MLL Amplification Research Articles

Genomic Analysis of NPM1 Mutation and KMT2A(MLL)-Rearrangement/Amplification in Japanese Patients with Acute Myeloid Leukemia: Hematologic Malignancies (HM)-Screen-Japan 01

Genomic Analysis of NPM1 Mutation and KMT2A(MLL)-Rearrangement/Amplification in Japanese Patients with Acute Myeloid Leukemia: Hematologic Malignancies (HM)-Screen-Japan 01

Microdeletion in Chromosome 17 Cytoband q21.31 Identified As Located within the KANSL1 Gene Prevailed in Flt3-ITD Negative Normal Karyotype AML Patients As Opposed to Their AML Counterparts and Influenced the Phenotype of the Disease

Recent advances in genetic tools for leukemic diagnosis have facilitated studies on genetic traits which in concert with a driving mutation if present may influence the phenotype of the disease. Flt3-ITD is present in about 30% of AML patients and tends to be associated with a poor prognosis. Currently the use of inhibitors of mutated and in consequence activated kinases is undergoing clinical trials. The dim prognosis of Flt3-ITD positive AML and the hope associated with kinases inhibitors prompted us to evaluate in some depth the genetics of AML cases in relation to the presence of Flt3-ITD .Ninety-two AML patients (diagnosed according to the FAB/WHO criteria, F/M=49/43, age median: 57, range: 21-81 years) who were seen in our institution were recruited to the present study which was conducted as follows:The patients underwent GTG karyotyping and/or FISH for X or Y deletion, inv (3), -5/5q-, -7/7q-,+8, MLL, RUNX1, PML/RARA or RARA, inv(16). 52 lacking GTG and/or FISH abnormalities were called as normal karyotype (NK) patients, among those, 20 were Flt3-ITD positive. The other 40 patients had karyotype abnormalities or were FISH positive for AML-associated abnormalities among which 10 had Flt3-ITD .In all 92 patients microarray analysis was performed using Agilent - Catalog Agilent Cancer CGH+SNP 180K or Roche - WG Catalog NimbleGene 12x270K microarrays.We found (Partec Genomics Suite and Biodiscovery Nexus 8 software) that NK Flt3-ITD negative had, when the whole genome was analyzed, a higher number of total copy number variation (CNV) aberrations (amplifications and/or deletions, vs 32.21±8.70 vs 16.15±2.60, p=0.008) including one copy losses, (2.79±0.57 vs 1.20±0.35 p=0.019)as opposed to NK patients having Flt3-ITD mutation .During the study, an interim analysis was performed at the time we switched from Roche to Agilent microarrays which revealed the significant prevalence of CNVs in AML Flt3-ITD negative patients over Flt3-ITD positive ones within the region 17q21.31 specifically in the positions from 44281092 to 4476549, 44702953 to 44745272 and 44745272 to 44765496. The same region appeared again as significantly discriminating Flt3-ITD negative and positive patients at the final analysis. Therefore, this region was analyzed in some depth. Hg19 annotation was used and according to that the region encompasses the KANSL1 gene among two others (NSFP1 and NSF). The NSF gene encodes a protein involved in transport to the Golgi and NSFP1 is a pseudogene. KANSL1, a component of the MLL1 complex (thought to contribute to leukemogenesis), was the focus of our interest, and the gene was further investigated to validate the microarray-based platform results and to localize the CNV area more precisely. In the region 44281092 to 4476549 within the KANSL1 gene Flt3-ITD negative patients had more deletions and amplifications as compared to those Flt3-ITD positive (31/62 vs 7/30, p=0.012). The same was valid when only deletions were considered (19/62 vs 4/30, p=0.058).Then to validate microarray platform results the same 80 samples were examined using the TaqMan Copy Number Assay (ThermoFisher) for the KANSL1 gene (primers and probe designed for the region 44297111-44297201 lying in chr17), and as a reference the RNaseP gene was employed. The results obtained using the microarray platform correlated well (r=0.671, p<0.001) with those obtained using RT-PCR copy number assay for KANSL1 .61 DNA samples obtained from the patients' solid organ biopsies were examined using the Copy Number Assay for KANSL1 gene CNV. In all instances except one the same CNP was observed in the marrow as that in the solid organ biopsies. It lends credit to the technology employed in this study, but still we do not have enough data to exclude the somatic origin of the microdeletions.The microdeletions are of biologic significance, as suggested by the following findings: 1.All three patients with FISH-documented MLL amplification had deletions within the KANSL1 gene (chr:17 44281092 to 4476549).2.Microdeletions within the KANSL1 gene were more frequently observed in the group M0-M2 patients than in other subtypes of AML (18/51 vs 5/35, p=0.026).In conclusion, a lack of Flt3-ITD and normal karyotype was associated with the frequent presence of microdeletions within the chr17 cytoband q21.31. The latter was identified as (i) present within the KANSL1 gene and (ii) influencing the phenotype of AML.Supported by the INNOMED/I/1/NCBR/2014 DisclosuresNo relevant conflicts of interest to declare.

Clinical implications of clonal chromosomal abnormalities in Philadelphia negative cells in CML patients after treated with tyrosine kinase inhibitors

Emergence of clonal chromosomal abnormalities in Philadelphia chromosome-negative (CCA/Ph-) cells in chronic myeloid leukemia (CML) patients during the treatment with tyrosine kinase inhibitors (TKIs) is an interesting phenomenon. Although previous studies revealed some potential impact of CCA/Ph- on CML patients’ outcome, clinical significance of CCA/Ph- in CML patients remains to be further elucidated. We retrospectively reviewed the patients with CML evaluated at Genoptix Medical Laboratory in Carlsbad, California from 2005 to 2015. Twenty-four CML patients with CCA/Ph- cells were identified. These include 18 patients with single chromosomal abnormality, 4 patients with double chromosomal abnormalities, and two patients with complex cytogenetic abnormalities. In addition to trisomy 8 and monosomy 7, we identified that 20q- was also a common abnormality in CCA/Ph- cells. Most of the patients with CCA/Ph- cells demonstrated no significant dysplasia or increased blasts with two exceptions: one patient with persistent 7q- exhibiting mild dysmegakaryopoiesis, suggestive of an early evolving myelodysplastic syndrome, and another patient with complex cytogenetic abnormalities who developed acute myeloid leukemia after gained MLL amplification. One patient with complex cytogenetic abnormalities showed optimal response to TKI treatment, no overt dysplasia, and no disease progression during almost 4-years of follow-up. More interestingly, FISH tests could identify more cases with double chromosomal abnormalities and these cases showed suboptimal responses to TKI treatments. Our observation indicates that 20q- was also a common abnormality in CCA/Ph- cells, further FISH tests revealed additional CCA/Ph-, and the majority of CML patients with two or more chromosomal abnormalities in Ph- cells showed inferior response to TKI treatments. The results of our study suggest that CML cases with CCA/Ph- may represent a group of patients with heterogeneous genetic alterations.

Double minute chromosomes in acute myeloid leukemia, myelodysplastic syndromes, and chronic myelomonocytic leukemia are associated with micronuclei, MYC or MLL amplification, and complex karyotype

Double minute chromosomes (dmin) are small, paired chromatin bodies that lack a centromere and represent a form of extrachromosomal gene amplification. Dmin are rare in myeloid neoplasms and are generally associated with a poor prognosis. Most studies of dmin in myeloid neoplasms are case reports or small series. In the current study, we present the clinicopathologic and cytogenetic features of 22 patients with myeloid neoplasms harboring dmin. These neoplasms included acute myeloid leukemia (AML) (n = 18), myelodysplastic syndrome (MDS) (n = 3), and chronic myelomonocytic leukemia (CMML) (n = 1). The AML cases consisted of AML with myelodysplasia-related changes (n = 13) and therapy-related AML (n = 5). Dmin were detected in initial pre-therapy samples in 14 patients with AML or CMML; they were acquired during the disease course in 8 patients who had AML or MDS. The presence of dmin was associated with micronuclei (18/18; 100%), complex karyotype (17/22; 77.3%), and amplification of MYC (12/16; 75%) or MLL (4/16; 25%). Immunohistochemical staining for MYC performed on bone marrow core biopsy or clot sections revealed increased MYC protein in all 19 cases tested. Except for one patient, most patients failed to respond to risk-adapted chemotherapies. At last follow up, all patients had died of disease after a median of 5 months following dmin detection. In conclusion, dmin in myeloid neoplasms commonly harbor MYC or MLL gene amplification and manifest as micronuclei within leukemic blasts. Dmin are often associated with myelodysplasia or therapy-related disease, and complex karyotypes.

Is amplification of c-MYC, MLL and RUNX1 genes in AML and MDS patients with trisomy 8, 11 and 21 a factor for a clonal evolution in the karyotype?

The aim of our study was 1) to define if the amplification of c-MYC, MLL and RUNX1 genes is related to the progressive changes of the karyotype in patients with AML and MDS with trisomy 8, 11 and 21 (+8, +11 and +21) in bone marrow and 2) can that amplification be accepted as part of the clonal evolution (CE). Karyotype analysis was performed in 179 patients with AML or MDS with the different chromosomal aberrations (CA) aged 16-81. The findings were distributed as follow: initiating balanced CA (n = 60), aneuploidia (n = 55), unbalanced CA (n = 64). Amplification of c-MYC, MLL and RUNX1 genes by means of fluorescence in situ hybridization (FISH) was found in 35% (7 out of 20) of AML and MDS patients with +8, +11 u +21 as single CA in their karyotype; in 63.6% of pts (7 out of 11)--with additional numerical or structural CA and in 75% (9 out of 12)--with complex karyotype. We assume that the amplification of the respective chromosomal regions in patients with +8, +11 and +21 is related to CE. Considering the amplification as a factor of CE, we established 3 patterns of karyotype development depending on the type of the initiating CA in it. Significant statistical differences were found between the three patterns regarding the karyotype distribution in the different stages of progression (p < 0.001).

MLL gene amplification in acute myeloid leukemia and myelodysplastic syndromes is associated with characteristic clinicopathological findings and TP53 gene mutation.

MLL gene rearrangements are well-recognized aberrations in acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS). In contrast, MLL gene amplification in AML/MDS remains poorly characterized. Here, we report a series of 21 patients with myeloid neoplasms associated with MLL gene amplification from 1 institution. This series included 13 men and 8 women, with a median age of 64 years. Eleven patients presented as AML with myelodysplasia-related changes, 6 as therapy-related AML, and 4 as therapy-related MDS. All patients had a highly complex karyotype, including frequent -5/del(5q), -18, and -17/del(17p) abnormalities; 16 patients were hypodiploid. TP53 mutations were detected in all 12 patients tested, and 3 patients showed TP53 mutation before MLL amplification. Morphologically, the leukemic cells frequently showed cytoplasmic vacuoles, bilobed nuclei, and were associated with background dyspoiesis. Immunophenotypically, 15 patients had a myeloid and 4 had myelomonocytic immunophenotype. Laboratory coagulopathies were common; 7 patients developed disseminated intravascular coagulopathy, and 3 died of intracranial bleeding. All patients were refractory to therapy; the median overall survival was 1 month, after MLL gene amplification was detected. We concluded that AML/MDS with MLL gene amplification is likely a subset of therapy-related AML/MDS or AML with myelodysplasia-related changes, associated with distinct clinicopathological features, frequent disseminated intravascular coagulopathy, a highly complex karyotype, TP53 deletion/mutation, and an aggressive clinical course.

Mixed lineage leukemia protein in normal and leukemic stem cells.

Transcription factors critical for normal hematopoietic stem cell functions are frequently mutated in acute leukemia leading to an aberrant re-programming of normal hematopoietic progenitor/stem cells into leukemic stem cells. Among them, re-arrangements of the mixed lineage leukemia gene (MLL), including chimeric fusion, partial tandem duplication (PTD), amplification and internal exonic deletion, represent one of the most common recurring oncogenic events and associate with very poor prognosis in human leukemias. Extensive research on wild type MLL and MLL-fusions has significant advanced our knowledge about their functions in normal and malignant hematopoiesis, which also provides a framework for the underlying pathogenic role of MLL re-arrangements in human leukemias. In contrast, research progress on MLL-PTD, MLL amplification and internal exonic deletion remains stagnant, in particular for the last two abnormalities where mouse model is not yet available. In this article, we will review the key features of both wild-type and re-arranged MLL proteins with particular focuses on MLL-PTD and MLL amplification for their roles in normal and malignant hematopoiesis.

Therapy-related pro-B cell acute lymphoblastic leukemia: report of two patients with MLL amplification

Improvements in chemotherapy and medical support of patients treated with chemotherapy and radiation have led to an ever-increasing number of cancer survivors. Unfortunately, a small fraction of these patients develop secondary hematologic malignancies as a consequence of their exposure to genotoxic anti-cancer regimens. Most of these are myeloid malignancies, therapy-related acute myeloid leukemia (t-AML) or myelodysplasia (t-MDS); however, a small but growing body of literature exists, which describes therapy-related acute lymphoblastic leukemias (t-ALL). Nearly all these cases are reportedly associated with translocations involving chromosome 11q23, the site of the MLL gene. We herein report two cases of ALL occurring after chemotherapy for other malignancies that showed complex karyotypic abnormalities and distinct MLL amplification by fluorescence in situ hybridization analysis. Immunophenotypic analysis showed that both cases expressed a pro-B cell (CD10-) phenotype with aberrant myeloid antigen expression. Although MLL amplification has been reported in therapy-related myeloid disease, to our knowledge this is the first report of MLL amplification occurring in therapy-related B cell ALL.

Six Cases of Rare Gene Amplifications and Multiple Copy of Fusion Gene in Childhood Acute Lymphoblastic Leukemia

Cytogenetic aberrations are very important factors in risk assessment of childhood hematological malignancies. We report six childhood acute lymphoid leukemia (ALL) cases with rare cytogenetic aberrations: five with RUNX1, ABL1 or MLL proto-oncogene amplification and one case of multiple copies of ETV6/RUNX1 fusion genes. The simultaneous presence of two adverse genetic aberrations is of special interest: ETV6-RUNX1 fusion gene is associated with good prognosis and intrachromosomal amplification of the homologue RUNX1 gene is associated with poor prognosis. We also report a patient with MLL amplification, a unique finding in childhood T-ALL. Report of these subtle rearrangements contributes to our understanding of diagnostic and prognostic significance of these rare cytogenetic abnormalities.

Therapy-Related Pro-B Cell Acute Lymphoblastic Leukemia: Report of Two Patients with MLL Amplification

Abstract 4911Recent advances in cancer treatment have led to an ever increasing number of cancer survivors. Unfortunately, a small fraction of these patients develop secondary hematologic malignancies as a consequence of their exposure to genotoxic anti-cancer regimens. Most of these are myeloid malignancies, therapy-related acute myeloid leukemia (t-AML) or myelodysplasia (t-MDS). Current classification schemes separate therapy-related myeloid disorders into two classes, those related to prior exposure to alkylating agents or radiation and those with prior exposure to DNA topoisomerase II inhibitors. The alkylating/radiotherapy group often contain chromosomal abnormalities involving chromosomes 5 and 7. In contrast, those with DNA topoisomerase II inhibitor exposure frequently possess chromosomal translocations involving chromosome 11q23, where the MLL gene locus resides. MLL, or “mixed lineage leukemia”, is also frequently rearranged in lymphoblastic leukemias and mixed phenotype acute leukemias. Interestingly, MLL also can be altered in myeloid disease through partial tandem duplication or amplification of the genomic region encompassing MLL [amp(MLL)]. Amp(MLL) of an unrearranged MLL locus has also been described in therapy-related myeloid disease.Recently, there has been a growing appreciation that therapy-related leukemias are not limited to the myeloid lineage and that therapy-related acute lymphoblastic leukemias (t-ALL) make up a small but significant percentage of therapy-related acute leukemias. Translocations involving the MLL locus have been found in a high percentage of t-ALLs. However, to our knowledge, amp(MLL) has not been reported previously in t-ALL. Herein we report 2 cases of ALL following chemotherapy for other malignancies that showed complex karyotypic abnormalities and distinct MLL amplification by FISH analysis.Patient 1 is an 80 year old male with a past medical history significant for a stage IV diffuse large B cell lymphoma two years prior. The patient received 8 cycles of R-CHOP chemotherapy and achieved a complete remission. One year later, he had a recurrence and was treated with 7 cycles of RICE chemotherapy. Eleven months later, he presented with leukopenia. A bone marrow examination revealed involvement by B cell ALL. Cytogenetic analysis showed a hypodiploid complex karyotype which included a homogeneously staining region (hsr) at chr11(q23). Metaphase FISH analysis showed amp(MLL) in the hsr.Patient 2 is a 62 year old male who five years prior had been diagnosed with a pleiomorphic sarcoma of the left hip. The patient received four cycles of doxorubicin and ifosfamide as well as radiation therapy. The patient remained well for five years but then presented with bleeding and a petechial rash. A complete blood count revealed anemia and thrombocytopenia. A bone marrow examination was performed which showed involvement by B cell ALL. Cytogenetic analysis showed multiple numerical and structural abnormalities including an hsr at chr11(q23) in all lines as well as a second hsr in two sidelines. Metaphase FISH analysis showed amp(MLL) in all cells and in both hsrs. Immunophenotypic analysis showed both cases to express a Pro-B cell (CD10-) phenotype with aberrant myeloid antigen expression.Frequent molecular perturbation makes the MLL locus one of the most common genetic targets in hematologic malignancies, particular in therapy-related neoplasms. The majority of MLL genetic alterations result in translocations involving the MLL gene. These translocations result in fusion proteins with heterologous functions of MLL that result in a block of differentiation. Similarly, amp(MLL), while not creating an abnormal fusion protein involving MLL, also is considered to be a “gain of function” mutation caused by increased expression of MLL. There seems to be an increased frequency of aml(MLL) in therapy-related myeloid disorders.In summary, we report, to our knowledge, the first cases of therapy-related pro B cell (CD10-) ALL associated with amp(MLL). The shared phenotype between B cell ALLs with MLL rearrangements and amp(MLL) suggests that both lesions may exert similar influences on the transcriptional programs of these leukemias. This observation extends the family of MLL-associated and therapy-related hematologic disorders. Disclosures:No relevant conflicts of interest to declare.

Oncogene Amplification as an Incidental Finding in FISH Testing for Gene Rearrangements in Lymphoid Hematopoietic Neoplasms

Abstract 2505Oncogene amplification resulting in overexpression, although common in solid tumors, is rare in hematopoietic neoplasms. This is particularly true in lymphoid neoplasms compared to AML where MYC, MLL or RUNX1 (AML1) amplification has been mostly seen, and to CML where BCR/ABL fusion gene amplification has been also reported. Typically, lymphoid neoplasms are tested at diagnosis by FISH for specific reciprocal chromosome translocations that lead to overexpression of deregulated oncogenes such as BCL1, BCL2, BCL6 and MYC in B-cell lymphoma and myeloma or BCR/ABL gene fusion in ALL. Nevertheless, we have unexpectedly seen oncogene amplification from time to time in our FISH lab. To study the incidence of gene amplification and its diagnostic and clinical implications, we retrospectively analyzed FISH results routinely performed on paraffin embedded lymphoma tissues, lymph nodes, bone marrow aspirates and peripheral bloods in the past three and half years using translocation probes for BCL2/IGH, BCL1/IGH, MYC/IGH and BCR/ABL and break apart probes for BCL6 and MYC. The highest amplification rate seen was in BCL2/IGH testing: 11 of the 1,710 cases were positive (0.643%). In 2 cases with follicular lymphoma (FL), BCL2 amplification presented as homogenously staining regions (hsr), one case with diffuse large B-cell lymphoma (DLBCL) showed double minutes (dmin), and two cases with FL had a pattern of combined hsr and dmin. Five cases with low grade FL intriguingly showed a similar pattern of BCL2/IGH translocation and concomitant amplification of the rearranged BCL2 as a small hsr. The remaining case diagnosed as Burkitt lymphoma (BL) was positive for MYC/IGH translocation and for BCL2 amplification present as large hsr. BCL6 amplification was observed in 4 of 1,537 cases tested (0.26%). In all these cases, amplification presented as hsr. One case diagnosed as EBV+ DLBLC was positive for MYC/IGH translocation and 3’ BCL6 high level amplification. Another case with FL showed BCL6 rearrangement and 5’ BCL6 amplification. BCL1 amplification with translocation was seen as hsr in 1 case with mantle cell lymphoma out of 2,898 tested. BCL1 amplification was observed in another case with plasma cell myeloma as hsr out of 3,413 tests performed. MYC gene amplification was positive in 3 of 2,186 (0.137%). One case with BL showed MYC rearrangement with a concomitant 3’ deletion and 5’ amplification. The second case diagnosed as B-cell lymphoma with features intermediate between DLBCL and BL showed highly amplified MYC gene as hsr. The third case was DLBCL with 15–50 copies of MYC gene per cell as dmin. Among 530 BCR/ABL tests ordered for acute leukemia or ALL, 2 (0.377%) cases with T cell lymphoblastic leukemia/lymphoma (T-ALL/LBL) showed ABL amplification as episomes (4–8 copies in one case; 6–30 in another). Both were abnormal by cytogenetics analysis, but negative for t(9;22)(q34;q11.2) and without dmin or hsr identified. Although detection rates of oncogene amplification seem to be very low with limited specific translocation probes applied to lymphoid neoplasms, they may well be higher when FISH signal patterns are analyzed more carefully. In our study, 5 of the 11 FL cases tested for IGH/BCL2 showed a peculiar pattern positive for the t(14;18)-IGH/BCL2 and a concomitant red signal amplification of BCL2, the size of which is usually small and may be overlooked under microscope. All these 5 cases had low grade FL, suggesting that oncogene amplification can be an early genomic event in lymphomagenesis. MYC gene amplification is generally considered a late stage genomic alteration. When MYC is rearranged, amplification of BCL2, BCL6 or BCL1 may need to be taken into consideration for disease stratification, or vice versa. For instance, so called “double-hit” or “triple-hit” lymphomas currently require two or three concurrent translocations for diagnosis. Oncogene amplification, equally important for deregulation/overexpression as a translocation, should be considered as a second or a third hit in diagnosis and differential diagnosis of B-cell lymphoma unclassifiable with features intermediate between DLBCL and BL. FISH is a very useful tool to identify episomal oncogene amplification. Episomes, unlike cytogenetically evident dmin and hrs, are invisible by chromosome analysis, but their detection is important as reported for integration of targeted tyrosine kinase inhibitors into chemotherapeutic regimens. Disclosures:No relevant conflicts of interest to declare.

MLL amplification in leukemia

The exact incidence of MLL amplification in myeloid malignancies is difficult to establish. In large studies, MLL amplification has been found in < 1% of AML/MDS with abnormal karyotype. Amplification of MLL gene is the second most amplified gene in AML/MDS after CMYC oncogene. In one study, MLL amplification was found in 8/27 (29%) AML cases exhibiting homogenously staining region (hsr) and double minutes (dmin). AML/MDS with MLL amplification is associated with an elderly age, a median age at presentation of 72 years, ranged from 4 to 91 years, often prior history of therapy with alkylating agents or topoisomerase II, and slight female predominance. In therapy-related MDS/AML amplification of MLL gene was found in 12% patients.

Molekularne i cytogenetyczne czynniki prognostyczne w ostrej białaczce szpikowej (OBS)

Acute myeloid leukemia (AML) constitutes a group of diseases that are very heterogeneous with regard to clinical course, response to therapy as well as cytogenetic aberrations and gene mutations. Such lesions are of prognostic value. Patients with t(8;21), inv(16)/t(16;16) or t(15;17) have a favorable prognosis. Patients with normal karyotype and aberrations +6, +8 –Y, t(9;11) and del(12p) are classified in the group of intermediate prognosis. In the case of patients with complex karyotype or with the aberrations inv(3)/t(3;3), t(6;9), –5, –7, del(5q), del(7q) and 11q23 rearrangements, the prognosis is poor. Unfavorable molecular factors include C-MYC amplification, MLL amplification and rearrangement, FLT3-ITD, WT1 mutation and overexpression of BAALC, ERG or MN1. The changes in miRNA expression may also be important for AML prognosis. That is why the cases with normal karyotype (CN-AML) and cases with complex aberrations remain to be better characterized at the genetic level. Array technology enables the analysis of genomic DNA and gene expression. This approach may be used in the search for new prognostic and predictive markers in AML.

Gene amplification in myeloid leukemias elucidated by fluorescence in situ hybridization

Gene amplification in hematologic malignancies is uncommon. When karyotyping leukemia cells, gene amplification is generally seen as double-minute (dmin) chromosomes and homogeneously staining regions (hsr). One of the more commonly amplified regions is MYC at 8q24.21, but amplification of MLL at 11q23 and regions on 9p, 19q, and elsewhere on 11q have been reported. Increased copy number of these genes has been associated with poor prognosis. Over an 11-year period, we identified 31 cases of possible gene amplification, 27 of which had enough sample material for further investigations. A total of 17 cases had dmin only, 13 cases had hsr only, and 1 case had both dmin and hsr in the karyotype. Fluorescence in situ hybridization (FISH) analysis identified amplification of MYC in 12 cases, all on dmin, and amplification of MLL in eight cases, all on hsr. Regions other than MYC and MLL were amplified in eight cases and, using multicolor FISH and multicolor banding, we identified a number of novel regions of amplification: 13q11∼q12.1, 15q26.1∼q26.3, and 17q12. We also identified one case where two different chromosomal regions were simultaneously amplified in the same cell line.

Association of MLL amplification with poor outcome in acute myeloid leukemia

Chromosomal rearrangements and amplification of the MLL gene at 11q23 are common abnormalities found in patients with severe myelodysplastic disorders and lymphoid and acute myeloid leukemias. MLL rearrangements are associated with aggressive disease in both children and adults, with current evidence suggesting that MLL alterations are associated with a poor prognosis. We report the clinical, cytogenetic and histologic findings of a patient who presented with a de novo diagnosis of AML-M4 and who fits the profile of patients presenting with MLL alterations, such as old age at presentation, rapid progression, therapeutic refractoriness, and poor outcome. Two bone marrow specimens taken 1 month apart show the rapid deterioration of the patient's cytogenetic abnormalities at the 11q23 locus, with amplification of MLL that was originally seen as a homogeneously staining region (hsr) on chromosome 11. In the second biopsy the hsr and MLL amplification appears as nonreciprocal translocation of multiple copies in the form of marked amplification of MLL on chromosome 16 in a background of increasing chromosomal aberrations. This case suggests that either the MLL amplification and translocation alone or in conjunction with other flanking oncogenes may have played an important role in poor patient outcome.

AML/MDS with 11q/MLL amplification show characteristic gene expression signature and interplay of DNA copy number changes

AML/MDS patients carrying 11q amplifications involving the mixed lineage leukemia gene (MLL) locus are characterized by a complex aberrant karyotype (CAK) frequently including deletions within 5q, 17p, and 7q, older age and fast progression of the disease with extremely poor prognosis. MLL has been shown to be overexpressed in cases with 11q amplification. However, in most of the cases, the amplified region is not restricted to the MLL locus. In this study, we investigated 19 patients with AML/MDS and MLL gain/amplification. By means of array CGH performed in 12 patients, we were able to delineate the minimal deleted regions within 5q and 17p and identified three independent regions 11q/I-III that were amplified in all cases. Gene expression profiles established in 15 cases were used to identify candidate genes within these regions. Notably, analysis of our data suggests a correlation of loss of 5q and 17p and expression of genes present in 11q23-25. Furthermore, we demonstrate that the gene expression signature can be used to discriminate AML/MDS with MLL amplification from several other types of AML.

A Case of Acute Myeloid Leukemia with Maturation (AML-M2) with C-MYC Amplification in Homogenous Staining Region (hsr) and Double Minutes (dmin)

Homogeneous staining region (hsr) and double minutes (dmin), cytogenetic indicators of gene amplification, are rare findings in hematologic malignancies, and most frequently associated with acute myeloid leukemias (AML). Herein, we report a 74-year-old female presenting AML with maturation (AML-M2) with c-MYC amplification in hsr and dmin. Conventional cytogenetic techniques showed 46, XX, hsr(4)(q25)[15]/46, XX,~20dmin[5]. The c-MYC amplification in hsr was suspected by multicolor fluorescence in situ hybridization (mFISH) using 24XCyte Human mFISH Painting Kit. Amplification of c-MYC in hsr and dmin were confirmed by fluorescence in situ hybridization (FISH) using LSI MYC(8q24) Dual color, Break Apart Rearrangement Probe. The presentation of both dmin and hsr was rare in AML cases. Majority of AML cases with c-MYC or MLL amplification in dmin or hsr were women older than 50 years, had de novo AML with complex karyotypes and had a short survival. But, this case had no chromosomal aberration besides c-MYC amplification and showed a good response to chemotherapy with a complete remission for 21 months.

MLL amplification in acute myeloid leukemia

The chromosomal alterations at 11q23 that involve the mixed-lineage leukemia gene ( MLL, HTRX1, HRX, ALL1) are one of the most common cytogenetic abnormalities in acute leukemia and have been associated with a poor prognosis. Given that not all MLL alterations are seen under conventional cytogenetics or fluorescence in situ hybridization (FISH), it is very important to use molecular techniques to determine the cause of alteration. In this study, we describe two cases of AML in which FISH analysis showed a high-level 11q23 amplification, to confirm if this overexpression may be accompanied by partial tandem duplication of the MLL gene ( MLL-PTD). Both patients showed complex karyotype and an unfavorable clinical course. The 11q23 region characterization included conventional cytogenetics, FISH, and comparative genomic hybridization analysis to study the expression patterns of several oncogenes located within the amplified region and detection of partial tandem duplication of the MLL gene by reverse-transcription polymerase chain reaction (RT-PCR) and sequencing. MLL-PTD were detected in the two patients. Moreover, patient 1 showed amplification of the MLL flanking region. Our data suggest that molecular methods such as RT-PCR or sequencing should be used to detect MLL alterations, and that amplification of MLL locus may be extended to its flanking region.

Analysis of MLL Amplification and 5q Deletions in AML/MDS Cell Lines Identifies Novel Targets.

Genomic amplifications of the 11q23 region occur in acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) where MLL and a few neighboring genes, notably DDX6, are deemed salient targets. However, the extent to which amp(lified)-MLL and translocated MLL share effector targets remains to be established. Even less is known about the target(s) of deletions affecting the long arm of chromosome 5 (5q-) which reportedly partner amp-MLL. We analyzed three AML/MDS cell lines by cytogenetics (conventional and FISH) in parallel with real time q(uantitative)-PCR at both 11q23 and 5q2 to measure copy number and expression of salient target genes together with putative downstream targets. The cell lines comprised: MOLM-17 (transforming-MDS), SAML-2 (therapy-related AML), and UOC-M1 (AML-M1). All three cell lines exhibited approximately four-fold genomic amplification of 11q23 including MLL and DDX6, while the amplicon extended telomerically to include FLI1 (11q24) and HNT (11q25) in MOLM-17 and UOC-M1 only. Expression, quantified relative to AML/MDS cell lines without MLL rearrangement, revealed that of the genomically amplified genes only MLL was generally overexpressed, namely by 9.5x (MOLM-17), 5.1x (UOC-M1), and 4.6x (SAML-2). In addition to the highest MLL expression, in MOLM-17 FLI1 (3.8x) and DDX6 (2.8x) were significantly upregulated. Expression was also quantified among reputed MLL target genes, and showed that in the three cell lines MEIS1 was upregulated in MOLM-17 only (by 6x), and CDKN2C in all cell lines (by about 2x), while HOXA9 and CDKN1B showed near-normal levels of expression. All three cell lines carried 5q- with a common deleted region at 5q31 extending from 134.2–137.5 Mbp. Of a panel of genes recently identified as 5q- deletion targets (centromere-TIGA1, CAMLG, C5orf15, C5orf14, BRD8, HARS, KIAA0141, CSNK1A1, RBM22-telomere), only C5orf15 (function unknown) and BRD8 (a component of the nua4 histone acetyltransferase complex involved in transcriptional activation) were generally downregulated - to about 0.25x, and about 0.4x normalized expression levels, respectively. Both genes lie within the common deleted region. In summary, we have characterized amp-MLL and 5q- in MOLM-17, the first MDS cell line to be described with these rearrangements, together with two AML cell lines with similar cytogenetic profiles. Our data suggest that MLL is the only clear object of 11q23 amplification hitherto identified and CDKN2C its sole unequivocal target in AML/MDS cell lines. It is possible that MEIS1 is also targeted for activation in specific cell types or disease phases in MDS. These findings also highlight C5orf15 and/or BRD8 as possible leukemogenic accomplices targeted for downregulation in accompanying 5q-. These findings may point to differences in signalling pathways targeted by amp-MLL in AML and MDS.

Del(5q) and MLL amplification in homogeneously staining region in acute myeloblastic leukemia: a recurrent cytogenetic association

We report here a 71 year-old female presenting with acute myeloblastic leukemia (FAB-M1) after treatment of essential thrombocythemia with Vercyte. Conventional cytogenetic techniques showed a complex karyotype, 44,XX,-5,-7,-11,add(11)(q23),-14,+mar,+r. The use of several fluorescent in situ hybridizations (FISH) lead to the identification of these complex rearrangements. The marker was found to be tricentric, with pericentromeric material of chromosome 7 inserted in the short arm of chromosome 5, resulting in monosomy 5q and 7q. The derivative chromosome 11 was dicentric and had subtelomeric sequences of 11p on both ends; several copies of the MLL gene were located in two different regions separated by a centromere of chromosome 11. Twenty-one cases, including ours, of myelodysplastic syndromes and acute myelogenous leukemia with MLL amplification present in hsr or dmin were found in the literature. Most of these patients shared some characteristics: they were old, they had de novo acute myeloid leukemia (AML) with a complex karyotype and a short survival, 90% of them having also a del(5q). Therefore, the simultaneous presence of MLL amplification and del(5q) appears to be a nonrandom association that could be the signature of AML in elderly patients with a poor prognosis.