children with 1 year or less showed a statistically significant better overall survival when compared with both older children and adults. Conclusions: The use of a combined strategy in the initial genetic evaluation of acute leukemia patients allowed us to characterize the pattern of MLL rearrangements in our institution, including our previous discovery of two novel MLL fusion partners, the SEPT2 and CT45A2 genes, and a very rare MLL-MLLT4 fusion variant. Disclosures: No relevant conflicts of interest to declare.

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Heatshockprotein-60/90 Are Overexpressed in Leukemia Stem Cells and Stabilize Oncogene Signaling.

Blood ◽

10.1182/blood.v120.21.2472.2472 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2472-2472

Author(s):

Kerstin M Kampa-Schittenhelm ◽

Lothar Kanz ◽

Marcus M Schittenhelm

Keyword(s):

Stem Cells ◽

Flow Cytometry ◽

Acute Leukemia ◽

Cellular Proliferation ◽

Hsp90 Inhibitor ◽

Cell Fraction ◽

Leukemia Cells ◽

Leukemia Stem Cells ◽

Acute Leukemias ◽

Induction Of Apoptosis

Abstract Abstract 2472 Introduction: Using a global phosphoproteome screen, we have recently identified heat shock proteins (HSPs) to be crucial effectors to stabilize KIT oncoprotein function in the presence of tyrosine kinase inhibitors in mutant-KIT leukemia models (Kampa-Schittenhelm et al., ASH annual meeting 2010). We now show that HSP60 and (p)HSP90 are frequently expressed in acute leukemia, arguing for an integrative function of HSPs to stabilize leukemia-driving oncoproteins. Importantly, HSPs are highly expressed in leukemia stem cells (LSCs), which provides a rationale for therapy insensitivity. Targeting HSPs may therefore be an attractive strategy to target the LSC clone. Methods: Intracellular protein levels of (p)HSPs in leukemic blasts were studied by flow cytometry. Three dimensional detection of the putative CD34+/CD38-/HSP+ leukemia stem/progenitor cell fraction was performed using -FITC, -PE and APC-coupled secondary antibodies using standard techniques. Cellular proliferation and induction of apoptosis in leukemia cells treated with the HSP90 inhibitor IPI-504 was determined by XTT- and annexin V-based assays. Immunoblot experiments to assess IPI-504 interaction on the chaperone level were set up using standard protocols including a LICOR imaging system. Results: Of 16 evaluated patients with AML, 50% and 56% demonstrated significant HSP90 and HSP60 expression levels (with a threshold level set as > 3× of the relative mean fluorescence compared to IgG controls), respectively. Interestingly, HSP expression was predominantly seen in patients with core–binding factor leukemia (associated with KIT mutations). Moreover, five evaluable freshly harvested patient samples were used to assess the putative CD34+/CD38-/HSP90 and HSP60 population by flow cytometry. Tantalizingly, all tested CD34+/CD38- samples revealed high co-expression of HSPs. Consequently, an HSP90 inhibitor (IPI-504) potently inhibited cellular proliferation in in vitro leukemia models as well as freshly harvested leukemia cells at highly variable doses starting in low nanomolar ranges up to lower micromolar concentrations with regard to IC50s. Several cell lines with defined leukemia-driving oncogenes (MOLM14 – FLT3 ITD, HMC1.1 – KIT V560G, Kasumi1 – KIT N822H) were treated with IPI-504, and degradation of FLT3 and KIT was confirmed by immunoblotting. Consequently, oncogene-degradation led to induction of apoptosis in the nanomolar range which was confirmed in ex vivo blasts. Noteworthy, treating isolated freshly harvested CD34+/CD38- progenitor cells lead to potent reduction of the viable cell fraction. Conclusion: HSPs are frequently expressed in acute leukemias, including LSCs, arguing for a function as mechanism to protect and uphold LSC function in the presence of cell stress such as antileukemic treatment. Targeting the HSP-expressing stem cell pool may be an attractive novel therapeutic strategy in acute leukemia. Disclosures: No relevant conflicts of interest to declare.

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Human homologue of the rat chondroitin sulfate proteoglycan, NG2, detected by monoclonal antibody 7.1, identifies childhood acute lymphoblastic leukemias with t(4;11)(q21;q23) or t(11;19)(q23;p13) and MLL gene rearrangements

Blood ◽

10.1182/blood.v87.3.1134.bloodjournal8731134 ◽

1996 ◽

Vol 87 (3) ◽

pp. 1134-1139 ◽

Cited By ~ 92

Author(s):

FG Behm ◽

FO Smith ◽

SC Raimondi ◽

CH Pui ◽

ID Bernstein

Keyword(s):

Monoclonal Antibody ◽

Chondroitin Sulfate ◽

Lymphoblastic Leukemia ◽

Central Nervous System Involvement ◽

Surface Expression ◽

Chondroitin Sulfate Proteoglycan ◽

Cell Surface Expression ◽

Gene Rearrangements ◽

Sulfate Proteoglycan ◽

Mll Gene

Monoclonal antibody 7.1, which recognizes the chondroitin sulfate proteoglycan molecule NG2, was used to screen prospectively blast cells from 104 consecutive children at initial presentation with acute lymphoblastic leukemia (ALL). Reactivity with this antibody was found in 9 cases (8.6%), of whom 5 had a t(4;11)(q21;q23) and 4 had a t(11;19)(p13;q23). None of the NG2- cases had either translocation. Southern blot analysis disclosed MLL gene rearrangement in only the 9 cases with 7.1 reactivity plus the t(4;11)(q21;q23) or t(11;19)(q23;p13) translocation. MLL gene rearrangements were not detected in 89 patient leukemic samples that did not express NG2, including 7 patients with del(11)(q23) or inv(11)(p13q23). As expected from the association with t(4;11) and t(11;19), NG2+ cases were significantly more likely to be infants, to have hyperleukocytosis and central nervous system involvement, to be CD10-, and to express myeloid- associated antigens CD15 and CD65. Despite short follow-up duration, 3 of the NG2+ cases have relapsed while the other 101 patients remain in remission. Thus, blast cell surface expression of NG2 is useful for identifying patients with ALL having t(4;11) or t(11;19) translocations that are associated with poor prognosis, especially in the infant age group.

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The Study of Acute Leukemia Cells by Means of Acridine Orange Staining and Flow Cytometry

Leukemia & Lymphoma ◽

10.3109/10428199409051653 ◽

1994 ◽

Vol 13 (1-2) ◽

pp. 61-73 ◽

Cited By ~ 2

Author(s):

H. D. Preisler ◽

A. Raza ◽

V. Gopal ◽

S. D. Banavali ◽

J. Bokhari ◽

...

Keyword(s):

Flow Cytometry ◽

Acute Leukemia ◽

Acridine Orange ◽

Leukemia Cells ◽

Acridine Orange Staining

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Alterations of p16 and p15 genes in acute leukemia with MLL gene rearrangements and their correlation with clinical features

Leukemia ◽

10.1038/sj.leu.2400872 ◽

1997 ◽

Vol 11 (12) ◽

pp. 2120-2124 ◽

Cited By ~ 5

Author(s):

H Ohnishi ◽

SX Guo ◽

K Ida ◽

T Taki ◽

S Naritaka ◽

...

Keyword(s):

Acute Leukemia ◽

Clinical Features ◽

Gene Rearrangements ◽

Mll Gene

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Study on Clinical and Biological Characteristics of Childhood Acute Leukemia with MLL Gene Rearrangements.

Blood ◽

10.1182/blood.v106.11.4473.4473 ◽

2005 ◽

Vol 106 (11) ◽

pp. 4473-4473

Author(s):

Jun He ◽

Zi-xing Chen ◽

Yong-quan Xue ◽

Jin-lan Pan ◽

Hai-long He ◽

...

Keyword(s):

Acute Leukemia ◽

Fusion Gene ◽

Molecular Genetic ◽

Chromosome Translocation ◽

Fish Analysis ◽

Gene Rearrangements ◽

Older Children ◽

Chromosome 11 ◽

Rt Pcr ◽

Mll Gene

Abstract The rearrangement of MLL gene is reported in 70%~80% of infant and in 5%~10% of older children (under the age of 15) with acute leukemia (AL). The biological features associated with alterations in MLL gene are hyperleukocytosis, CD10−/CD19+ phenotype and very poor prognosis. To explore the MLL rearrangement in details in our AL children patients and obtain more information on the relationship between the MLL gene abnormality and clinical outcomes. The following study has been conducted. A total of 298 patients with AL attended The Affiliated Children’s Hospital of Soochow University, including 16 cases with MLL rearrangements, were recruited in this study. Of the cohort, 11 were diagnosed as ALL, 5 were AML. 9 of 16 patients were in infant age (up to 2 year) and the rest were between the age of 2 to 13 years. Fluorescence in situ hybridization (FISH) analysis using LSI MLL dual color probe. Multiplex reverse transcriptase- polymerase chain reaction (multiplex RT-PCR) were used to discriminate 13 different fusion transcripts. These results were analyzed together with R banding karyotyping and immunolphenotyping determined by flow cytometry. We have found MLL rearrangements in 16 cases of childhood AL which were accounted for 5.4% of 298 AL patients, and 56.3% of infant AL. Among 106 cases analyzed by multiplex RT-PCR, MLL gene rearrangement was found in 11 cases, including MLL/AF4 fusion gene in 2, MLL/AF6 fusion gene in 1, MLL/AF6, MLL/ELL combined with MLL/AFX or HOX11 in one of each, MLL/AF9 in 2, MLL/AF10 in 1, MLL/ELL in 2. MLL partial tandem duplication in 1. In addition an activated HOX11 gene was found in 1 case.. In 27 cases assayed by FISH, MLL gene rearrangements have been detected in 9 cases (36.0%). In 16 patients with MLL gene rearrangements, 14 (87.5%) exhibited clonal chromosome abnormalities involved chromosome 11 in 11 cases, presenting as t(4;11) in 2, t(6;11), t(8;11), t(7;8;11), and t(9;11) in one of each, respectively, trisomy 11 in 2 and 11q- in 3 cases. Among these 16 patients, 11 were B-ALL, including Pro-B and Pre-B ALL; 5 of AML-M5, 3 of these 5 M5 patients were CD7+ and CD2+. Of these 16 patients 8 received chemotherapy and 7 of them achieved complete remission, while the other 8 patients eventually gave up treatment. Our results demonstrated that multiplex RT-PCR combined with FISH provided a more accurate and sensitive method for detection of MLL gene rearrangements, including chromosome translocation, deletion and duplication. Our findings lead to the detection of novel rearrangements at molecular genetic level. These findings regarding the MLL rearrangement provide most important information in guiding therapy and predicting prognosis in childhood AL. Besides our results also provide evidence in support of the value of 11q23/MLL in WHO classification categories.

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Unique Familial MLL-rearranged Precursor B Cell Infant Acute Lymphoblastic Leukemia (ALL) in Non-Twin Siblings

Blood ◽

10.1182/blood.v118.21.2417.2417 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2417-2417

Author(s):

Karen A. Urtishak ◽

Blaine W. Robinson ◽

Jaclyn A. Biegel ◽

Kim E. Nichols ◽

Julie W. Stern ◽

...

Keyword(s):

Risk Factor ◽

B Cell ◽

Snp Array ◽

Leukemia Cells ◽

Upstream Region ◽

Gene Rearrangements ◽

Breakpoint Junction ◽

Fusion Transcripts ◽

Mll Gene ◽

Monozygous Twin

Abstract Abstract 2417 Leukemia is the commonest malignancy in infants, the most frequently occurring form of which is infant ALL. When ALL occurs in infants the disease is clinically aggressive and associated with poor outcome. MLL gene rearrangements producing transforming fusion oncoproteins are found in 75% of infant ALL and they are adverse prognostic factors. Infant ALL has never occurred in families except in monozygous twins, where concordance in leukemia occurrence is nearly 100%. Identical but non-germline genomic breakpoint junction sequences have pointed to an in utero origin of MLL gene rearrangements in these twin cases, where it is believed that metastasis of cells with the rearrangement occurs from one twin to the other via the placental circulation. Here we describe two highly novel siblings both deceased from precursor B cell infant ALL (ages at diagnosis: proband 160 d, sibling 121 d). Remarkably, the second of these two decedents is survived by a now 3 year-old monozygous twin who is as yet unaffected. MLLrearrangements in the leukemia blasts of both affected siblings were characterized by conventional cytogenetics and/or FISH, M-FISH, high resolution Illumina 610K Bead Chip SNP array and Southern blot analysis. MLL genomic breakpoint junction sequences and fusion transcripts were defined using panhandle PCR approaches, PCR with gene-specific primers and reverse transcriptase PCR. The twins were confirmed to be monozygous using the genotype calls from SNP array analysis of the peripheral blood and bone marrow of the unaffected and affected twins, respectively. Quantitative real-time PCR analysis of leukemia DNA was used to determine the allele specific sequences of the NQO1 (NADPH quinone oxidorecutase 1) gene, an inactivating polymorphism in which previously was implicated as a risk factor for MLL-rearranged infant ALL. The complex karyotype in the leukemia cells of the proband was 46, XX, der(2) t(2;3) (q3?;?), der(3) ?t(3;4;11), del(4) (q21), der(11) ?del(11) (p11.2) t(3;11) (?;q23).ish der(3) (5'MLL+), der(11) (3'MLL+) [14]/46, XX[8], suggesting extensive damage to the genome. Consistent with a three-way t(3;4;11) translocation, two alternately spliced 5'-MLL-MLLT2(AF-4)-3' fusion transcripts were identified, indicating disruption of the chromosome band 4q21 partner gene MLLT2. Also consistent with the three-way translocation, reverse panhandle PCR detected a 5'-partner-MLL-3' genomic breakpoint junction fusing 3' MLL to the upstream region of a highly novel chromosome 3 gene encoding a nucleotidyltransferase fold protein C3ORF31 (Accession no. NM_138807; Kuchta, 2009). Unlike in the proband, the ALL of the affected twin exhibited a simple t(4;11)(q21;q23) translocation, the reciprocal genomic breakpoint junctions of which fusing MLL and MLLT2 also have been characterized. Similar to non-familial infant ALL, the genomic breakpoint junctions in both infants contained sequence features of nonhomologous end joining DNA repair. The different MLL gene rearrangements in the leukemia cells in the affected siblings indicate that the translocations were not hereditary. Even though the NQO1 inactivating polymorphism is one genetic risk factor for infant ALL, the NQO1 genotype was wild-type in both affected siblings. Further studies in this uniquely afflicted family with two siblings who succumbed to infant ALL and a monozygous twin of one of the decedents surviving beyond infancy unaffected, will provide a one-time opportunity to capture novel mutations predisposing to the development of, and cooperating with, MLL translocations in infant ALL. The MLLT2 involvement in both cases has even further implications for the knowledge to be gained because the MLL-MLLT2 rearrangement occurs in 50% of infant ALL and adversely impacts outcome. Disclosures: Felix: Children's Hospital of Philadelphia: Methods and Kits for Analysis of Chromosomal Rearrangements Associated with Leukemia - U.S. Patent # 6,368,791 issued April 9, 2002.

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Acute Bilineal Leukemia Is a Very Rare Entity in Childhood

Blood ◽

10.1182/blood.v118.21.4871.4871 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4871-4871

Author(s):

Ester Mejstrikova ◽

Lucie Slamova ◽

Eva Fronkova ◽

Jana Volejnikova ◽

Katerina Muzikova ◽

...

Keyword(s):

Complete Remission ◽

Acute Leukemia ◽

Conflicts Of Interest ◽

Treatment Modalities ◽

Mixed Chimerism ◽

Induction Treatment ◽

Gene Rearrangements ◽

Rare Entity ◽

Hematopoietic Stem ◽

Mll Gene

Abstract Abstract 4871 Recent WHO 2008 classification introduced a new category named Mixed Phenotype Acute Leukemia (MPAL) for leukemias in which primary lineage cannot be determined by morphology, cytochemistry and/or flow cytometry (FC). Acute bilineal leukemia (ABL) is a subtype of MPAL and is defined by presence of distinct myeloid and lymphoid clonal populations simultaneously at diagnosis. No epidemiological data on ABL have been published so far and there are also few data on the origin of distinct leukemic clones. We examined the incidence and biology of ABL cases among children with primary acute leukemia in the period between 1996 and 2011 in the Czech Republic. Morphology and FC were centrally evaluated in all patients. In total 1065 patients were diagnosed (919 ALL, 146 AML); out of them 3 patients were classified as ABL. Two cases had simultaneous presence of distinct B-cell precursor (BCP) and myeloid clones (BCP-My) at diagnosis, one patient had discrete T ALL and myeloid population (T-My). All ABL patients were screened for immunoglobulin (Ig) and T-cell receptor (TCR) clonality, BCR-ABL, MLL gene rearrangements and FLT3-ITD. All three patients had detectable clonality in lymphoid-specific Ig/TCR rearrangements. In pt1 (BCP-My), FLT3-ITD abnormality and in pt3 (BCP-My), BCR-ABL fusion gene were found. Both patients with BCP-My ABL had Ikaros (IKZF1) gene deletion. In pt2 complex karyotype with MLL gene translocation was identified. Using high speed cell sorting we evaluated the presence or absence of previously mentioned changes in separated subpopulations. In patients with BCP-My ABL, identical clonal Ig rearrangements were found in both lymphoid and myeloid clones. Also FLT3-ITD and BCR-ABL aberrations were present in both clones of respective patients. In pt2 (T-My) TCR gene rearrangement was absent in myeloid population. All three patients achieved complete remission (CR) by lymphoid-directed induction treatment followed by switch to myeloid-oriented blocks according Interfant 99, resp. 2006 protocol in pt 1 and 2 and ALL treatment combined with tyrosine kinase inhibitor (TKI) in pt3. Finally all patients underwent allogeneic hematopoietic stem cell transplantation (SCT) in first CR. Pt1 relapsed after SCT as “typical” cALL. However, the plasticity was maintained and the myeloid clone reappeared at day 28 of relapse therapy. Pt2 relapsed as AML with undetectable TCR gene rearrangements. Pt3 is in complete remission 23 months after SCT with detectable low-level MRD and mixed chimerism with repeated lowering MRD after re-administered TKI dasatinib. Conclusions: ABL is an extremely rare entity in childhood accounting for less than 0.3 % of all acute leukemia cases. Surprisingly, the same genetic changes can be identified in both clonal populations making the “true” ABL even rarer. FC in combination with morphology is the basic method for identifying ABL and should be followed by detailed genetic analysis. The prognosis of ABL patients in our cohort was poor, despite SCT preceded by the application of treatment modalities targeting both lymphoid and myeloid clones. Disclosures: No relevant conflicts of interest to declare.

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MLL Gene Rearrangements in 174 Infants with Acute Leukemia.

Blood ◽

10.1182/blood.v120.21.2537.2537 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2537-2537

Author(s):

Grigory Tsaur ◽

Alexander Popov ◽

Elena Fleishman ◽

Olga Sokova ◽

Anna Demina ◽

...

Keyword(s):

Acute Leukemia ◽

Fusion Gene ◽

Fish Analysis ◽

Gene Rearrangements ◽

Gene Transcript ◽

Rt Pcr ◽

Long Distance ◽

Mll Gene ◽

Breakpoint Location ◽

Breakpoint Position

Abstract Abstract 2537 Background. MLL gene rearrangements are the most common genetic events in infant leukemia. Up to date more than 100 various MLL rearrangements were described. Purpose. To evaluate the distribution of MLL rearrangements among infants (aged from 1 to 365 days) with both acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). Methods. 174 infants (117 ALL and 57 AML cases) were included in the current study. 11q23/MLL rearrangements were detected by chromosome banding analysis (CBA), fluorescence in-situ hybridization (FISH) and reverse-transcriptase PCR (RT-PCR). CBA was done according to standard procedure. FISH analysis using LSI MLL Dual Color, Break Apart Rearrangement Probe (Abbott Molecular, USA) was performed on at least 200 interphase nuclei and on all available metaphases. RT-PCR was performed as previously described (A. Borkhardt et al.,1994, N. Palisgaard et al., 1998, J. van Dongen et al., 1999). In 39 cases genomic DNA breakpoint was detected in MLL and translocation partner genes by long-distance inverse PCR (LDI-PCR). Exon-intron numbering of MLL gene was done according to I. Nilson et al, 1996. Results. 11q23/MLL rearrangements were revealed in 74 ALL patients (63.2%). Among this group MLL-AF4 was detected in the majority of cases (53.5%), less frequently were found MLL-MLLT1, MLL-MLLT3, MLL-MLLT10 and others (fig. 1a). Children with ALL under 6 months of age had significantly higher incidence of MLL rearrangements in comparison with older infants (84.0% vs. 47.8%, p<0.001). MLL-positive patients more frequently had BI-ALL and less frequently BII-ALL than infants without these rearrangements (p<0.001 for both). Fusion gene transcripts were sequenced in 26 MLL-rearranged ALL cases. Depending on breakpoint position within MLL and partner genes we detected 7 different types of MLL-AF4 fusion gene transcript, 3 types of MLL-MLLT1, 2 types of MLL-EPS15. The most common fusion site within MLL gene in ALL patients was exon 11, detected in 14 cases (53.8%). It was confirmed by LDI-PCR, that in addition to common breakpoint location in MLL gene (18 out of 27 cases in intron 11, 4 cases in intron 9) allowed to reveal less frequent breakpoint sites, like intron 12 (1 case), intron 10 (3 cases) and intron 7 (1 case). Interestingly, in the last case where LDI-PCR showed presence of MLL-AF4, this fusion gene transcript was not initially found by RT-PCR, because applied primer set did not cover exon 7. Moreover, due to lack of metaphases this patient was primary misclassified as MLL-rearranged, but MLL-AF4-negative. MLL rearrangements were found in 28 AML cases (49.1%). In AML patients the most common MLL rearrangements were MLL-MLLT10 (32% of cases) and MLL-MLLT3 (28%). Other ones were detected less frequently (fig. 1b). In AML patients frequency of MLL rearrangements was similar in children younger and older than 6 months (p=0.904). Among MLL-positive cases AML M5 were detected significantly more often and AML M7 significantly less frequent than in MLL-negative patients (p=0.024 and p=0.001, correspondingly). The most common breakpoint location within MLL gene in AML patients was intron 9, detected in 6 out of 12 cases (50%). Additional chromosomal abnormalities were revealed in 7 out of 21 MLL-positive AML patients with known karyotype (33%), while complex karyotype was detected in 5 cases (24%). Application of LDI-PCR allowed to verify rare MLL rearrangements, including MLL-AFF3 (1 ALL case), MLL-MYO1F (2 AML cases), MLL-SEPT6 (1 AML case), MLL-SEPT9 (1 AML case) In 4 ALL and 3 AML patients MLL rearrangements with concurrent 3'-deletion of MLL gene were found. 3'-deletion of MLL was not associated with breakpoint position in MLL gene and type of translocation partner gene. None of the patients with 3'-deletions had reciprocal fusion gene. Based on LDI-PCR data we assessed several mechanisms of fusion gene formation. Reciprocal translocations were detected in 29 cases, 3-way translocations in 3 cases, inversions in 5 cases, combination of inversion and insertion in 2 cases. Conclusion. In the current study we precisely characterized large cohort of MLL-rearranged infant acute leukemia patients. Combination of all available techniques, including cytogenetics, FISH, RT-PCR and LDI-PCR can lead to detailed verification of every single MLL rearrangement. Disclosures: No relevant conflicts of interest to declare.

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Detection of Rearrangements of Mixed Lineage Leukemia Gene and Its clinical relevance

Blood ◽

10.1182/blood.v124.21.5339.5339 ◽

2014 ◽

Vol 124 (21) ◽

pp. 5339-5339

Author(s):

Weihong Chen ◽

Jiacai Zhuo ◽

Xin Du

Keyword(s):

Acute Leukemia ◽

Clinical Relevance ◽

Fusion Gene ◽

Leukemia Cell ◽

Mixed Lineage Leukemia ◽

The Other ◽

Fusion Genes ◽

Cell Leukemia ◽

Rt Pcr ◽

Mll Gene

Abstract Background: The mixed lineage leukemia (MLL) gene located on chromosome 11 band q23 normally functions as a transcription regulator of the HOX genes and is essential for normal mammalian development and hematopoiesis. Chromosomal translocations involving MLL gene represent frequent cytogenetic abnormalities found in aggressive acute leukemia, both lymphoblastic and myeloid. AL MLL rearrangements have the unique clinical, hematological and prognostic features. We aimed to study the incidence and the types of fusion genes and the clinical relevance. Results: Study samples were from 60 acute leukemia (AL) patients from Sep. 2003 to Dec. 2005. There were 28 males and 32 females. The ages were from 3 months to 54 years old. 17 patients were less than 15 years old and 43 patients were more than 15 years old. All patients were diagnosed based on FAB diagnostic criteria. The patients included 30 with ALL (10 of ALL-L1 patients, 15 of ALL-L2, 5 of ALL-L3), 28 with AML (2 of AML-M1 patients, 5 of AML-M2, 5 of AML-M4, 13 of AML-M5, 1 of AML-M6, 2 of AML-M7), 1 with mixed cell leukemia (AMLL), and 1 with NK cell leukemia. 59 patients were newly diagnosed and 1 patient was refractory. The rearrangements of MLL gene were detected by fluorescence in situ hybridization (FISH) and 6 types of common fusion genes (MLL / AF4, MLL / ENL, MLL / AF9, MLL / ELL MLL / AF6, MLL / AF10) resulting from the rearrangements of MLL gene were detected by nested RT-PCR. There arrangements of MLL gene was found in 7 out of 60 AL patients, (11.67%). Among these 7 patients, 2 were diagnosed with AML- M5 and 5 patients were diagnosed with B-ALL. The fusion genes of the 2 AML-M5 patients who had the rearrangements of MLL gene were MLL/AF9. Among 5 B-ALL patients, 2 patients were confirmed to express MLL/ENL, 1 patient was confirmed to express MLL/AF4, and the other 2 patients did not express the fusion genes. 1 of 2 AML-M5 MLL fusion gene positive patients had invasion of leukemia cell in the left leg and CR was achieved after the first course of chemotherapy. The central nervous system leukemia was got after 1 year and died. The other achieved CR with the third of chemotherapy. 1 of 5 patients died from DIC and the cerebral hemorrhage on the second day after diagnosis B-ALL of MLL fusion gene positive. 2 of 5 patients were not in remission and died from multi-organ failures and infection 3 weeks after diagnosis with positive MLL fusion gene. 1 of 5 patients B-ALL of MLL fusion gene positive got invasion of leukemia cell in thoracic and achieved CR after the second course of chemotherapy. 1 of 5 patients of B-ALL of MLL fusion gene positive achieved CR after the first course of chemotherapy and was relapse 1 year after the bone marrow transplant and then died. Conclusions: We conclude that nested RT-PCR is convenient and feasible method to detect the types of fusion genes resulting from the rearrangements of MLL gene. The clinical features of AL with MLL fusion gene include high white blood cell, invasion of multiple organs, resistant to conventional chemotherapy, easy to relapse after remission, and poor prognosis. The detection of MLL gene rearrangement is of great importance in predicting prognosis and guiding therapy in AL. Disclosures No relevant conflicts of interest to declare.

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