The Conundrum of Diagnosing Cutaneous Composite Lymphoma in the Molecular Age.

INTRODUCTION: Cutaneous composite lymphoma (CCL) is extremely rare. When 2 potentially distinct lymphoid lesions occur at one skin site, distinguishing between one neoplastic clone and a secondary reactionary lymphoid response versus a second neoplasm is difficult. In this study, we describe a unique case of CCL along with a review of reported cases in literature to identify clues and discuss issues that are relevant to the diagnosis of CCL. DESIGN: Review of a CCL case from our institution and a systematic review of reported cases of CCL in the literature. RESULTS: A total of 18 studies describing 22 cases and a case report from our institution are included. The mean age at diagnosis was 68 years. Most cases herein presented with multiple skin lesions (67%) and reported a history of immune suppression (76%). Nineteen cases (83%) had a combination of T-cell and B-cell neoplasms, whereas the remaining cases had 2 distinct B-cell clones. Clonal differentiation was confirmed based on morphology and immunohistochemistry in all cases, and by polymerase chain reaction studies in 19 cases. Complete remission was achieved in only one quarter of reported cases. CONCLUSION: Diagnosing CCL can be challenging because accurate differentiation of 2 or more clonal populations at 1 site is tedious. A stepwise approach and integration of clinical, morphologic, immunohistochemistry, and molecular data along with an understanding of the prognosis of the lymphomas in question is essential for an accurate diagnosis and necessary because of therapeutic and prognostic implications.

Author Correction: RNA cytosine methylation and methyltransferases mediate chromatin organization and 5-azacytidine response and resistance in leukaemia.

In the originally published version of this Article, the GAPDH loading control blot in Fig. 1a was inadvertently replaced with a duplicate of the DNMT2 blot in the same panel during assembly of the figure. This has now been corrected in both the PDF and HTML versions of the Article.

RNA cytosine methylation and methyltransferases mediate chromatin organization and 5-azacytidine response and resistance in leukaemia.

The roles of RNA 5-methylcytosine (RNA:m5C) and RNA:m5C methyltransferases (RCMTs) in lineage-associated chromatin organization and drug response/resistance are unclear. Here we demonstrate that the RCMTs, namely NSUN3 and DNMT2, directly bind hnRNPK, a conserved RNA-binding protein. hnRNPK interacts with the lineage-determining transcription factors (TFs), GATA1 and SPI1/PU.1, and with CDK9/P-TEFb to recruit RNA-polymerase-II at nascent RNA, leading to formation of 5-Azacitidine (5-AZA)-sensitive chromatin structure. In contrast, NSUN1 binds BRD4 and RNA-polymerase-II to form an active chromatin structure that is insensitive to 5-AZA, but hypersensitive to the BRD4 inhibitor JQ1 and to the downregulation of NSUN1 by siRNAs. Both 5-AZA-resistant leukaemia cell lines and clinically 5-AZA-resistant myelodysplastic syndrome and acute myeloid leukaemia specimens have a significant increase in RNA:m5C and NSUN1-/BRD4-associated active chromatin. This study reveals novel RNA:m5C/RCMT-mediated chromatin structures that modulate 5-AZA response/resistance in leukaemia cells, and hence provides a new insight into treatment of leukaemia.

Instructive Role of MLL-Fusion Proteins Revealed by a Model of t(4;11) Pro-B Acute Lymphoblastic Leukemia.

The t(4;11)(q21;q23) fuses mixed-lineage leukemia (MLL) to AF4, the most common MLL-fusion partner. Here we show that MLL fused to murine Af4, highly conserved with human AF4, produces high-titer retrovirus permitting efficient transduction of human CD34+ cells, thereby generating a model of t(4;11) pro-B acute lymphoblastic leukemia (ALL) that fully recapitulates the immunophenotypic and molecular aspects of the disease. MLL-Af4 induces a B ALL distinct from MLL-AF9 through differential genomic target binding of the fusion proteins leading to specific gene expression patterns. MLL-Af4 cells can assume a myeloid state under environmental pressure but retain lymphoid-lineage potential. Such incongruity was also observed in t(4;11) patients in whom leukemia evaded CD19-directed therapy by undergoing myeloid-lineage switch. Our model provides a valuable tool to unravel the pathogenesis of MLL-AF4 leukemogenesis.

Does Taking the Fellowship In-Service Hematopathology Examination and Performance Relate to Success on the American Board of Pathology Hematology Examination?

OBJECTIVES: The biannual Fellow In-Service Hematopathology Examination (FISHE) assesses knowledge in five content areas. We examined the relationship between taking the FISHE and performance on it with outcomes on the first attempted American Board of Pathology Hematology subspecialty certifying examination (ABP-HE). METHODS: The pass rate between the ABP-HE candidates who took the spring FISHE and those who did not were compared. The likelihood of fellows passing the ABP-HE based on their percentiles on the FISHE was also assessed. RESULTS: ABP-HE candidates who took the spring FISHE had a higher pass rate (96.4%) than those who did not (76.1%, P < .001). Spring FISHE performance, including total percentile and percentiles in four of five FISHE content areas, was only a weak predictor of passing the ABP-HE. CONCLUSIONS: Candidates who take the spring FISHE do better on the ABP-HE than those who do not. Most fellows passed the first attempted ABP-HE regardless of FISHE performance. Whether this is due to fellows making use of the FISHE as a self-evaluation tool to help identify and then correct their knowledge deficiencies remains to be determined.

Reduced DOCK4 expression leads to erythroid dysplasia in myelodysplastic syndromes.

Anemia is the predominant clinical manifestation of myelodysplastic syndromes (MDS). Loss or deletion of chromosome 7 is commonly seen in MDS and leads to a poor prognosis. However, the identity of functionally relevant, dysplasia-causing, genes on 7q remains unclear. Dedicator of cytokinesis 4 (DOCK4) is a GTPase exchange factor, and its gene maps to the commonly deleted 7q region. We demonstrate that DOCK4 is underexpressed in MDS bone marrow samples and that the reduced expression is associated with decreased overall survival in patients. We show that depletion of DOCK4 levels leads to erythroid cells with dysplastic morphology both in vivo and in vitro. We established a novel single-cell assay to quantify disrupted F-actin filament network in erythroblasts and demonstrate that reduced expression of DOCK4 leads to disruption of the actin filaments, resulting in erythroid dysplasia that phenocopies the red blood cell (RBC) defects seen in samples from MDS patients. Reexpression of DOCK4 in -7q MDS patient erythroblasts resulted in significant erythropoietic improvements. Mechanisms underlying F-actin disruption revealed that DOCK4 knockdown reduces ras-related C3 botulinum toxin substrate 1 (RAC1) GTPase activation, leading to increased phosphorylation of the actin-stabilizing protein ADDUCIN in MDS samples. These data identify DOCK4 as a putative 7q gene whose reduced expression can lead to erythroid dysplasia.

Epigenetic Control of Apolipoprotein E Expression Mediates Gender-Specific Hematopoietic Regulation.

Epigenetic alterations play a central role in the control of normal and malignant blood cell development. We demonstrate here that expression of a truncated DNA methyltransferase 3B isoform DNMT3B7, which has been shown to alter cellular epigenetic patterns, decreases the overall number of hematopoietic stem and progenitor cells (HSPCs), and markedly diminishes blood cell reconstitution within the female hormonal microenvironment. Gene expression profiling of HSPCs isolated from DNMT3B7 transgenic embryos identified Apolipoprotein E (Apoe) as overexpressed. The CpG island controlling Apoe expression had lower levels of modified cytosines in DNMT3B7 transgenic HSPCs, corresponding with the observed increase in gene expression. Furthermore, we observed that spleens and bone marrows of female mice transplanted with DNMT3B7 transgenic HSPCs express very high levels of Apoe. Finally, the introduction of Apoe-overexpressing HSPCs into male recipients decreased bone marrow engraftment, recapitulating our original observations in female recipients. Our work reveals a dynamic interplay between the intrinsic epigenetic changes in HSPCs and extrinsic endocrine factors acting on these cells to regulate the efficiency of HSPC engraftment and reconstitution. We have identified a novel mechanism by which gender-specific hormones modulate HSPC function, which could serve as a target for augmenting hematopoiesis in cases with limited HSC functionality.

Acute Myeloid Leukemia With Recurrent Cytogenetic Abnormalities.

OBJECTIVES: Session 1 of the 2013 Society for Hematopathology/European Association for Hematopathology Workshop was devoted to the cases of acute myeloid leukemia (AML) with recurrent cytogenetic abnormalities. METHODS: Based on World Health Organization 2008 criteria, seven specific translocations are defined as "recurrent" in AML. Of these seven, three are considered to be AML defining regardless of blast percentage. Workshop cases provided the opportunity to consider potential new AML-defining cytogenetic mutations, as well as other unique aspects of AML with cytogenetic abnormalities. RESULTS: Most of the 38 cases submitted were acute promyelocytic leukemia (APL) with t(15;17)(q24.1;q21.1) and so-called variants (12 cases), AML with t(8;21)(q22;q22) (seven cases), AML with inv(3)(q11q26.2) (six cases), and AML with 11q23 translocations (five cases). CONCLUSIONS: This review focuses on providing updated recommendations for the rapid diagnosis of APL, discussing the types and significance of variant RARA mutations in APL-like leukemias, and refining low-blast-count (oligoblastic) AML. In addition, the significance of unique morphologic, immunophenotypic, and genetic variations in AML defined by a recurrent cytogenetic abnormality is included.

The spectrum of somatic mutations in high-risk acute myeloid leukaemia with -7/del(7q).

-7/del(7q) occurs in half of myeloid malignancies with adverse-risk cytogenetic features and is associated with poor survival. We identified the spectrum of mutations that co-occur with -7/del(7q) in 40 patients with de novo or therapy-related myeloid neoplasms. -7/del(7q) leukaemias have a distinct mutational profile characterized by low frequencies of alterations in genes encoding transcription factors, cohesin and DNA-methylation-related proteins. In contrast, RAS pathway activating mutations occurred in 50% of cases, a significantly higher frequency than other acute myeloid leukaemias and higher than previously reported. Our data provide guidance for which pathways may be most relevant in the treatment of adverse-risk myeloid leukaemia.

Haploinsufficiency of del(5q) genes, Egr1 and Apc, cooperate with Tp53 loss to induce acute myeloid leukemia in mice.

An interstitial deletion of chromosome 5, del(5q), is the most common structural abnormality in primary myelodysplastic syndromes (MDS) and therapy-related myeloid neoplasms (t-MNs) after cytotoxic therapy. Loss of TP53 activity, through mutation or deletion, is highly associated with t-MNs with a del(5q). We previously demonstrated that haploinsufficiency of Egr1 and Apc, 2 genes lost in the 5q deletion, are key players in the progression of MDS with a del(5q). Using genetically engineered mice, we now show that reduction or loss of Tp53 expression, in combination with Egr1 haploinsufficiency, increased the rate of development of hematologic neoplasms and influenced the disease spectrum, but did not lead to overt myeloid leukemia, suggesting that altered function of additional gene(s) on 5q are likely required for myeloid leukemia development. Next, we demonstrated that cell intrinsic loss of Tp53 in hematopoietic stem and progenitor cells haploinsufficient for both Egr1 and Apc led to the development of acute myeloid leukemia (AML) in 17% of mice. The long latency (234-299 days) and clonal chromosomal abnormalities in the AMLs suggest that additional genetic changes may be required for full transformation. Thus, loss of Tp53 activity in cooperation with Egr1 and Apc haploinsufficiency creates an environment that is permissive for malignant transformation and the development of AML.

Complex or monosomal karyotype and not blast percentage is associated with poor survival in acute myeloid leukemia and myelodysplastic syndrome patients with inv(3)(q21q26.2)/t(3;3)(q21;q26.2): a Bone Marrow Pathology Group study.

Acute myeloid leukemia and myelodysplastic syndrome with inv(3)(q21q26.2)/t(3;3)(q21;q26.2) have a poor prognosis. Indeed, the inv(3)(q21q26.2)/t(3;3)(q21;q26.2) has been recognized as a poor risk karyotype in the revised International Prognostic Scoring System. However, inv(3)(q21q26.2)/t(3;3)(q21;q26.2) is not among the cytogenetic abnormalities pathognomonic for diagnosis of acute myeloid leukemia irrespective of blast percentage in the 2008 WHO classification. This multicenter study evaluated the clinico-pathological features of acute myeloid leukemia/myelodysplastic syndrome patients with inv(3)(q21q26.2)/t(3;3)(q21;q26.2) and applied the revised International Prognostic Scoring System to myelodysplastic syndrome patients with inv(3)(q21q26.2)/t(3;3)(q21;q26.2). A total of 103 inv(3)(q21q26.2)/t(3;3)(q21;q26.2) patients were reviewed and had a median bone marrow blast count of 4% in myelodysplastic syndrome (n=40) and 52% in acute myeloid leukemia (n=63) (P<0.001). Ninety-one percent of patients showed characteristic dysmegakaryopoiesis. There was no difference in overall survival between acute myeloid leukemia and myelodysplastic syndrome patients with inv(3)(q21q26.2)/t(3;3)(q21;q26.2) (12.9 vs. 7.9 months; P=0.16). Eighty-three percent of patients died (median follow up 7.9 months). Complex karyotype, monosomal karyotype and dysgranulopoiesis (but not blast percentage) were independent poor prognostic factors in the entire cohort on multivariable analysis. The revised International Prognostic Scoring System better reflected overall survival of inv(3)(q21q26.2)/t(3;3)(q21;q26.2) than the International Prognostic Scoring System but did not fully reflect the generally dismal prognosis. Our data support consideration of myelodysplastic syndrome with inv(3)(q21q26.2)/t(3;3)(q21;q26.2) as an acute myeloid leukemia with recurrent genetic abnormalities, irrespective of blast percentage.

GFI1B, EVI5, MYB--additional genes that cooperate with the human BCL6 gene to promote the development of lymphomas.

The BCL6 gene, which is expressed in certain B- and T-cell human lymphomas, is involved with chromosomal rearrangements and mutations in a number of these neoplasms. Lymphomagenesis is believed to evolve through a multi-step accumulation of genetic alterations in these tumors. We used retroviral insertional mutagenesis in transgenic mice expressing the human BCL6 transgene in order to identify genes that cooperate with BCL6 during lymphomatous transformation. We previously reported PIM1 as the most frequently recurring cooperating gene in this model. We now report three newly identified cooperating genes-GFI1B, EVI5, and MYB-that we identified in the lymphomas of retroviral-injected BCL6 transgenic mice (but not in retroviral-injected non-transgenic controls); mRNA and protein expression of GFI1B and EVI5 were decreased in the murine tumors, whereas MYB mRNA and protein expression were increased or decreased. These findings correlated with protein expression in human lymphomas, both B- and T-cell. Improved therapy of lymphomas may necessitate the development of combinations of drugs that target the alterations specific to each neoplasm.

Cell intrinsic and extrinsic factors synergize in mice with haploinsufficiency for Tp53, and two human del(5q) genes, Egr1 and Apc.

Therapy-related myeloid neoplasms (t-MN) are a late complication of the successful use of cytotoxic therapy for patients with cancer. A heterozygous deletions of the long arm of chromosome 5 [del(5q)], observed in 40% of patients, is associated with prior exposure to alkylating agents, and a high frequency of TP53 loss or mutation. In previous studies, we demonstrated that haploinsufficiency of 2 del(5q) genes, Egr1, and Apc, individually play a role in the pathogenesis of hematologic disease in mice. We now show that loss of one copy of Egr1 or Tp53 in an Apc haploinsufficient background (Apc (del/+)) accelerated the development of a macrocytic anemia with monocytosis, early features of t-MN. The development of anemia was significantly accelerated by treatment of mice with the alkylating agent, N-ethyl-N-nitrosourea (ENU), regardless of the levels of expression of Egr1 and Tp53. Transplantation of either wild type; Egr1(+/-); Tp53(+/-); Apc(del/+); or Egr1(+/-), Apc(del/+) bone marrow cells into lethally irradiated Apc(del/+) recipients resulted in rapid development of anemia that was further accelerated by administration of ENU to recipients, demonstrating that the Apc(del/+)-induced anemia was cell extrinsic and potentiated by ENU mutagenesis. These data emphasize the synergistic role of cell intrinsic and cell extrinsic (microenvironment) factors in the pathogenesis of t-MN, and raise awareness of the deleterious effects of cytotoxic therapy on the stromal microenvironment.

Dnmt3b is a haploinsufficient tumor suppressor gene in Myc-induced lymphomagenesis.

The drivers of abnormal DNA methylation in human cancers include widespread aberrant splicing of the DNMT3B gene, producing abnormal transcripts that encode truncated proteins that may act as dominant negative isoforms. To test whether reduced Dnmt3b dosage can alter tumorigenesis, we bred Dnmt3b(+/-) mice to Eµ-Myc mice, a mouse model susceptible to B-cell lymphomas. Eµ-Myc/Dnmt3b(+/-) mice showed a dramatic acceleration of lymphomagenesis, greater even than that observed in Eµ-Myc mice that express a truncated DNMT3B isoform found in human tumors, DNMT3B7. This finding indicates that Dnmt3b can act as a haploinsufficient tumor suppressor gene. Although reduction in both Dnmt3b dosage and expression of DNMT3B7 within the Eµ-Myc system had similar effects on tumorigenesis and DNA hypermethylation, different molecular mechanisms appear to underlie these changes. This study offers insight into how de novo DNA methyltransferases function as tumor suppressors and the sensitivity of Myc-induced lymphomas to DNA methylation.

Immune reconstitution after combined haploidentical and umbilical cord blood transplant.

Umbilical cord blood (UCB) stem cells are frequently employed for allogeneic stem cell transplant, but delayed myeloid and lymphoid immune reconstitution leads to increased risk of infections. We recently reported the clinical results of 45 patients enrolled on a pilot study combining UCB with a human leukocyte antigen (HLA)-haploidentical donor with reduced-intensity conditioning who showed rapid neutrophil and platelet recovery. We report here preliminary immune reconstitution data of these patients. Patients were assessed for lymphocyte subsets, T-cell diversity, Cylex ImmuKnow assay and serological response to pneumococcal vaccination. Natural killer (NK)-cell and B-cell reconstitution were rapid at 1 month and 3 months, respectively. T-cell recovery was delayed, with a gradual increase in the number of T-cells starting around 6 months post-transplant, and was characterized by a diverse polyclonal T-cell repertoire. Overall, immune reconstitution after haplo-cord transplant is similar to that seen after cord blood transplant, despite infusion of much lower cord blood cell dose.

CUX1 is a haploinsufficient tumor suppressor gene on chromosome 7 frequently inactivated in acute myeloid leukemia.

Loss of chromosome 7 and del(7q) [-7/del(7q)] are recurring cytogenetic abnormalities in hematologic malignancies, including acute myeloid leukemia and therapy-related myeloid neoplasms, and associated with an adverse prognosis. Despite intensive effort by many laboratories, the putative myeloid tumor suppressor(s) on chromosome 7 has not yet been identified.We performed transcriptome sequencing and SNP array analysis on de novo and therapy-related myeloid neoplasms, half with -7/del(7q). We identified a 2.17-Mb commonly deleted segment on chromosome band 7q22.1 containing CUX1, a gene encoding a homeodomain-containing transcription factor. In 1 case, CUX1 was disrupted by a translocation, resulting in a loss-of-function RNA fusion transcript. CUX1 was the most significantly differentially expressed gene within the commonly deleted segment and was expressed at haploinsufficient levels in -7/del(7q) leukemias. Haploinsufficiency of the highly conserved ortholog, cut, led to hemocyte overgrowth and tumor formation in Drosophila melanogaster. Similarly, haploinsufficiency of CUX1 gave human hematopoietic cells a significant engraftment advantage on transplantation into immunodeficient mice. Within the RNA-sequencing data, we identified a CUX1-associated cell cycle transcriptional gene signature, suggesting that CUX1 exerts tumor suppressor activity by regulating proliferative genes. These data identify CUX1 as a conserved, haploinsufficient tumor suppressor frequently deleted in myeloid neoplasms.