The mutational oncoprint of recurrent cytogenetic abnormalities in adult patients with de novo acute myeloid leukemia.

Recurrent chromosomal abnormalities and gene mutations detected at the time of diagnosis of acute myeloid leukemia (AML) are associated with particular disease features, treatment response and survival of AML patients, and are used to denote specific disease entities in the World Health Organization classification of myeloid neoplasms and acute leukemia. However, large studies that integrate cytogenetic and comprehensive mutational information are scarce. We created a comprehensive oncoprint of mutations associated with recurrent cytogenetic findings by combining the information on mutational patterns of 80 cancer- and leukemia-associated genes with cytogenetic findings in 1603 adult patients with de novo AML. We show unique differences in the mutational profiles among major cytogenetic subsets, identify novel associations between recurrent cytogenetic abnormalities and both specific gene mutations and gene functional groups, and reveal differences in cytogenetic and mutational features between patients younger than 60 years and those aged 60 years or older. The identified associations between cytogenetic and molecular genetic data may help guide mutation testing in AML, and result in more focused application of targeted therapy in patients with de novo AML.

Mutations in the CCND1 and CCND2 genes are frequent events in adult patients with t(8;21)(q22;q22) acute myeloid leukemia.

Core-binding factor acute myeloid leukemia (CBF-AML) is defined by the presence of either t(8;21)(q22;q22)/RUNX1-RUNX1T1 or inv(16)(p13.1q22)/t(16;16)(p13.1;q22)/CBFB-MYH11. The resulting fusion genes require a 'second hit' to initiate leukemogenesis. Mutation assessment of 177 adults with CBF-AML, including 68 with t(8;21) and 109 with inv(16)/t(16;16), identified not only mutations well known in CBF-AML but also mutations in the CCND1 and CCND2 genes, which represent novel frequent molecular alterations in AML with t(8;21). Altogether, CCND1 (n=2) and CCND2 (n=8) mutations were detected in 10 (15%) patients with t(8;21) in our cohort. A single CCND2 mutation was also found in 1 (0.9%) patient with inv(16). In contrast, CCND1 and CCND2 mutations were detected in only 11 (0.77%) of 1426 non-CBF-AML patients. All CCND2 mutations cluster around the highly conserved amino-acid residue threonine 280 (Thr280). We show that Thr280Ala-mutated CCND2 leads to increased phosphorylation of the retinoblastoma protein, thereby causing significant cell cycle changes and increased proliferation of AML cell lines. The identification of CCND1 and CCND2 mutations as frequent mutational events in t(8;21) AML may provide further justification for cell cycle-directed therapy in this disease.

Maintenance therapy with decitabine in younger adults with acute myeloid leukemia in first remission: a phase 2 Cancer and Leukemia Group B Study (CALGB 10503).

In this prospective phase 2 clinical trial conducted by Cancer and Leukemia Group B (CALGB, now the Alliance), we studied decitabine as maintenance therapy for younger adults with acute myeloid leukemia (AML) who remained in first complete remission (CR1) following intensive induction and consolidation. Given that decitabine is clinically active in AML and with hypomethylating activity distinct from cytotoxic chemotherapy, we hypothesized that 1 year of maintenance therapy would improve disease-free survival (DFS) for AML patients <60 years, who did not receive allogeneic stem cell transplantation in CR1. After blood count recovery from final consolidation, patients received decitabine at 20 mg/m2 intravenously daily for 4-5 days, every 6 weeks for eight cycles. One hundred and thirty-four patients received decitabine and 85 (63%) had favorable risk AML. The median number of cycles received was 7 (range: 1-8) and the primary reason for discontinuation was relapse. DFS at 1 year and 3 years was 79% and 54%, respectively. These results are similar to the outcomes in the historical control comprising similar patients treated on recent CALGB trials. Thus, maintenance with decitabine provided no benefit overall. Standard use of decitabine maintenance in younger AML patients in CR1 is not warranted. This trial was registered at www.clinicaltrials.gov as NCT00416598.

Prognostic and biologic significance of DNMT3B expression in older patients with cytogenetically normal primary acute myeloid leukemia.

DNMT3B encodes a DNA methyltransferase implicated in aberrant epigenetic changes contributing to leukemogenesis. We tested whether DNMT3B expression, measured by NanoString nCounter assay, associates with outcome, gene and microRNA expression and DNA methylation profiles in 210 older (⩾60 years) adults with primary, cytogenetically normal acute myeloid leukemia (CN-AML). Patients were dichotomized into high versus low expressers using median cut. Outcomes were assessed in the context of known CN-AML prognosticators. Gene and microRNA expression, and DNA methylation profiles were analyzed using microarrays and MethylCap-sequencing, respectively. High DNMT3B expressers had fewer complete remissions (CR; P=0.002) and shorter disease-free (DFS; P=0.02) and overall (OS; P<0.001) survival. In multivariable analyses, high DNMT3B expression remained an independent predictor of lower CR rates (P=0.04) and shorter DFS (P=0.04) and OS (P=0.001). High DNMT3B expression associated with a gene expression profile comprising 363 genes involved in differentiation, proliferation and survival pathways, but with only four differentially expressed microRNAs (miR-133b, miR-148a, miR-122, miR-409-3p) and no differential DNA methylation regions. We conclude that high DNMT3B expression independently associates with adverse outcome in older CN-AML patients. Gene expression analyses suggest that DNMT3B is involved in the modulation of several genes, although the regulatory mechanisms remain to be investigated to devise therapeutic approaches specific for these patients.

A stem cell-like gene expression signature associates with inferior outcomes and a distinct microRNA expression profile in adults with primary cytogenetically normal acute myeloid leukemia.

Acute myeloid leukemia (AML) is hypothesized to be sustained by self-renewing leukemia stem cells (LSCs). Recently, gene expression signatures (GES) from functionally defined AML LSC populations were reported, and expression of a 'core enriched' (CE) GES, representing 44 genes activated in LCSs, conferred shorter survival in cytogenetically normal (CN) AML. The prognostic impact of the CE GES in the context of other molecular markers, including gene mutations and microRNA (miR) expression alterations, is unknown and its clinical utility is unclear. We studied associations of the CE GES with known molecular prognosticators, miR expression profiles, and outcomes in 364 well-characterized CN-AML patients. A high CE score (CE(high)) associated with FLT3-internal tandem duplication, WT1 and RUNX1 mutations, wild-type CEBPA and TET2, and high ERG, BAALC and miR-155 expression. CE(high) patients had a lower complete remission (CR) rate (P=0.003) and shorter disease-free (DFS, P<0.001) and overall survival (OS, P<0.001) than CE(low) patients. These associations persisted in multivariable analyses adjusting for other prognosticators (CR, P=0.02; DFS, P<0.001; and OS, P<0.001). CE(high) status was accompanied by a characteristic miR expression signature. Fifteen miRs were upregulated in both younger and older CE(high) patients, including miRs relevant for stem cell function. Our results support the clinical relevance of LSCs and improve risk stratification in AML.

Increased anti-leukemic activity of decitabine via AR-42-induced upregulation of miR-29b: a novel epigenetic-targeting approach in acute myeloid leukemia.

Histone deacetylase (HDAC) inhibitors either alone or in combination with hypomethylating agents have limited clinical effect in acute myeloid leukemia (AML). Previously, we demonstrated that AML patients with higher miR (microRNA)-29b expression had better response to the hypomethylating agent decitabine. Therefore, an increase in miR-29b expression preceding decitabine treatment may provide a therapeutic advantage. We previously showed that miR-29b expression is suppressed by a repressor complex that includes HDACs. Thus, HDAC inhibition may increase miR-29b expression. We hypothesized that priming AML cells with the novel HDAC inhibitor (HDACI) AR-42 would result in increased response to decitabine treatment via upregulation of miR-29b. Here, we show that AR-42 is a potent HDACI in AML, increasing miR-29b levels and leading to downregulation of known miR-29b targets (that is, SP1, DNMT1, DNMT3A and DNMT3B). We then demonstrated that the sequential administration of AR-42 followed by decitabine resulted in a stronger anti-leukemic activity in vitro and in vivo than decitabine followed by AR-42 or either drug alone. These preclinical results with AR-42 priming before decitabine administration represent a promising, novel treatment approach and a paradigm shift with regard to the combination of epigenetic-targeting compounds in AML, where decitabine has been traditionally given before HDACIs.

Escalation of daunorubicin and addition of etoposide in the ADE regimen in acute myeloid leukemia patients aged 60 years and older: Cancer and Leukemia Group B Study 9720.

Untreated de novo (n=421) and secondary (n=189) acute myeloid leukemia (AML) patients ≥60 years received intensified chemotherapy, including daunorubicin 60 mg/m(2) and etoposide 100 mg/m(2) during days 1, 2, 3 with cytarabine 100 mg/m(2) during days 1-7, with a second induction if needed and one consolidation course with these drugs and doses for 2, 2 and 5 days, respectively. In all, 287 (47%) achieved complete remission (CR), 136 (22%) died and 187 (31%) were non-responders. CR rates were 27, 44 and 52% for complex karyotypes, rare aberrations and neither (P<0.001), 52 and 37% for de novo and secondary AML (P=0.003), and 53 and 42% for age 60-69 and ≥70 years (P=0.015). In multivariable analysis, CR predictors included non-complex/non-rare karyotypes (P<0.001), de novo AML (P<0.001), better performance status (PS) (P<0.001) and younger age (P=0.001). Disease-free (DFS) and overall (OS) survival medians were 6.8 (95% CI: 6.2, 7.8) and 7.2 (95% CI: 6.4, 8.6) months. In multivariable analysis, DFS was shorter for complex karyotypes (P<0.001) and increasing white blood count (WBC) (P<0.001) and age (P=0.038), and OS for complex karyotypes (P<0.001), increasing WBC (P=0.001) and age (P<0.001), poorer PS (P<0.001) and secondary AML (P=0.010). Outcomes and prognostic factors were similar to those in previous Cancer and Leukemia Group B studies.

BAALC, the human member of a novel mammalian neuroectoderm gene lineage, is implicated in hematopoiesis and acute leukemia.

The molecular basis of human leukemia is heterogeneous. Cytogenetic findings are increasingly associated with molecular abnormalities, some of which are being understood at the functional level. Specific therapies can be developed based on such knowledge. To search for new genes in the acute leukemias, we performed a representational difference analysis. We describe a human gene in chromosome 8q22.3, BAALC (brain and acute leukemia, cytoplasmic), that is highly conserved among mammals but evidently absent from lower organisms. We characterized BAALC on the genomic level and investigated its expression pattern in human and mouse, as well as its complex splicing behavior. In vitro studies of the protein showing its subcellular localization suggest a function in the cytoskeleton network. Two isoforms are specifically expressed in neuroectoderm-derived tissues, but not in tumors or cancer cell lines of nonneural tissue origin. We show that blasts from a subset of patients with acute leukemia greatly overexpress eight different BAALC transcripts, resulting in five protein isoforms. Among patients with acute myeloid leukemia, those overexpressing BAALC show distinctly poor prognosis, pointing to a key role of the BAALC products in leukemia. Our data suggest that BAALC is a gene implicated in both neuroectodermal and hematopoietic cell functions.

Suppression of p53 function in normal human mammary epithelial cells increases sensitivity to extracellular matrix-induced apoptosis.

Little is known about the fate of normal human mammary epithelial cells (HMECs) that lose p53 function in the context of extracellular matrix (ECM)-derived growth and polarity signals. Retrovirally mediated expression of human papillomavirus type 16 (HPV-16) E6 and antisense oligodeoxynucleotides (ODNs) were used to suppress p53 function in HMECs as a model of early breast cancer. p53+ HMEC vector controls grew exponentially in reconstituted ECM (rECM) until day 6 and then underwent growth arrest on day 7. Ultrastructural examination of day 7 vector controls revealed acinus-like structures characteristic of normal mammary epithelium. In contrast, early passage p53- HMEC cells proliferated in rECM until day 6 but then underwent apoptosis on day 7. p53- HMEC-E6 passaged in non-rECM culture rapidly (8-10 passages), lost sensitivity to both rECM-induced growth arrest and polarity, and also developed resistance to rECM-induced apoptosis. Resistance was associated with altered expression of alpha3-integrin. Treatment of early passage p53- HMEC-E6 cells with either alpha3- or beta1-integrin function-blocking antibodies inhibited rECM-mediated growth arrest and induction of apoptosis. Our results indicate that suppression of p53 expression in HMECs by HPV-16 E6 and ODNs may sensitize cells to rECM-induced apoptosis and suggest a role for the alpha3/beta1-heterodimer in mediating apoptosis in HMECs grown in contact with rECM.

Absence of the wild-type allele predicts poor prognosis in adult de novo acute myeloid leukemia with normal cytogenetics and the internal tandem duplication of FLT3: a cancer and leukemia group B study.

The FLT3 gene is mutated by an internal tandem duplication (ITD) in 20-25% of adults with acute myeloid leukemia (AML). We studied 82 adults <60 years of age with primary AML and normal cytogenetics, who received uniform high-dose therapy and found FLT3 ITD in 23 (28%) patients. When the 23 FLT3 ITD+ cases were compared with the 59 cases with wild-type (WT) FLT3, disease-free survival (DFS) was inferior (P = 0.03), yet overall survival (OS) was not different (P = 0.14). However, 8 (35%) of 23 FLT3 ITD/+ cases also lacked a FLT3 WT allele (FLT3(ITD-R)) as determined by PCR and loss of heterozygosity. Thus, three genotypic groups were identified: normal FLT3(WT/WT), heterozygous FLT3(ITD/WT), and hemizygous FLT3(ITD/-). DFS and OS were significantly inferior for patients with FLT3(ITD/-) (P = 0.0017 and P = 0.0014, respectively). Although DFS and OS for FLT3(WT/WT) and FLT3(ITD/WT) groups did not differ (P = 0.32 and P = 0.98, respectively), OS of the FLT3(ITD/-) group was worse than the FLT3(WT/WT) (P = 0.0005) and FLT3(ITD/WT) (P = 0.008) groups. We propose a model in which FLT3(ITD/-) represents a dominant positive, gain-of-function mutation providing AML cells with a greater growth advantage compared with cells having the FLT3(WT/WT) or FLT3(ITD/WT) genotypes. In conclusion, we have identified the FLT3(ITD/-) genotype as an adverse prognostic factor in de novo AML with normal cytogenetics. A poor prognosis of the relatively young FLT3(ITD/-) adults (median age, 37 years), despite treatment with current dose-intensive regimens, suggests that new treatment modalities, such as therapy with a FLT3 tyrosine kinase inhibitor, are clearly needed for this group of patients.

Clinical importance of cytogenetics in acute myeloid leukaemia.

Acquired chromosome aberrations are present in the marrow of most patients with acute myeloid leukaemia (AML) at diagnosis. Cytogenetically, AML is a very heterogeneous disease with over 160 structural chromosome abnormalities observed recurrently to date. Molecular dissection of many reciprocal translocations and inversions has resulted in cloning of the genes involved in leukaemogenesis. Some recurrent aberrations and the resulting gene rearrangements, namely inv(16)/t(16;16) and CBFbeta- MYH11, t(8;21) and CBFA2-CBFA2T1, t(15;17) and PML-RARalpha, and rearrangements of band 11q23 and the MLL gene, are now used to help define distinct disease entities within AML in the new World Health Organization classification of haematological malignancies. Moreover, cytogenetic abnormalities, whether molecularly characterized or not, are among the most important, independent prognostic factors in AML, and are being used in the management of AML patients. This review presents current information on chromosome abnormalities in AML, and on associations between karyotype and clinical characteristics and outcome of AML patients.

Comparison of cytogenetic and molecular genetic detection of t(8;21) and inv(16) in a prospective series of adults with de novo acute myeloid leukemia: a Cancer and Leukemia Group B Study.

PURPOSE: To prospectively compare cytogenetics and reverse transcriptase-polymerase chain reaction (RT-PCR) for detection of t(8;21)(q22;q22) and inv(16)(p13q22)/t(16;16)(p13;q22), aberrations characteristic of core-binding factor (CBF) acute myeloid leukemia (AML), in 284 adults newly diagnosed with primary AML. PATIENTS AND METHODS: Cytogenetic analyses were performed at local laboratories, with results reviewed centrally. RT-PCR for AML1/ETO and CBFbeta/MYH11 was performed centrally. RESULTS: CBF AML was ultimately identified in 48 patients: 21 had t(8;21) or its variant and AML1/ETO, and 27 had inv(16)/t(16;16), CBFbeta/MYH11, or both. Initial cytogenetic and RT-PCR analyses correctly classified 95.7% and 96.1% of patients, respectively (P =.83). Initial cytogenetic results were considered to be false-negative in three AML1/ETO-positive patients with unique variants of t(8;21), and in three CBFbeta/MYH11-positive patients with, respectively, an isolated +22; del(16)(q22),+22; and a normal karyotype. The latter three patients were later confirmed to have inv(16)/t(16;16) cytogenetically. Only one of 124 patients reported initially as cytogenetically normal was ultimately RT-PCR-positive. There was no false-positive cytogenetic result. Initial RT-PCR was falsely negative in two patients with inv(16) and falsely positive for AML1/ETO in two and for CBFbeta/MYH11 in another two patients. Two patients with del(16)(q22) were found to be CBFbeta/MYH11-negative. M4Eo marrow morphology was a good predictor of the presence of inv(16)/t(16;16). CONCLUSION: Patients with t(8;21) or inv(16) can be successfully identified in prospective multi-institutional clinical trials. Both cytogenetics and RT-PCR detect most such patients, although each method has limitations. RT-PCR is required when the cytogenetic study fails; it is also required to determine whether patients with suspected variants of t(8;21), del(16)(q22), or +22 represent CBF AML. RT-PCR should not replace cytogenetics and should not be used as the only diagnostic test for detection of CBF AML because of the possibility of obtaining false-positive or false-negative results.

High frequency of immunophenotype changes in acute myeloid leukemia at relapse: implications for residual disease detection (Cancer and Leukemia Group B Study 8361).

Multiparameter flow cytometry (MFC) has the potential to allow for sensitive and specific monitoring of residual disease (RD) in acute myeloid leukemia (AML). The use of MFC for RD monitoring assumes that AML cells identified by their immunophenotype at diagnosis can be detected during remission and at relapse. AML cells from 136 patients were immunophenotyped by MFC at diagnosis and at first relapse using 9 panels of 3 monoclonal antibodies. Immunophenotype changes occurred in 124 patients (91%); they consisted of gains or losses of discrete leukemia cell populations resolved by MFC (42 patients) and gains or losses of antigens on leukemia cell populations present at both time points (108 patients). Antigen expression defining unusual phenotypes changed frequently: CD13, CD33, and CD34, absent at diagnosis in 3, 33, and 47 cases, respectively, were gained at relapse in 2 (67%), 15 (45%), and 17 (36%); CD56, CD19, and CD14, present at diagnosis in 5, 16, and 20 cases, were lost at relapse in 2 (40%), 6 (38%), and 8 (40%). Leukemia cell gates created in pretreatment samples using each 3-antibody panel allowed identification of relapse AML cells in only 68% to 91% of cases, but use of 8 3-antibody panels, which included antibodies to a total of 16 antigens, allowed identification of relapse AML cells in all cases. Thus, the immunophenotype of AML cells is markedly unstable; nevertheless, despite this instability, MFC has the potential to identify RD in AML if multiple antibody panels are used at all time points. (Blood. 2001;97:3574-3580)

Spectral karyotyping reveals 17;22 fusions in a cytogenetically atypical dermatofibrosarcoma protuberans with a large marker chromosome as a sole abnormality.

The presence of an extra ring chromosome containing material from 17q and 22q, or, less frequently, a t(17;22)(q22;q13), is a cytogenetic hallmark of dermatofibrosarcoma protuberans (DFSP). However, occasionally tumors with other, atypical karyotypes are encountered. We describe a case of recurrent DFSP without a ring chromosome or a t(17;22) on standard cytogenetic analysis. In all cells analyzed by G-banding, an additional, large marker chromosome was present as a sole abnormality. This chromosome apparently included chromosome 8 or the 8q arm, but the origin of its remaining part could not be determined with certainty. To characterize further the abnormal chromosome, we applied spectral karyotyping (SKY). SKY confirmed the presence of an extra chromosome 8 or arm 8q in the marker and showed that its remaining part was composed of segments from chromosomes 7, 17, 21, and 22, with two copies of a 17;22 fusion. Our results and the literature data suggest that, in addition to a specific 17;22 fusion, amplification of material from chromosomes 17, 22, 8, 5, 7, and 21 may play a role in DFSP development and/or progression. Furthermore, our case demonstrates the usefulness of SKY in detection of a diagnostically relevant 17;22 fusion in DFSP patients who have unusual karyotypic features.

Human papillomavirus type 16 E6 inactivation of p53 in normal human mammary epithelial cells promotes tamoxifen-mediated apoptosis.

Aberrant p53 expression is frequently observed in mammary epithelial cells obtained from women at high risk for developing breast cancer and is a predictor for the subsequent development of malignancy. Tamoxifen has recently been shown to reduce the incidence of noninvasive breast cancer in high-risk women, but the molecular mechanism of tamoxifen chemoprevention in mammary epithelial tissue that does not overexpress the estrogen receptor is poorly understood. We suppressed p53 expression by retroviral-mediated expression of human papillomavirus type-16 E6 protein (HPV-16 E6) in human mammary epithelial cells (HMECs) to develop an in vitro model of tamoxifen chemoprevention in the context of p53 loss. Early passage p53(-) HMEC-E6-transduced cells treated with 1.0 microM tamoxifen rapidly underwent apoptosis. In contrast, early passage p53(+) HMEC-LXSN vector controls treated with 1.0 microM tamoxifen underwent G1-G0-phase arrest but did not undergo apoptosis. p53(-) HMEC-E6 cells rapidly acquired resistance to tamoxifen-mediated apoptosis after 10 passages in culture (in the absence of tamoxifen). Both p53(+) and p53(-) HMECs exhibited a low level of estrogen receptor staining and minimal estrogen binding, characteristic of proliferating normal luminal mammary epithelial cells. Tamoxifen-mediated apoptosis in p53(-) HMEC-E6 cells was not blocked by inhibitors of transcription and protein synthesis. These data suggest that the acute loss of p53 function in HMECs by expression of HPV-16 E6 results in marked sensitivity to tamoxifen-mediated apoptosis but that resistance to apoptosis rapidly develops within 10 passages in vitro. Observations in our model system predict a critical role for the early institution of tamoxifen chemoprevention.