Rajko Kusec

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.

B and CTL responses to the ALK protein in patients with ALK‐positive ALCL

Anaplastic lymphoma kinase (ALK)‐positive anaplastic large cell lymphoma (ALCL) has a good prognosis compared to ALK‐negative ALCL, possibly as a result of the immune recognition of the ALK proteins. The aim of our study was to investigate the presence of both a B and cytotoxic T cell (CTL) response to ALK in ALK‐positive ALCL. We confirmed the presence of an antibody response to ALK in all 9 ALK‐positive ALCL patients investigated. An ELISpot assay was used to detect a γ‐interferon (IFN) T cell response after short term culture of mononuclear blood cells with 2 ALK‐derived HLA‐A*0201 restricted peptides: ALKa and ALKb. A significant γ‐IFN response was identified in all 7 HLA‐A*0201‐positive ALK‐positive ALCL patients but not in ALK‐negative ALCL patients (n = 2) or normal subjects (n = 6). CTL lines (>95% CD8‐positive) raised from 2 ALK‐positive ALCL patients lysed ALK‐positive ALCL derived cell lines in a MHC‐Class I restricted manner. This is the first report of both a B cell and CTL response to ALK in patients with ALK‐positive ALCL. This response persisted during long‐term remission. The use of modified vaccinia virus Ankara (MVA) to express ALK is also described. Our findings are of potential prognostic value and open up therapeutic options for those ALK‐positive patients who do not respond to conventional treatment.

Cryptic splicing events in the iron transporter ABCB7 and other key target genes in SF3B1 -mutant myelodysplastic syndromes

The splicing factor SF3B1 is the most frequently mutated gene in myelodysplastic syndromes (MDS), and is strongly associated with the presence of ring sideroblasts (RS). We have performed a systematic analysis of cryptic splicing abnormalities from RNA sequencing data on hematopoietic stem cells (HSCs) of SF3B1-mutant MDS cases with RS. Aberrant splicing events in many downstream target genes were identified and cryptic 3′ splice site usage was a frequent event in SF3B1-mutant MDS. The iron transporter ABCB7 is a well-recognized candidate gene showing marked downregulation in MDS with RS. Our analysis unveiled aberrant ABCB7 splicing, due to usage of an alternative 3′ splice site in MDS patient samples, giving rise to a premature termination codon in the ABCB7 mRNA. Treatment of cultured SF3B1-mutant MDS erythroblasts and a CRISPR/Cas9-generated SF3B1-mutant cell line with the nonsense-mediated decay (NMD) inhibitor cycloheximide showed that the aberrantly spliced ABCB7 transcript is targeted by NMD. We describe cryptic splicing events in the HSCs of SF3B1-mutant MDS, and our data support a model in which NMD-induced downregulation of the iron exporter ABCB7 mRNA transcript resulting from aberrant splicing caused by mutant SF3B1 underlies the increased mitochondrial iron accumulation found in MDS patients with RS.

Treatment of chronic lymphocytic leukemia in early and stable phase of the disease : long-term results of a randomized trial

Abstract: In 1982 the IGCI CLL cooperative group decided to investigate the usefulness of treating, at diagnosis B‐cell chronic lymphocytic leukemia (CLL) in early and stable phase of the disease. From January 1982 to December 1986, 148 patients were randomized either to receive immediate treatment with chlorambucil (CLB) or to defer therapy to the time of progression. The early and stable phase of the disease was defined by a total tumor mass (TTM) score < 9, the absence of anemia or thrombocytopenia and a doubling time > 12 months. The main end‐point of the study was survival. At the last evaluation in April 1993, after a median follow‐up of 75 months, no significant difference was found in overall survival between early vs. deferred treatment patients from every cause of death as well as from death due to CLL‐related causes only. The same results were obtained when the patients in more favorable stages, such as Binet stage A and TTM < 4.5, were considered. Interestingly, the incidence of epithelial cancer was similar in the two groups. Early treatment was associated with a significantly better response and a lower progression rate. From this long‐term experience, it can be concluded that immediate chemotherapy with CLB is not beneficial for CLL patients in early and stable phase of the disease in terms of survival.

The FOXP1 transcription factor is expressed in the majority of follicular lymphomas but is rarely expressed in classical and lymphocyte predominant Hodgkin's lymphoma.

SummaryThe expression of the FOXP1 forkhead/winged helix transcription factor has been investigated in normal and neoplastic lymphoid tissues using the FOXP1-specific monoclonal antibody, JC12. Using single and double immunoenzymatic staining, FOXP1 expression has been studied in a series of classical and lymphocyte predominant Hodgkin’s lymphomas, follicle centre lymphomas and Hodgkin’s lymphoma-derived cell lines. Neoplastic cells in the majority of classical and lymphocyte predominant Hodgkin’s lymphoma were FOXP1-negative. In two cases of classical Hodgkin’s lymphoma, the tumour cells showed mislocalisation of FOXP1 to the cytoplasm, while in one case of lymphocyte predominant Hodgkin’s lymphoma, and in the Hodgkin’s lymphoma cell line KMH2, scattered tumour cells showed nuclear expression of FOXP1. In contrast, the tumour cells in the majority of follicle centre lymphomas showed strong nuclear FOXP1 reactivity. Double labelling studies of lymphoid tissue indicated that a variable proportion of CD20-positive germinal centre B cells express FOXP1 while the vast majority of CD30-positive cells are FOXP1-negative. The heterogeneity of FOXP1 expression in germinal centre-derived lymphomas, may have more to do with the transforming events underlying these distinct types of lymphoma than with their common origin.

Aberrant expression of the neuronal transcription factor FOXP2 in neoplastic plasma cells.

FOXP2 mutation causes a severe inherited speech and language defect, while the related transcription factors FOXP1, FOXP3 and FOXP4 are implicated in cancer. FOXP2 mRNA and protein expression were characterised in normal human tissues, haematological cell lines and multiple myeloma (MM) patients’ samples. FOXP2 mRNA and protein were absent in mononuclear cells from different anatomical sites, lineages and stages of differentiation. However, FOXP2 mRNA and protein was detected in several lymphoma (8/20) and all MM‐derived cell lines (nu2003=u20034). FOXP2 mRNA was expressed in bone marrow samples from 96% of MM patients (24/25), 66·7% of patients with the pre‐neoplastic plasma cell proliferation monoclonal gammopathy of undetermined significance (MGUS) (6/9), but not in reactive plasma cells. The frequency of FOXP2 protein expression in CD138+ plasma cells was significantly higher in MGUS (Pu2003=u20030·0005; mean 46·4%) and MM patients (Pu2003≤u20030·0001; mean 57·3%) than in reactive marrows (mean 2·5%). FOXP2 (>10% nuclear positivity) was detectable in 90·2% of MM (55/61) and 90·9% of MGUS (10/11) patients, showing more frequent expression than CD56 and labelling 75% of CD56‐negative MM (9/12). FOXP2 represents the first transcription factor whose expression consistently differentiates normal and abnormal plasma cells and FOXP2 target genes are implicated in MM pathogenesis.

Bone morphogenetic proteins and receptors are over-expressed in bone-marrow cells of multiple myeloma patients and support myeloma cells by inducing ID genes

We assessed the expression pattern and clinical relevance of BMPs and related molecules in multiple myeloma (MM). MM bone-marrow samples (n=32) had increased BMP4, BMP6, ACVR1 and ACVR2A, and decreased NOG expression compared with controls (n=15), with BMP6 having the highest sensitivity/specificity. Within MM bone-marrow, the source of BMPs was mainly CD138(+) plasma-cell population, and BMP6 and ACVR1 expression correlated with plasma-cell percentage. Using myeloma cell lines NCI H929 and Thiel we showed that BMPs induced ID1, ID2 and IL6, and suppressed CDKN1A and BAX gene expression, and BAX protein expression. Finally, BMPs partially protected myeloma cells from bortezomib- and TRAIL-induced apoptosis. We concluded that BMPs may be involved in MM pathophysiology and serve as myeloma cell biomarkers.

Exacerbation of Psoriasis after Treatment with Alpha-Interferon

Rajko Kušec, MD, Slobodanka Ostojić, MD, Ana Planinc-Peraica, MD, Hrvoje Minigo, MD Branimir Jakšić, MD, PhD, Department of Hematology, Clinical Hospital ‘O Novosel’ Zajčeva 19, 41000 Zagreb (Yugoslavia) Dear Sir, At the International Symposium on Interferons and Related Lym-phokines held in Berlin 1989, Harrison [1] described the exacerbation of psoriasis after treatment with α-interferon. As we have observed a similar behaviour of the psoriatic process under treatment with α-interferon we would like to briefly present our experience with a comment on the possible explanation for this phenomenon. We have treated a 54-year-old male patient with hairy cell leukaemia and psoriasis with recombinant α2b-interferon. Therapy with α-interferon (Intron A; Schering) in doses of 3 MU s.c. thrice weekly was started because of the activation of malignant disease. The condition had been stable previously for 9 years not requiring specific or supportive therapy (splenec-tomy was performed early at the time of diagnosis). A psoriasis with skin and joint involvement was established 3 years from the diagnosis of hairy cell leukaemia. The disease was stationary with usual topical therapy and occasionally nonsteroid analgesics. A month after starting α-interferon treatment, which was already showing haematological improvement, the exacerbation of psoriatic skin manifestations occurred with arthralghias, limitations of movement in affected joints and swelling of small finger joints. This condition did not conform with a pattern of typical interferon-related side-effects. Interferon treatment was discontinued for a month with a substantial improvement of psoriasis entering again a stationary phase. After this period α-interferon was restarted, and again exacerbation of psoriasis was noted. This resulted in the definite exclusion of the patient from αinterferon treatment for hairy cell leukaemia. The cause for this finding is not clear. At the same symposium a study detecting various interferons in psoriatic skin was presented providing data on the presence of α-interferon in the active disease, while it was absent in the stable phase or normal skin [2]. Searching for other mechanisms that could mediate/regulate a psoriatic process, an increased adrenal activity during interferon therapy should be considered [3], which, we believe, deserves further clarification. References Harrison P: Exacerbation of psoriasis with alpha interferon (abstract). J Invest Dermatol 1989;93:555.

Methionine enkephalin suppresses metabolic activity of a leukemic cell line (NALM-1) and enhances CD10 expression.

NALM-1 cells (a cell line derived from human pre-B leukemia) were exposed to the opioid pentapeptide methionine-enkephalin (Met-enkephalin) and/or to thiorphan, an inhibitor of the enzyme that degrades the enkephalins (membrane endopeptidase EC 3.4.24.11, CALLA, the CD10 marker). Metabolic and proliferative activity was assessed after 6, 24 and 48 h in microplates using a colorimetric assay with vital dye MTT. CD10 expression was determined by means of semi-quantitative RT-PCR. Exposure to the Met-enkephalin at concentrations of 10(-8)-10(-6) M for 6 h reduced the MTT-activity, and after 24 and 48 h the suppression waned. Thiorphan (5 x 10(-6) M) abrogated the suppressive effect of the enkephalin, and after 6 h converted suppression into stimulation. Met-enkephalin (10(-6) M) increased and thiorphan (2.5 x 10(-6)-10(-6) M) decreased expression of CD10 at the RNA level. Suppression of the MTT uptake was attributed to the products of Met-enkephalin degradation caused by the enzymatic activity of CD10.

CSNK1A1 mutations and gene expression analysis in myelodysplastic syndromes with del(5q).

Mutations of CSNK1A1, a gene mapping to the commonly deleted region of the 5q‐ syndrome, have been recently described in patients with del(5q) myelodysplastic syndromes (MDS). Haploinsufficiency of Csnk1a1 in mice has been shown to result in β‐catenin activation and expansion of haematopoietic stem cells (HSC). We have screened a large cohort of 104 del(5q) MDS patients and have identified mutations of CSNK1A1 in five cases (approximately 5%). We have shown up‐regulation of β‐catenin target genes in the HSC of patients with del(5q) MDS. Our data further support a central role of CSNK1A1 in the pathogenesis of MDS with del(5q).