Differential expression of ribosomal proteins in myelodysplastic ...

JCP Online First, published on September 25, 2015 as 10.1136/jclinpath-2015-203093 Short report

Differential expression of ribosomal proteins in myelodysplastic syndromes Elizabeth B Rinker,1,2 Julie C Dueber,1,3 Julianne Qualtieri,1,4 Jason Tedesco,1,5 Begum Erdogan,1 Amma Bosompem,1 Annette S Kim1,6 For numbered affiliations see end of article. Correspondence to Dr Annette S Kim, Brigham and Women’s Hospital, 75 Francis Street, Thorn 613A, Boston, MA 02115, USA; [email protected] Received 22 April 2015 Accepted 3 September 2015

ABSTRACT Aberrations of ribosomal biogenesis have been implicated in several congenital bone marrow failure syndromes, such as Diamond–Blackfan anaemia, Shwachman–Diamond syndrome and Dyskeratosis Congenita. Recent studies have identified haploinsufficiency of RPS14 in the acquired bone marrow disease isolated 5q minus syndrome, a subtype of myelodysplastic syndromes (MDS). However, the expression of various proteins comprising the ribosomal subunits and other proteins enzymatically involved in the synthesis of the ribosome has not been explored in non5q minus MDS. Furthermore, differences in the effects of these expression alterations among myeloid, erythroid and megakaryocyte lineages have not been well elucidated. We examined the expression of several proteins related to ribosomal biogenesis in bone marrow biopsy specimens from patients with MDS (5q minus patients excluded) and controls with no known myeloid disease. Specifically, we found that there is overexpression of RPS24, DKC1 and SBDS in MDS. This overexpression is in contrast to the haploinsufficiency identified in the congenital bone marrow failure syndromes and in acquired 5q minus MDS. Potential mechanisms for these differences and aetiology for these findings in MDS are discussed. INTRODUCTION

To cite: Rinker EB, Dueber JC, Qualtieri J, et al. J Clin Pathol Published Online First: [ please include Day Month Year] doi:10.1136/jclinpath-2015203093

The myelodysplastic syndromes (MDS) are a group of haematopoietic disorders typically affecting the elderly. These syndromes are characterised by ineffective haematopoiesis, which is fatal within 3 years in 65% of patients either from cytopenias or from transformation to acute myeloid leukaemia (AML).1 The cause of MDS is largely unknown, although the spectrum of the disorders is so broad that multiple aetiologies are possible. A wide range of genetic and epigenetic aberrations have been implicated in MDS, including numerous recurrent genetic mutations, global hypermethylation, gross karyotypic changes, alterations in transcriptional profiling and miRNA expression and pro-inflammatory changes. While less common, inherited bone marrow failure syndromes share a propensity for the development of MDS and have been studied extensively. Recent work has shown that several of these syndromes demonstrate dysregulated biosynthesis of ribosomes.2 Haploinsufficiencies for ribosomal proteins have been linked to defects in pre-rRNA processing and have been associated with decreased expression of ribosomal genes in in vitro and in vivo studies.2 Many patients with Diamond– Blackfan anaemia (DBA) have mutations in a number of genes that encode for proteins directly

incorporated into the small and large subunits of the ribosome. The two most common of these are RPS19 and RPS24 in addition to numerous others. Proteins involved in ribosome synthesis have also been implicated in other bone marrow failure syndromes. Shwachman-Diamond syndrome (SDS) is caused by mutations in the SBDS gene, which encodes a protein that acts as a shuttle for ribosomal subunits, carrying them to the cytoplasm, and also stabilises the mitotic spindle.2 3 X-linked Dyskeratosis Congenita is caused by mutations in DKC1, whose product dyskerin catalyses the requisite pseudo-uridylation of ribosomal RNA and is also involved in telomerase activity.2 Abnormal ribosome synthesis in erythrocytes may lead directly to the anaemia seen in many of these diseases. With the inability of these diseased cells to appropriately translate the globins, unbound haem and iron accumulate, triggering p53 signalling pathways and thereby stimulating the apoptosis of the erythrocytes and causing the anaemia seen in many of the bone marrow failure syndromes.4 5 Interestingly, anaemia is the most common presenting cytopenia in MDS as well. Of particular interest is the observation that patients with the 5q minus subtype of MDS are also haploinsufficient for a gene encoding a protein in the small ribosomal subunit, RPS14.6 Murine models have shown that this haploinsufficiency alone gives rise to a macrocytic anaemia.7 While 5q minus syndrome is a distinct diagnostic entity within the broad category of MDS, this disease is grouped with the other MDS subtypes due to certain commonalities of ineffective haematopoiesis, manifested pathologically as dysplasia, and clinically with cytopenias (in particular anaemia). Transcriptional profiling studies on DBA, SDS and non-5q minus MDS have been performed previously.8–10 When the discriminatory ribosomerelated genes that characterise the individual disease entities are compared, several ribosomal genes are commonly identified as markers in all three conditions. These genes are: RPS14, RPS10, RPS24, RPL36 and RPL14. In this study, we investigate the role of altered ribosomal protein expression as a possible phenomenon associated with the ineffective haematopoiesis that characterises MDS. We hypothesise that acquired ribosomopathies may also contribute to the pathogenesis of non-5q minus MDS. Using immunohistochemistry (IHC), we examined the expression of several ribosomal proteins to determine if there is an abnormal expression of these proteins in the bone marrows of patients with myelodysplasia as compared to normal controls. Unlike previous transcriptional

Rinker EB, et al. J Clin Pathol 2015;0:1–5. doi:10.1136/jclinpath-2015-203093

Copyright Article author (or their employer) 2015. Produced by BMJ Publishing Group Ltd under licence.

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Short report profiling studies, IHC enables the examination of the protein expression in the entire marrow while morphologically distinguishing the specific cells in which the expression occurs.

METHODS Patient samples All patient samples were obtained with approval from the Institutional Review Board at the Vanderbilt University Medical Center. Samples were identified by querying the electronic laboratory information system (LIS) at the Vanderbilt University Medical Center (Triple G) for the terms ‘myelodysplastic syndrome’, ‘refractory cytopenia’ or ‘refractory anaemia’ in the diagnostic lines from 2004 through 2011. Study samples were limited to well-documented cases of non-5q minus MDS. A total of 22 archival paraffin-embedded bone marrow biopsy specimens from patients with MDS were obtained. This included two cases of refractory anaemia (RA), 14 cases of refractory cytopenia with multilineage dysplasia (RCMD), one case of RCMD-ring sideroblasts (RS), four cases of RA with excess blasts 1 (RAEB-1), and one case of therapy-related MDS. There were 16 males and six females with a median age of 64.5 (range 44–85 years). For comparison, 22 control bone marrow samples were also obtained (eight males, 14 females with a median age of 60.5, range 14–80 years) from patients with no known myeloid disease. Since this is a retrospective study utilising archival pathology materials, bone marrow samples from healthy donors were not available. The majority of these cases were negative bone marrows obtained for the purposes of staging for lymphoma. These cases were all obtained and processed identically to the MDS samples. Two additional samples were obtained from patients with no known haematologic malignancies undergoing orthopaedic procedures. Thirteen of the 22 patients did have at least one cytopenia (the rest had no cytopenias), which was attributed both clinically and pathologically to causes other than MDS (anaemia of chronic disease, renal insufficiency, cirrhosis). Unfortunately, since these samples were obtained from 2008 through 2010 and the blocks utilised for this study have been decalcified, mutational analysis was never performed to confirm definitively that early myeloid clones were not present.

Selection of ribosomal proteins Using published gene expression profiling studies of SDS,9 DBA8 and MDS,10 we chose to examine three of the five dysregulated ribosomal proteins, which represented a union between the three sets of data and could be assessed by commercially available IHC-compatible antibodies: RPS14, RPS24 and RPL36. In order to further probe the involvement of potential pathways of ribosomal biogenesis, we also selected DKC1 and SBDS as other proteins that regulate the synthesis of the ribosomes. We included CASP3 and Ki67 to attempt to address the effect of ribosomal dysregulation on apoptosis and proliferation, respectively. Corresponding bone marrow biopsy sections were interrogated via immunohistochemical staining.

Immunohistochemistry Immunohistochemical analysis was performed on 4 mm sections from the bone marrow preparations. Staining was performed on the EnVision+horseradish peroxidase system (DAKO, Carpinteria, California, USA) using antibodies against RPS11 (#sab1402345; Sigma-Aldrich, St. Louis, Missouri, USA), RPS14 (#16683-1-ap; Proteintech Group, Chicago, Illinois, USA), RPS24 (#hpa003364; Sigma-Aldrich, St.Louis, Missouri, USA), SBDS (#LS-C40532; LifeSpan Biosciences, Seattle, 2

Washington DC, USA), DKC1 (#nbp1-40097; Novus Biologicals, Littleton, Colorado, USA), RPL36 (#ab74737; Abcam, Cambridge, Massachusetts, USA), CASP3 (#mbs440008; MyBioSource, San Diego, California, USA), and Ki67 (#M7240, Dako, Carpenteria, California, USA).

Data analysis Expression levels of the proteins were counted in 200 nucleated cells in a blinded fashion by two independent reviewers. A standard 0–3 point scale for stain intensity was used for scoring. Stain indices for each sample were determined based on the percentage of positive staining cells per haematologic lineage ( per cent of positive cells), with any staining intensity greater than 0 considered positive. The stain indices of the two reviewers were considered individually as well as averaged for each sample. The cellular localisation pattern (nuclear vs cytoplasmic) was also documented based on the counterstain morphology. Unpaired, two-tailed, Student’s t-test analysis was used to ascertain the significance of stain index ( per cent of positive cells) differences between MDS and normal control samples. Since the majority of the cases fell into the category of RCMD (14 of 22) or ‘no increase in blasts’ (17 or 22), the study was not sufficiently powered to analyse the stain index with regards to either MDS subclassification or blast percentage.

RESULTS Immunohistochemical studies performed on paraffin-embedded tissue demonstrate statistically significant differences in expression of the ribosomal proteins RPS24, DKC1 and SBDS (22 MDS, 22 control samples each) in MDS bone marrow core biopsies compared to normal controls (table 1 and figure 1). There was no significant difference in the staining intensity of a given antibody in a given cell lineage between MDS and control samples. However, the stain index ( per cent of positive cells per lineage) calculated by two independent reviewers identified that cytoplasmic expression of RPS24 in maturing myeloid precursors in MDS biopsies is increased compared with normal controls ( p value=0.0025). There were no significant expression differences in the erythroid or megakaryocytic lineages. DKC1 was expressed in the nucleus in a greater percentage of cells in MDS cases compared with normal controls, with evidence of differential expression within immature and mature myeloid cells, immature erythroid precursors and megakaryocytes (total myeloid p value=0.00017, erythroid p value=0.00056 and megakaryocytic p value=0.027). SBDS was found in the cytoplasm of increased numbers of immature erythroid precursors in MDS cases compared with normal controls ( p value=0.013). RPS14 and RPL36 demonstrated expression within an increased number of cells in the myeloid lineage on an initial subset of cases. However, these findings did not reach significance on the full data set. By contrast, the expression of RPS14 found in a decreased number of megakaryocytes ( p value=0.0093). There was no statistical significance in stain index or intensity per lineage for CASP3 or Ki67 ( performed on a limited sample set, data not shown).

DISCUSSION These immunohistochemical studies demonstrate that there is increased expression of selected ribosomal proteins in the maturing cells of MDS as compared with control samples. It should be noted that control samples from healthy donors were not available in this retrospective study. Accordingly, the study is limited by the use of bone marrow samples from patients that Rinker EB, et al. J Clin Pathol 2015;0:1–5. doi:10.1136/jclinpath-2015-203093

Short report Table 1 Immunohistochemical scoring results of ribosomal proteins from two independent pathologist reviewers by lineage, scored as percent positive RPS14

RPS24

RPL36

DKC1

SBDS

Myelo Eryth Eryth Myelo Myelo % Eryth % Megak % % % Megak % Myelo % % Megak % % Eryth % Megak % Myelo % Eryth % Megak % Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Controls Average SD

29 20

MDS Average SD p Value

31 25 0.71

5 8 7 10 0.43

28 42

14 12

51 37

10 23 0.0093

26 56 23 35 0.0025 0.47

47 49

42 29

5 5

72 35

59 27

6 8

75 41

44 47 0.74

48 19 0.53

6 5 0.52

83 37 0.36

84 14 93 15 17 16 0.00017 0.00056 0.027

56 22

3.18 4.46

66.43 30.44

61 22 0.38

8.36 9.55 0.013

81.03 31.88 0.12

Eryth, erythroid lineage; MDS, myelodysplastic syndromes; Megak, megakaryocytic lineage; Myelo, myeloid lineage.

did require a bone marrow study for their clinical state. However, control patients were screened to have no known myeloid neoplasm, and their marrows were without involvement by any overt malignancies. Three ribosomal proteins (RPS24, DKC1 and SBDS) are expressed in the MDS cases in an increased percentage of cells compared with the control cases. This increase in expression is found specifically in the myeloid lineage for RPS24, all three lineages for DKC1, and in immature erythroid cells for SBDS. Importantly, this is unlike congenital bone marrow failure syndromes, which are associated with

ribosomal protein loss of function (haploinsufficiency). Decreased expression of RPS14 was noted only in the megakaryocytic lineage of MDS patients. The functions of DKC1, RPS24 and SBDS are summarised in figure 2. DKC1 encodes dyskerin, which acts as a nucleolar protein associated with the snoRNPs involved in rRNA modification.2 Dyskerin associates with a specific group of snoRNPs known as H/ACA, which function in the pseudo-uridylation of rRNAs.11 Mutations in DKC1 have been implicated in the pathogenesis of the X-linked form of Dyskeratosis Congenita

Figure 1 Examples of RPS24 staining on (A) myelodysplastic syndromes (MDS) marrow (400×) and (B) normal marrow (400×). Examples of DKC1 staining on (C) MDS marrow (400×) and (D) normal marrow (400×). Examples of SBDS staining on (E) MDS marrow (400×) and (F) normal marrow (400×).


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Short report

Figure 2 Schematic demonstrating the functions of DKC1, RPS24 and SBDS. (DKC), which is associated with a more severe phenotype compared with the autosomal dominant form of DKC. Mutations in RPS24 impair pre-rRNA processing of the 18S rRNA, which leads to decreased production of the 40S ribosomal subunit.2 A prior study has demonstrated that the depletion of a single ribosomal subunit protein causes a reduction in the amount of free 40S subunits and a significant reduction in the amount of mature 80S ribosomes.12 RPS24 is a ribosomal protein-encoding gene that along with at least 12 other genes, nearly all of which encode for ribosomal proteins,13 has been implicated in the pathogenesis of a subset of patients with DBA. Mutations in different ribosomal genes have been linked to distinct clinical phenotypes.2 Previous studies using murine models demonstrated that SBDS promotes eIF6 release from isolated 60S ribosomal subunits.14 More recent studies indicate that the loss of SBDS protein expression in human cells impairs association of the 40S and 60S subunits.3 However, SBDS is a multifunctional protein, and nonribosomal activities may play a role in producing the clinical phenotype of SDS.15 16 Mutations in the SBDS gene have been implicated as the cause of SDS, with approximately 90% of the patients with SDS found to have biallelic SBDS mutations.17 SDS is an autosomal recessive disease characterised by exocrine pancreatic insufficiency, ineffective haematopoiesis and an increased risk of myelodysplasia and leukaemia. Neutropenia is the most common haematologic problem in SDS. In this study, the increased expression of ribosomal proteins within MDS cases compared with normal controls was found typically on morphologically more mature/maturing cells of the three lineages rather than on the blasts. This is in contrast to previous studies performed on CD34-positive cells isolated from patients with MDS that found decreases in ribosomal protein expression in cases with MDS compared with controls by gene expression profiling.10 Sohal et al10 conducted a meta-analytical comparison of the transcriptional profiles of CD34-positive cells from non-5q minus cases of MDS along with normal CD34-positive marrow precursors. In this study, CD34-positive precursors were isolated using magnetic cell separation columns. They observed that multiple ribosomal protein 4

genes, including RPS14, RPS24 and RPL36, were downregulated in their non-5q minus MDS study set.10 Some downregulated ribosomal protein genes were associated with genomic deletions when a subset of MDS cases was examined by high resolution array comparative genome hybridisation (aCGH), including RPL36 and RPS14.10 By contrast, our study did not examine expression levels, but rather the percentage of positive cells per lineage since IHC is an insensitive method to assess subtle changes in expression, particularly from low baseline levels. In addition, as our study did not differentiate the immunohistochemical staining patterns of immature CD34-positive haematopoietic precursors from more mature cells, we cannot exclude the possibility that there is differential expression of ribosomal proteins between CD34-positive and more mature precursors. The discrimination between ribosomal protein expressions specifically in CD34-positive cells versus more mature cells may be a potential avenue for future study. In a separate study, RPS14 transcription was noted to be downregulated in 53% of MDS patients without 5q minus associated with higher platelet counts, lenalidomide response, and higher 2-year survival.18 Although the differences between this study and our study are similar to those described in the previous paragraph, we also identified fewer megakaryocytes, which expressed RPS14. However, where data were available, there was no significant correlation with platelet count (data not shown) and outcome data were too limited to permit the analysis of this variable. Several of the ribosomal proteins demonstrating increased expression levels by IHC in cases with MDS in the present study are predicted to be regulated by microRNAs (miRNAs) previously shown to be underexpressed in MDS (RPS24: miR-342, DKC1: miR-150, SBDS: miR-378, miR-140, and miR-103).19–21 Since miRNAs most typically regulate protein expression by sequence-specific translational repression, underexpression of MDS-associated miRNAs could result in the overexpression of their mRNA targets, in accordance with the findings presented here. Notably, miR-103 and miR-150 are implicated in the shift from erythroid to megakaryocytic differentiation.22–25 The decreased expression of these miRNAs in the cases of non-5q minusMDS may contribute to the erythroid hyperplasia that is often seen in MDS.19 In contrast, an increase in miR-150 expression is seen in the 5q minus subtype of MDS, which may feature megakaryocytic hyperplasia.24 However, there is not always a clear relationship between miRNA levels and mRNA levels, and there are numerous mechanisms for differential transcriptional expression that do not involve miRNA-associated pathways.19 Therefore, there may be other unelucidated mechanisms for the observed overexpression of these ribosomal proteins in MDS. Our evidence supports the hypothesis that acquired ribosomopathies may contribute to the pathogenesis of non-5q minus MDS, although possibly not in the same manner as that seen in inherited bone marrow failure syndromes. This study demonstrates the evidence of increased expression of ribosomal proteins in the maturing cells of higher grade MDS (RCMD and RAEB-1). These results are unlike the expression of these proteins in congenital bone marrow failure syndromes, where they are associated with ribosomal protein loss of function, highlighting the different aetiologies of acquired MDS and the inherited bone marrow failure syndromes. In addition, the markers may serve as important gateways to understand the specific pathobiology of MDS. Lastly, DKC1 in particular may prove to be a useful biomarker in the diagnosis of MDS. The relevance of these genes in the prognosis of MDS and response to therapy remains to be elucidated. Rinker EB, et al. J Clin Pathol 2015;0:1–5. doi:10.1136/jclinpath-2015-203093

Short report 6

Take home message 7

▸ Several ribosomal proteins are dysregulated in myelodysplastic syndrome (MDS), with increased expression of RPS24, DKC1 and SBDS and decreased expression of RPS14, each in specific maturing bone marrow lineages. ▸ These findings highlight the different aetiologies of acquired MDS and the inherited bone marrow failure syndromes, despite their clinical similarities.

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Author affiliations 1 Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee, USA 2 Department of Pathology, Louisiana State University Health Sciences Center, New Orleans, Louisiana, USA 3 Department of Pathology, University of Kentucky, Lexington, Kentucky, USA 4 Department of Pathology, University of Cincinnati, Cincinnati, Ohio, USA 5 Sarasota Pathology, Sarasota, Florida, USA 6 Department of Pathology, Brigham and Women’s Hospital, Boston, Massachusetts, USA Handling editor Mary Frances McMullin Contributors Design of study was conducted by EBR, JCD, JQ, JT and ASK. Experiments were conducted by all authors. The manuscript was developed by EBR, JCD and ASK. All authors have approved the manuscript. Competing interests None declared.

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Ethics approval Institutional Review Board of Vanderbilt University. Provenance and peer review Not commissioned; externally peer reviewed.

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