A CpG island methylator phenotype in acute myeloid leukemia independent of IDH mutations and associated with a favorable outcome.

Genetic changes are infrequent in acute myeloid leukemia (AML) compared with other malignancies and often involve epigenetic regulators, suggesting that an altered epigenome may underlie AML biology and outcomes. In 96 AML cases including 65 pilot samples selected for cured/not-cured, we found higher CpG island (CGI) promoter methylation in cured patients. Expanded genome-wide digital restriction enzyme analysis of methylation data revealed a CGI methylator phenotype independent of IDH1/2 mutations we term AML-CGI methylator phenotype (CIMP) (A-CIMP+). A-CIMP was associated with longer overall survival (OS) in this data set (median OS, years: A-CIMP+=not reached, CIMP-=1.17; P=0.08). For validation we used 194 samples from The Cancer Genome Atlas interrogated with Illumina 450k methylation arrays where we confirmed longer OS in A-CIMP (median OS, years: A-CIMP+=2.34, A-CIMP-=1.00; P=0.01). Hypermethylation in A-CIMP+ favored CGIs (OR: CGI/non-CGI=5.21), and while A-CIMP+ was enriched in CEBPA (P=0.002) and WT1 mutations (P=0.02), 70% of cases lacked either mutation. Hypermethylated genes in A-CIMP+ function in pluripotency maintenance, and a gene expression signature of A-CIMP was associated with outcomes in multiple data sets. We conclude that CIMP in AML cannot be explained solely by gene mutations (for example, IDH1/2, TET2), and that curability in A-CIMP+ AML should be validated prospectively.

Comprehensive gene expression profiling and immunohistochemical studies support application of immunophenotypic algorithm for molecular subtype classification in diffuse large B-cell lymphoma: a report from the International DLBCL Rituximab-CHOP Consortium Program Study.

Gene expression profiling (GEP) has stratified diffuse large B-cell lymphoma (DLBCL) into molecular subgroups that correspond to different stages of lymphocyte development-namely germinal center B-cell like and activated B-cell like. This classification has prognostic significance, but GEP is expensive and not readily applicable into daily practice, which has lead to immunohistochemical algorithms proposed as a surrogate for GEP analysis. We assembled tissue microarrays from 475 de novo DLBCL patients who were treated with rituximab-CHOP chemotherapy. All cases were successfully profiled by GEP on formalin-fixed, paraffin-embedded tissue samples. Sections were stained with antibodies reactive with CD10, GCET1, FOXP1, MUM1 and BCL6 and cases were classified following a rationale of sequential steps of differentiation of B cells. Cutoffs for each marker were obtained using receiver-operating characteristic curves, obviating the need for any arbitrary method. An algorithm based on the expression of CD10, FOXP1 and BCL6 was developed that had a simpler structure than other recently proposed algorithms and 92.6% concordance with GEP. In multivariate analysis, both the International Prognostic Index and our proposed algorithm were significant independent predictors of progression-free and overall survival. In conclusion, this algorithm effectively predicts prognosis of DLBCL patients matching GEP subgroups in the era of rituximab therapy.

Loss of SHIP-1 protein expression in high-risk myelodysplastic syndromes is associated with miR-210 and miR-155.

The myelodysplastic syndromes (MDSs) comprise a group of disorders characterized by multistage progression from cytopenias to acute myeloid leukemia (AML). They display exaggerated apoptosis in early stages, but lose this behavior during evolution to AML. The molecular basis for loss of apoptosis is unknown. To investigate this critical event, we analyzed phosphatidylinositol (PI) 3'kinase signaling, implicated as a critical pathway of cell survival control in epithelial and hematological malignancies. PI 3'kinase activates Akt through its production of 3' phosphoinositides. In turn, the phosphoinositides are dephosphorylated by two lipid phosphatases, PTEN and SHIP-1, in myeloid cells. We studied primary MDS-enriched bone marrow cells and bone marrow sections by western blotting, immunohistochemistry, immunocytochemistry and quantitative PCR for components of the SHIP/PTEN/PI 3'kinase signaling circuit. We reported constitutively activated Akt, variable levels of PTEN and uniformly decreased SHIP-1 expression in MDS progenitor cells. Overexpression of SHIP-1, but not the phosphatase-deficient form, inhibited myeloid leukemic growth. Levels of microRNA (miR)-210 and miR-155 transcripts, which target SHIP-1, were increased in CD34(+) MDS cells compared with their normal counterparts. Direct binding of miR-210 to the 3' untranslated region of SHIP-1 was confirmed by luciferase reporter assay. Transfection of a myeloid cell line with miR-210 resulted in loss of SHIP-1 protein expression. These data suggest that miR-155 and miR-210/SHIP-1/Akt pathways could serve as clinical biomarkers for disease progression, and that miR-155 and miR-210 might serve as novel therapeutic targets in MDS.

A randomized trial of a single-dose rasburicase versus five-daily doses in patients at risk for tumor lysis syndrome.

BACKGROUND: Tumor lysis syndrome (TLS) is a life-threatening disorder characterized by hyperuricemia and metabolic derangements. The efficacy of rasburicase, administered daily for 5 days, has been well established. However, the optimal duration of therapy is unknown in adults. PATIENTS AND METHODS: We evaluated the efficacy of rasburicase (0.15 mg/kg) administered as single dose followed by as needed dosing (maximum five doses) versus daily dosing for 5 days in adult patients at risk for TLS. RESULTS: Eighty of the 82 patients enrolled received rasburicase; 40 high risk [median uric acid (UA) 8.5 mg/dl; range, 1.5-19.7] and 40 potential risk (UA = 5.6 mg/dl; range, 2.4-7.4). Seventy-nine patients (99%) experienced normalization in their UA within 4 h after the first dose; 84% to an undetectable level (<0.7 mg/dl). Thirty-nine of 40 (98%) patients in the daily-dose arm and 34 of 40 (85%) patients in single-dose arm showed sustained UA response. Six high-risk patients within the single-dose arm required second dose for UA >7.5 mg/dl. Rasburicase was well tolerated; one patient with glucose-6-phosphate dehydrogenase deficiency developed methemoglobinemia and hemolysis. CONCLUSIONS: Rasburicase is highly effective for prevention and management of hyperuricemia in adults at risk for TLS. Single-dose rasburicase was effective in most patients; only a subset of high-risk patients required a second dose.

Recent advances in the diagnosis and classification of myeloid neoplasms--comments on the 2008 WHO classification.

The fourth edition of the World Health Organization (WHO) classification of myeloid neoplasms refined the criteria for some previously described myeloid neoplasms and recognized several new entities based on recent elucidation of molecular pathogenesis, identification of new diagnostic and prognostic markers, and progress in clinical management. Protein tyrosine kinase abnormalities, including translocations or mutations involving ABL1, JAK2, MPL, KIT, PDGFRA, PDGFRB, and FGFR1, have been used as the basis for classifying myeloproliferative neoplasms (MPN). Two new entities - refractory cytopenia with unilineage dysplasia and refractory cytopenia of childhood have been added to the group of myelodysplastic syndromes (MDS), and 'refractory anemia with excess blasts-1' has been redefined to emphasize the prognostic significance of increased blasts in the peripheral blood. A list of cytogenetic abnormalities has been introduced as presumptive evidence of MDS in cases with refractory cytopenia but without morphologic evidence of dysplasia. The subgroup 'acute myeloid leukemia (AML) with recurrent genetic abnormalities' has been expanded to include more molecular genetic aberrations. The entity 'AML with multilineage dysplasia' specified in the 2001 WHO classification has been renamed 'AML with myelodysplasia-related changes' to include not only cases with significant multilineage dysplasia but also patients with a history of MDS or myelodysplasia-related cytogenetic abnormalities. The term 'therapy-related myeloid neoplasms' is used to cover the spectrum of disorders previously known as t-AML, t-MDS, or t-MDS/MPN occurring as complications of cytotoxic chemotherapy and/or radiation therapy. In this review, we summarize many of these important changes and discuss some of the diagnostic challenges that remain.

Accurate detection of uniparental disomy and microdeletions by SNP array analysis in myelodysplastic syndromes with normal cytogenetics.

Progress in the management of patients with myelodysplastic syndromes (MDS) has been hampered by the inability to detect cytogenetic abnormalities in 40-60% of cases. We prospectively analyzed matched pairs of bone marrow and buccal cell (normal) DNA samples from 51 MDS patients by single nucleotide polymorphism (SNP) arrays, and identified somatically acquired clonal genomic abnormalities in 21 patients (41%). Among the 33 patients with normal bone marrow cell karyotypes, 5 (15%) had clonal, somatically acquired aberrations by SNP array analysis, including 4 with segmental uniparental disomies (UPD) and 1 with three separate microdeletions. Each abnormality was detected more readily in CD34+ cells than in unselected bone marrow cells. Paired analysis of bone marrow and buccal cell DNA from each patient was necessary to distinguish true clonal genomic abnormalities from inherited copy number variations and regions with apparent loss of heterozygosity. UPDs affecting chromosome 7q were identified in two patients who had a rapidly deteriorating clinical course despite a low-risk International Prognostic Scoring System score. Further studies of larger numbers of patients will be needed to determine whether 7q UPD detected by SNP array analysis will identify higher risk MDS patients at diagnosis, analogous to those with 7q cytogenetic abnormalities.

Cytogenetic findings in lymphoplasmacytic lymphoma/Waldenström macroglobulinemia. Chromosomal abnormalities are associated with the polymorphous subtype and an aggressive clinical course.

We correlated bone marrow cytogenetic findings with morphologic and immunophenotypic data in 37 patients with lymphoplasmacytic lymphoma (LPL)/Waldenström macroglobulinemia (WM). Each LPL/WM case was classified as lymphoplasmacytoid (n = 18), lymphoplasmacytic (n = 10), or polymorphous (n = 9) using the Kiel criteria. Of 12 cases with chromosomal abnormalities, a single numeric abnormality was present in 4 and a complex karyotype in 8. The most common numeric abnormalities were and -8 in 3 cases each; the most common structural abnormality was del(6q) in 6 cases. Cytogenetic abnormalities were significantly less common in the lymphoplasmacytic and lymphoplasmacytoid groups (5/28 [18%]) compared with the polymorphous group (7/9 [78%]). Clinical follow-up was available for 28 patients for a median of 36 months. Six (67%) of 9 patients with aneuploid tumors, including 4 with polymorphous subtype, subsequently had clinical progression or developed high-grade lymphoma. In contrast, 4 (21%) of 19 patients with diploid tumors, including 1 of polymorphous type, developed clinical progression or high-grade lymphoma. We conclude that abnormal cytogenetic findings in LPL/WM correlate with the polymorphous subtype and poor prognosis.

BCR rearrangement-negative chronic myelogenous leukemia revisited.

PURPOSE: To document the characteristics of patients with major breakpoint cluster region (M-bcr) rearrangement-negative chronic myelogenous leukemia (CML). PATIENTS AND METHODS: The hematopathologist, who was blinded to patients' molecular status, reviewed the referral bone marrows and peripheral-blood smears from 26 patients with Philadelphia (Ph) translocation-negative CML who lacked Bcr rearrangement (and other evidence of a Bcr-Abl anomaly) and 14 patients (controls) with chronic-phase Ph-positive CML. Clinical data was ascertained by chart review. RESULTS: Among the 26 M-bcr rearrangement-negative CML patients, three pathologic subtypes emerged: (1) patients indistinguishable from classic CML (n = 9), (2) patients with atypical CML (n = 8), and (3) patients with chronic neutrophilic leukemia (n = 9). Among the 14 patients with Ph-positive CML who were included in the blinded review, 13 were classified as classic CML, and one was classified as atypical CML. The only statistically significant difference between M-bcr rearrangement-negative subgroups was in the proportion of patients having karyotypic abnormalities, an observation common only in patients with atypical CML (P = 0.008). However, the small number of patients in each subgroup limited our ability to differentiate between them. Interferon alfa induced complete hematologic remission in five of 14 patients; four of these remissions lasted more than 5 years. Only one of 26 patients developed blast crisis. The median survival of the 26 patients was 37 months. CONCLUSION: Patients with M-bcr rearrangement-negative CML fall into three morphologic subgroups. Disease evolution does not generally involve blastic transformation. Instead, patients show progressive organomegaly, leukocytosis, anemia, and thrombocytosis. Some patients in each subgroup can respond to interferon alfa.

Acute myelomonocytic leukemia with histologic features resembling sarcomatoid carcinoma in bone marrow.

We report a case of primary acute myelomonocytic leukemia involving the bone marrow that resembled sarcomatoid carcinoma. The neoplastic cells in bone marrow biopsy specimens formed cohesive-appearing clusters and cords separated by an immature fibroblastic proliferation and myxoid stroma. Blasts in the bone marrow aspirate smears formed clusters and sheets, and a subset of blasts exhibited erythrophagocytosis. Dysgranulopoiesis was also present. Lineage was confirmed by immunohistochemical analysis of formalin-fixed, paraffin-embedded tissue. The tumor cells showed strong reactivity for lysozyme, myeloperoxidase, CD45, and CD68 and were negative for keratin, S100, CD20, and CD3. The serum lysozyme concentration (110 microgram/mL) was 13 times greater than the normal value (8 microgram/mL). Cytogenetic studies performed on bone marrow aspirate material revealed a complex karyotype, including trisomy 8 and abnormalities of chromosome 11q. We report this case of acute myelomonocytic leukemia because the neoplastic cells appeared cohesive and spindled, resembling sarcomatoid carcinoma, and therefore caused diagnostic difficulty. Other monocytic neoplasms with similar resemblance to carcinoma or sarcoma have been reported in the literature, suggesting that the tendency to appear cohesive may be an inherent characteristic of neoplastic cells with monocytic differentiation.

Primary myeloid leukemia presenting concomitantly with primary multiple myeloma: two cases and an update of the literature.

We report one case of primary acute myelogenous leukemia (AML) and one case of refractory anemia with excess blasts in transformation (RAEB-T) each presenting concomitantly with multiple myeloma, an unusual finding. The twin diagnoses in each patient were confirmed by cytochemical and immunohistochemical studies, and in one of our cases, by ultrastructural, flow cytometric, and molecular studies. The last three methods have not been previously used to document this phenomenon.

Applicability of direct in situ reverse transcription-polymerase chain reaction on bone marrow smears.

In situ reverse transcription (RT)-polymerase chain reaction (PCR) is a promising laboratory tool for biomedical investigation at the molecular level in tissues. Direct in-cell amplification of the breakpoint cluster region (BCR)-Abelson (ABL) fusion transcript of chronic myeloid leukemia (CML) has recently been accomplished in Italy using bone marrow mononuclear cell suspensions. The goals of this study are to determine if in situ RT-PCR amplification is possible on bone marrow spirate smears and to demonstrate any unique factors in this procedure. A commercially available method was used because of the existence of published protocols for adaptation. Bone marrow (BM) aspirate smears (n = 17) from patients with CML in blast crisis (positive case material) or other hematological malignancies (negative case material) were evaluated. Satisfactory amplification of the BCR-ABL fusion transcript occurred, and distinct blue cytoplasmic granules that varied in intensity were found in most CML blasts. The negative case materials lacked the specifically amplified granular signals. Overall signal strength and backgrounds were readily affected by the quality of the specimen as well as by changes in assay parameters. In conclusion, the direct in situ RT-PCR technique is applicable for bone marrow aspirate smear evaluation. However, it remains an investigative tool until optimization for sensitivity, specificity, and accuracy can be achieved.

Expression profile of MDM-2 proteins in chronic lymphocytic leukemia and their clinical relevance.

The MDM-2 oncoprotein exists in an autoregulatory feedback loop with the tumor suppressor protein p53. Therefore, intracellular levels of these two proteins may play important roles in cell proliferation and tumorigenesis. Several MDM-2 proteins (Mr 35-100 Kd) have been demonstrated in human cell lines. We report here the expression profile of MDM-2 and p53 proteins in 87 cases of chronic lymphocytic leukemia (CLL) as detected by immunoblot analysis. The MDM-2 proteins (p57, p59, p67, and p90) were found to be overexpressed in different combinations in 56/87 (64%) of cases of CLL when compared with normal volunteers. The MDM-2 protein p57 was predominantly overexpressed 46/87 (53%) in CLL. In 22/87 (25%) cases of CLL p57 was overexpressed alone, and in 24/87 (28%) cases it was co-overexpressed with other MDM-2 proteins p59/p67/p90. Six of the 87 cases of CLL showed overexpression of the tumor suppressor protein p53 by immunoblot analysis, and five of those cases also co-overexpress MDM-2 protein p57. No statistically significant correlation of MDM-2 protein overexpression to clinical disease stage and history of previous chemotherapy of CLL patients has been found. However, considering the oncogenic potential of overexpressed MDM-2 proteins, a possible role of MDM-2 proteins in the promotion of CLL disease remains to be evaluated.

Abnormal expression of MDM-2 in breast carcinomas.

MDM-2 is a cellular oncoprotein that binds to the p53 protein and abrogates its growth-suppressing function. At least seven MDM-2 mRNAs and five proteins (p90, p85, p76, p74, and p57) have been reported in tissue culture. MDM-2 gene amplification occurs in human sarcomas and high-grade gliomas. MDM-2 overexpression without gene amplification has been reported in leukemias and lymphomas. Here we report MDM-2 mRNA overexpression in 24 (73%) out of 33 cases of human breast carcinoma as compared with normal breast tissue. The MDM-2 overexpression was seen in the absence of MDM-2 gene amplification. MDM-2 protein expression was studied by western blot analysis in 21 of these cases of carcinoma. We found complete concordance between MDM-2 mRNA overexpression and MDM-2 protein levels. MDM-2 proteins were overexpressed in 15 of 21 breast carcinoma tissue samples but not in normal breast tissue controls. Ten of these fifteen cases overexpressed MDM-2 p57 protein, two cases overexpressed both p57 and p90, and three cases overexpressed only p90. MDM-2 overexpression was confirmed by immunohistochemistry. p53 overexpression was also studied by immunohistochemistry, 69% of breast carcinomas that overexpressed the MDM-2 mRNA had detectable nuclear p53 protein. These findings demonstrate that MDM-2 oncoprotein expression is altered in primary human breast carcinomas at both mRNA and protein levels. In addition, our results suggest that MDM-2 p57 protein represents the main MDM-2 protein altered in breast carcinomas.

Multiple patterns of MDM-2 deregulation in human leukemias: implications in leukemogenesis and prognosis.

The human analogue of the mouse double minute-2 (MDM-2) protein binds to p53 protein and abrogates its tumor-suppressing activity. MDM-2 overexpression may represent an alternative mechanism to p53 mutation for escaping the p53-mediated growth control. Interestingly, multiple MDM-2 protein isoforms have been described and the possibility of functional differences between various isoforms has been raised. Previously, we demonstrated significant MDM-2 mRNA overexpression in human leukemias and suggested that MDM-2 overexpression may be a marker of aggressiveness of the disease. Polyclonal antibodies (Ab) have been generated to detect various isoforms of the MDM-2 protein. Using these Abs, we confirmed MDM-2 protein overexpression in leukemias. Furthermore, we observed heterogeneity in the isoforms expressed in various types of leukemias. In addition, we demonstrated that analysis by flow cytometry could be used as a diagnostic tool for detecting altered MDM-2 protein expression in leukemias. Here we review and expand our initial observations and confirm MDM-2 mRNA and protein overexpression by reverse transcription-polymerase chain reaction (RT-PCR), flow cytometry, and western blot analyses. Understanding the possible role of MDM-2 oncogene expression in leukemias may establish the scientific basis for new therapeutic approaches.

Anaplastic large cell lymphoma presenting as a soft-tissue mass mimicking sarcoma.

Anaplastic, sarcomatoid lymphomas appearing primarily in the soft tissue without peripheral lymphadenopathy create considerable diagnostic difficulties for the pathologist, as they may be mistaken for sarcomas. The clinical, pathologic, immunohistochemical, and molecular features of one such case are described.

The human MDM-2 oncogene is overexpressed in leukemias.

The human homologue of the mouse double minute 2 (MDM-2) gene codes for a cellular protein that forms a complex with the mutant and wild-type p53 protein and modulates its trans-activation activity. Overexpression of the MDM-2 gene in cells increases their tumorigenic potential and overcomes the growth-suppressive activity of p53. Previous reports have shown that the MDM-2 gene is amplified in approximately one third of human sarcomas. To examine the role of MDM-2 in leukemia, we analyzed MDM-2 gene amplification and mRNA expression in various types of leukemias. We did not detect gene amplification in any of the 48 cases of leukemia that we examined. In contrast, we observed significant MDM-2 mRNA overexpression in 34 of 64 cases (53%). The level of mRNA overexpression in some cases of leukemias was comparable to that observed in some cases of sarcomas, which demonstrate more than 50-fold MDM-2 gene amplification. Furthermore, we divided these cases into different prognostic groups according to their karyotypic abnormalities. MDM-2 overexpression seemed to be associated with unfavorable chromosomal abnormalities. These findings suggest that the expression of the MDM-2 gene is altered in a significant fraction of human leukemias and MDM-2 may play a significant role in leukemogenesis. In addition, these results suggest that mechanisms other than gene amplification may play a significant role in deregulating the MDM-2 expression.

Cytofluorometric analyses of human T cell CD2/CD4 inter-molecular interactions.

Incubation of human T lymphocytes with saturating concentrations of combinations of certain anti-CD2 and -CD4 mAb results in reciprocal down-regulation of the cell surface density expression of the respective CD molecules. Such reciprocal down-regulation occurs at 0 degrees C in the presence of sodium azide and appears selective for CD2 and CD4 molecules because mAb identifying various other CD T cell surface molecules (anti-Leu2a, -OK-CLL, -W6/32, -beta 2-microglobulin, -4B4) do not modulate CD2 or CD4 R density, and because anti-CD2 mAb (anti-OKT11 and -D66 clone-1) do not alter CD8 R density (anti-OKT8, -Leu2a) and vice versa. Down-regulation of CD2 by mAb specific to CD4 is epitope-specific but does not vary on the basis of the antibody isotype used. The anti-CD4 mAb, Leu3a, was the strongest CD2 down-regulator examined followed by OKT4F. mAb specific to other CD4 epitopes (B, C, D, and E) caused only slight down-regulation of CD2 expression whereas anti-OKT4 and -OKT4A mAb had no significant regulatory effect. Also, mAb specific to the 9.6 (anti-OKT11) and D66 (anti-D66 clone 1) epitopes of the CD2 molecule down-regulated CD4 density detectable with Leu3a, OKT4, and OKT4A anti-CD4 mAb. Down-regulation of CD2 by anti-CD4 mAb also occurred with the transformed T cell line, KE-37, which demonstrates that such effects can occur without mononuclear phagocytic accessory cells. From these data it can be concluded that important T cell immunoregulatory signals may be transmitted intramembranally between CD2 and CD4 glycoproteins.