Erica Ballabio

Novel markers of normal and neoplastic human plasmacytoid dendritic cells.

Plasmacytoid dendritic cells (pDCs) are involved in innate immunity (eg, by secreting interferons) and also give rise to CD4+CD56+ hematodermic neoplasms. We report extensive characterization of human pDCs in routine tissue samples, documenting the expression of 19 immunohistologic markers, including signaling molecules (eg, BLNK), transcription factors (eg, ICSBP/IRF8 and PU.1), and Toll-like receptors (TLR7, TLR9). Many of these molecules are expressed in other cell types (principally B cells), but the adaptor protein CD2AP was essentially restricted to pDCs, and is therefore a novel immunohistologic marker for use in tissue biopsies. We found little evidence for activation-associated morphologic or phenotypic changes in conditions where pDCs are greatly increased (eg, Kikuchi disease). Most of the molecules were retained in the majority of pDC neoplasms, and 3 (BCL11A, CD2AP, and ICSBP/IRF8) were also commonly negative in leukemia cutis (acute myeloid leukemia in the skin), a tumor that may mimic pDC neoplasia. In summary, we have documented a range of molecules (notably those associated with B cells) expressed by pDCs in tissues and peripheral blood (where pDCs were detectable in cytospins at a frequency of <1% of mononuclear cells) and also defined potential new markers (in particular CD2AP) for the diagnosis of pDC tumors.

Expression of microRNAs in diffuse large B cell lymphoma is associated with immunophenotype, survival and transformation from follicular lymphoma

MicroRNAs are naturally occurring small RNA species that regulate gene expression and are frequently abnormally expressed in cancers. However, the role of microRNAs in lymphoma is poorly understood. Therefore, we undertook a comprehensive study of microRNA expression in two of the most common lymphomas: diffuse large B‐cell lymphoma (DLBCL) (n= 80) and follicular lymphoma (FCL) (n= 18) using microarrays containing probes for 464 human microRNAs. Unsupervised cluster analysis revealed distinct expression patterns between these two lymphomas and specific microRNA signatures (including members of the miR‐17–92 cluster) were derived that correctly predicted lymphoma type in >95% of cases. Furthermore, we identified microRNAs in de novo DLBCL (n= 64) associated with germinal centre‐like and non‐germinal centre‐like immunophenotypes, international prognostic index status and event‐free survival in CHOP and rituximab (R)‐CHOP treated patients. Despite the indolent nature of FCL a significant proportion of cases undergo high‐grade transformation to more aggressive DLBCL. In order to see if transformation is associated with changes in microRNA expression we compared transformed DLBCL cases (n= 16) with de novo DLBCL, as well as FCL cases that underwent subsequent transformation (n= 7) with FCL cases that had not transformed at a median follow‐up of 60 months (n= 11). Differential expression of 12 microRNAs correctly predicted >85% of transformed versus de novo DLBCL cases; six microRNAs (miR‐223, 217, 222, 221 and let‐7i and 7b) were found which could similarly predict or transformation in FCL (P< 0.05). These data suggest that microRNAs have potential as diagnostic and prognostic markers in these lymphomas and may be used to identify FCL patients at risk of high‐grade transformation.

MicroRNA expression in Sezary syndrome: identification, function, and diagnostic potential.

MicroRNAs are commonly aberrantly expressed in many cancers. Very little is known of their role in T-cell lymphoma, however. We therefore elucidated the complete miRNome of purified T cells from 21 patients diagnosed with Sézary Syndrome (SzS), a rare aggressive primary cutaneous T-cell (CD4(+)) lymphoma. Unsupervised cluster analysis of microarray data revealed that the microRNA expression profile was distinct from CD4(+) T-cell controls and B-cell lymphomas. The majority (104 of 114) of SzS-associated microRNAs (P < .05) were down-regulated and their expression pattern was largely consistent with previously reported genomic copy number abnormalities and were found to be highly enriched (P < .001) for aberrantly expressed target genes. Levels of miR-223 distinguished SzS samples (n = 32) from healthy controls (n = 19) and patients with mycosis fungoides (n = 11) in more than 90% of samples. Furthermore, we demonstrate that the down-regulation of intronically encoded miR-342 plays a role in the pathogenesis of SzS by inhibiting apoptosis, and describe a novel mechanism of regulation for this microRNA via binding of miR-199a* to its host gene. We also provide the first in vivo evidence for down-regulation of the miR-17-92 cluster in malignancy and demonstrate that ectopic miR-17-5p expression increases apoptosis and decreases cell proliferation in SzS cells.

miRNA expression profiling of mycosis fungoides

MicroRNAs (miRNAs) are small RNA species that regulate gene expression post‐transcriptionally and are aberrantly expressed in many malignancies including lymphoma. However, the role of miRNAs in the pathogenesis of T‐cell lymphoid malignancies is poorly understood. Previously we examined the miRNA profile of Sézary syndrome (Sz), a leukemia of skin‐homing memory T cells. In this study we determined the complete miRNome of mycosis fungoides (MF), the most common type of cutaneous T cell lymphoma. The miRNA profile of skin biopsies from 19 patients with tumor stage MF and 12 patients with benign inflammatory dermatoses (eczema and lichen planus) were compared by microarray analysis. We identified 49 miRNAs that are differentially expressed in tumor stage MF compared to benign inflammatory dermatoses using ANOVA analysis (P < 0.05, Benjamini–Hochberg corrected). The majority of the differentially expressed miRNAs (30/49) were up‐regulated in tumor stage MF. The most significant differentially expressed were miR‐155 and miR‐92a (both up‐regulated in tumor stage MF), while miR‐93 showed the highest up‐regulation in tumor stage MF with a fold difference of 5.8. Differential expression of a selection of these miRNAs was validated by miRNA‐Q‐PCR on additional test groups (tumors and controls). None of the miRNAs up‐regulated in tumor stage MF was previously shown to be up‐regulated in Sz, and only 2 of the 19 miRNAs down‐regulated in tumor stage MF were also down‐regulated in Sz. Taken together this report is the first describing the miRNA signature of tumor stage MF.

RUNX1 Is a Key Target in t(4;11) Leukemias that Contributes to Gene Activation through an AF4-MLL Complex Interaction

Summary The Mixed Lineage Leukemia (MLL) protein is an important epigenetic regulator required for the maintenance of gene activation during development. MLL chromosomal translocations produce novel fusion proteins that cause aggressive leukemias in humans. Individual MLL fusion proteins have distinct leukemic phenotypes even when expressed in the same cell type, but how this distinction is delineated on a molecular level is poorly understood. Here, we highlight a unique molecular mechanism whereby the RUNX1 gene is directly activated by MLL-AF4 and the RUNX1 protein interacts with the product of the reciprocal AF4-MLL translocation. These results support a mechanism of transformation whereby two oncogenic fusion proteins cooperate by activating a target gene and then modulating the function of its downstream product.

The inducible T-cell co-stimulator molecule is expressed on subsets of T cells and is a new marker of lymphomas of T follicular helper cell-derivation

Background T follicular helper (TFH) cells reside in the light zone of germinal centers and are considered the cell of origin of angioimmunoblastic T-cell lymphoma. Recently, CXCL13, PD-1 and SAP were described as useful markers for TFH cells and angioimmunoblastic T-cell lymphoma but also reported in some peripheral T-cell lymphomas, not otherwise specified. Design and Methods In the present study the expression pattern of ICOS protein was investigated by immunohistochemistry-based techniques in routine sections of normal lymphoid tissues and 633 human lymphomas. Results Cells strongly positive for ICOS were restricted to the light zone of germinal centers and co-expressed TFH-associated molecules. In addition, weak to moderate ICOS expression was observed in a small proportion of FOXP3-positive cells. In lymphomas, ICOS expression was confined to angioimmunoblastic T-cell lymphoma (85/86), peripheral T-cell lymphomas of follicular variant (18/18) and a proportion of peripheral T-cell lymphomas, not otherwise specified (24/56) that also expressed other TFH-associated molecules. Conclusions ICOS is a useful molecule for identifying TFH cells and its restricted expression to angioimmunoblastic T-cell lymphoma and a proportion of peripheral T-cell lymphomas, not otherwise specified (showing a TFH-like profile) suggests its inclusion in the antibody panel for diagnosing TFH-derived lymphomas. Our findings provide further evidence that the histological spectrum of TFH-derived lymphomas is broader than previously assumed.

MicroRNA expression in multiple myeloma is associated with genetic subtype, isotype and survival

BackgroundMicroRNAs are small RNA species that regulate gene expression post-transcriptionally and are aberrantly expressed in many cancers including hematological malignancies. However, the role of microRNAs in the pathogenesis of multiple myeloma (MM) is only poorly understood. We therefore used microarray analysis to elucidate the complete miRNome (miRBase version 13.0) of purified tumor (CD138+) cells from 33 patients with MM, 5 patients with monoclonal gammopathy of undetermined significance (MGUS) and 9 controls.ResultsUnsupervised cluster analysis revealed that MM and MGUS samples have a distinct microRNA expression profile from control CD138+ cells. The majority of microRNAs aberrantly expressed in MM (109/129) were up-regulated. A comparison of these microRNAs with those aberrantly expressed in other B-cell and T-cell malignancies revealed a surprising degree of similarity (~40%) suggesting the existence of a common lymphoma microRNA signature. We identified 39 microRNAs associated with the pre-malignant condition MGUS. Twenty-three (59%) of these were also aberrantly expressed in MM suggesting common microRNA expression events in MM progression. MM is characterized by multiple chromosomal abnormalities of varying prognostic significance. We identified specific microRNA signatures associated with the most common IgH translocations (t(4;14) and t(11;14)) and del(13q). Expression levels of these microRNAs were distinct between the genetic subtypes (by cluster analysis) and correctly predicted these abnormalities in > 85% of cases using the support vector machine algorithm. Additionally, we identified microRNAs associated with light chain only myeloma, as well as IgG and IgA-type MM. Finally, we identified 32 microRNAs associated with event-free survival (EFS) in MM, ten of which were significant by univariate (logrank) survival analysis.ConclusionsIn summary, this work has identified aberrantly expressed microRNAs associated with the diagnosis, pathogenesis and prognosis of MM, data which will prove an invaluable resource for understanding the role of microRNAs in this devastating disease.ReviewersThis article was reviewed by Prof. Neil Smalheiser, Prof. Yuriy Gusev, and an unknown reviewer.

Self-Enforcing Feedback Activation between BCL6 and Pre-B Cell Receptor Signaling Defines a Distinct Subtype of Acute Lymphoblastic Leukemia

Studying 830 pre-B ALL cases from four clinical trials, we found that human ALL can be divided into two fundamentally distinct subtypes based on pre-BCR function. While absent in the majority of ALL cases, tonic pre-BCR signaling was found in 112 cases (13.5%). In these cases, tonic pre-BCR signaling induced activation of BCL6, which in turn increased pre-BCR signaling output at the transcriptional level. Interestingly, inhibition of pre-BCR-related tyrosine kinases reduced constitutive BCL6 expression and selectively killed patient-derived pre-BCR(+) ALL cells. These findings identify a genetically and phenotypically distinct subset of human ALL that critically depends on tonic pre-BCR signaling. In vivo treatment studies suggested that pre-BCR tyrosine kinase inhibitors are useful for the treatment of patients with pre-BCR(+) ALL.

Primary cutaneous anaplastic large cell lymphoma shows a distinct miRNA expression profile and reveals differences from tumor-stage mycosis fungoides.

The miRNA expression profiles of skin biopsies from 14 primary cutaneous anaplastic large cell lymphoma (C‐ALCL) patients were analysed with miRNA microarrays using the same control group of 12 benign inflammatory dermatoses (BID) as previously used to study the miRNA expression profile of tumor‐stage mycosis fungoides (MF). We identified 13 differentially expressed miRNAs between C‐ALCL and BID. The up‐regulation of miR‐155, miR‐27b, miR‐30c and miR‐29b in C‐ALCL was validated by miRNA‐Q‐PCR on independent study groups. Additionally, the miRNA expression profiles of C‐ALCL were compared with those of tumor‐stage MF. Although miRNA microarray analysis did not identify statistically significant differentially expressed miRNAs, miRNA‐Q‐PCR demonstrated statistically significantly differential expression of miR‐155, miR‐27b, miR‐93, miR‐29b and miR‐92a between tumor‐stage MF and C‐ALCL. This study, the first describing the miRNA expression profile of C‐ALCL, reveals differences with tumor‐stage MF, suggesting a different contribution to the pathogenesis of these lymphomas.

MLL-Rearranged Acute Lymphoblastic Leukemias Activate BCL-2 through H3K79 Methylation and Are Sensitive to the BCL-2-Specific Antagonist ABT-199

Summary Targeted therapies designed to exploit specific molecular pathways in aggressive cancers are an exciting area of current research. Mixed Lineage Leukemia (MLL) mutations such as the t(4;11) translocation cause aggressive leukemias that are refractory to conventional treatment. The t(4;11) translocation produces an MLL/AF4 fusion protein that activates key target genes through both epigenetic and transcriptional elongation mechanisms. In this study, we show that t(4;11) patient cells express high levels of BCL-2 and are highly sensitive to treatment with the BCL-2-specific BH3 mimetic ABT-199. We demonstrate that MLL/AF4 specifically upregulates the BCL-2 gene but not other BCL-2 family members via DOT1L-mediated H3K79me2/3. We use this information to show that a t(4;11) cell line is sensitive to a combination of ABT-199 and DOT1L inhibitors. In addition, ABT-199 synergizes with standard induction-type therapy in a xenotransplant model, advocating for the introduction of ABT-199 into therapeutic regimens for MLL-rearranged leukemias.