MicroRNA expression profiling identifies molecular signatures associated with anaplastic large cell...

doi:10.1182/blood-2012-08-447375Prepublished online June 25, 2013;

Wing C. Chan

andDennis D. Weisenburger, Patricia Aoun, Sherrie L. Perkins, Timothy W. McKeithan, Giorgio Inghirami M. Lachel, Can Kucuk, Chun-Sun Jiang, Xiaozhou Hu, Sharathkumar Bhagvati, Timothy C. Greiner,Dybkaer, Megan S. Lim, Roberto Piva, Antonella Barreca, Elisa Pellegrino, Elisa Spaccarotella, Cynthia Cuiling Liu, Javeed Iqbal, Julie Teruya-Feldstein, Yulei Shen, Magdalena Julia Dabrowska, Karen with anaplastic large cell lymphomaMicroRNA expression profiling identifies molecular signatures associated

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MicroRNA expression profiling identifies molecular signatures associated with anaplastic large cell lymphoma

Cuiling Liu1, 6#, Javeed Iqbal1#, Julie Teruya‐Feldstein2 , Yulei Shen1, Magdalena Julia Dabrowska3, Karen Dybkaer3 , Megan S Lim5, Roberto Piva4, Antonella Barreca4, Elisa Pellegrino4, Elisa Spaccarotella4, Cynthia M Lachel1, Can Kucuk1, Chun‐Sun Jiang1, Xiaozhou Hu1, Sharathkumar Bhagvati1, Timothy C Greiner1, Dennis D Weisenburger1, Patricia Aoun1, Sherrie L. Perkins7,Timothy W McKeithan1, Giorgio Inghirami4, Wing C Chan1*

1Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, NE, USA; 2 Department of Pathology, Memorial Sloan‐Kettering Cancer Center, New York, NY 10021, USA; 3Department of Hematology, Aalborg Hospital, University of Aarhus, Denmark; 4Department of Molecular Biotechnology and Health Sciences, and Center for Experimental Research and Medical Studies (CeRMS), University of Torino, Italy; 5Department of Pathology, University of Michigan Health System, Ann Arbor, MI 48109, USA; 6 Department of Pathology, Peking University Health Science Center, Beijing 100191, P.R.China, 7Department of Pathology,University of Utah, UT,USA * Correspondence to: Wing C. Chan. M. D. Co‐Director, Center for Research in Lymphoma and Leukemia Department of Pathology and Microbiology 983135 Nebraska Medical Center Omaha, NE 68198‐3135 Phone: (402) 559‐7684 Fax: (402) 559‐6018 E‐mail: [email protected] Running title: MicroRNA expression profiling of ALCL Keywords: Anaplastic large cell lymphoma, Anaplastic lymphoma kinase, Molecular classifier, miRNA expression signature, miRNA17~92 # contributed equally to this work Scientific category: Lymphoid Neoplasia

Blood First Edition Paper, prepublished online June 25, 2013; DOI 10.1182/blood-2012-08-447375

Copyright © 2013 American Society of Hematology




Key Points

• Anaplastic large cell lymphoma has unique miRNA signature

• Role of miRNA17~92 in ALK(+)ALCL pathogenesis Abstract Anaplastic large cell lymphomas (ALCLs) encompass at least two systemic diseases distinguished by the

presence or absence of anaplastic lymphoma kinase (ALK) expression. We performed genome‐wide

miRNA profiling on ALK(+)ALCLs (n=33), ALK(‐)ALCLs (n=25), angioimmunoblastic T‐cell lymphoma (n =

9), peripheral T‐cell lymphoma, not otherwise specified (n=11) and normal T‐cells and demonstrated

that ALCL express many of the miRNAs that are highly expressed in normal T‐cells with the prominent

exception of miR‐146a. Unsupervised hierarchical clustering demonstrated distinct clustering of ALCL,

PTCL‐NOS and the AITL subtype of PTCL. Cases of ALK(+)ALCL and ALK(‐) ALCL were interspersed in

unsupervised analysis suggesting a close relationship at molecular level. We identified a miRNA

signature of seven miRNAs (five upregulated: miR‐512‐3p, miR‐886‐5p, miR‐886‐3p, miR‐708, miR‐135b

and two down‐regulated: miR‐146a, miR‐155) significantly associated with ALK(+)ALCL cases. In

addition, we derived an 11 miRNA signature (four up‐regulated: miR‐210, miR‐197, miR‐191, miR‐512‐

3p and seven down‐regulated: miR‐451, miR‐146a, miR‐22, miR‐455‐3p, miR‐455‐5p, miR‐143, miR‐

494) that differentiates ALK(‐)ALCL from other PTCLs. Our in vitro studies identified a set of 32 miRNAs

that was associated with ALK expression. Of these the miR‐17~92 cluster and its paralogues were also

highly expressed in ALK(+)ALCL and may represent important downstream effectors of the ALK

oncogenic pathway.




Introduction Anaplastic large cell lymphomas (ALCLs) are aggressive T‐cell neoplasms typically composed of cohesive

clusters of large cells with abundant cytoplasm and eccentric, horseshoe or kidney shaped nuclei1. The

tumor cells show strong, uniform expression of CD30, one or more T‐cell antigens, epithelial membrane

antigen (EMA or MUC1) and cytotoxic cell associated antigens (e.g. TIA‐1, granzyme B, and/or

perforin)1. The two major subgroups of ALCL recognized by World Health Organization (WHO) show

similar morphologic and immunophenotypic features and are classified based on the presence or

absence of chromosomal translocations involving the anaplastic lymphoma kinase (ALK) gene located at

the chromosome 2p23 locus1. The translocations result in aberrant ALK expression with NPM1 as the

major fusion partner due to t(2;5)(p23;q35) in ALK(+)ALCL2 This genetic alteration leads to STAT3

activation via phosphorylation by the NPM‐ALK chimeric protein, which is critical for the maintenance

of the neoplastic phenotype3. The majority of the patients with ALK(+)ALCL are young and have a

significantly better clinical outcome than patients with systemic ALK(‐) ALCL4,5.

The systemic ALK(‐)ALCL1, 2,6subgroup lacks ALK expression, and has been included as a provisional

pathologic entity in the current WHO classification1. The lack of a defining biomarker or genetic

abnormality in ALK (‐) ALCL makes the diagnosis challenging5,7. Some investigators argue that ALK(‐)

ALCLs may represent a morphologic variant within the heterogeneous category of PTCL‐NOS based on a

lack of clear biological differences between them8,9. Although several gene expression profiling (GEP)

and comparative genomic hybridization (CGH) studies have shown that ALK(+)ALCL and ALK(‐)ALCL have

distinct patterns of expression signatures and genomic aberrations10‐14, there are many overlapping

molecular features10‐15 including shared expression of a number of genes16,19. Recently, recurrent

translocations t(6;14)(p25;q11.2) involving IRF417 and t(6;7)(p25.3;q32.3) involving DUSP2218 in ALK(‐)




ALCL have been identified supporting that ALK(‐)ALCL is a distinct entity5,11,19 . However, these

abnormalities are restricted to only a small subset (4‐10%) of the ALK(‐)ALCL cases17,18.

MicroRNAs (miRNA) are emerging as tissue‐specific biomarkers with the potential for clinical

application in identifying cancer subtypes20. More recently, several miRNAs such as miR‐10121, miR‐

1622, miR‐135b23 and miR‐29a24 have been demonstrated to have a role in the pathogenesis of

ALK(+)ALCL. However diagnostic signatures for the two subgroups of ALCL have not been defined.. We

have performed a large‐scale global analysis of miRNA expression in ALCL including 58 ALCL, 20

peripheral T‐cell lymphomas (PTCL), normal CD3+ T‐cells and stromal cells using a platform based on

high‐throughput Taqman® quantitative real‐time PCR (qRT‐PCR). We report here (i) the miRNA

signatures associated with the two systemic forms of ALCL, and (ii) functional attributes of miRNA

expression in ALCL.

Materials and Methods

Patient specimens, cell lines and normal cells

Tumor specimens from patients with ALK(+)ALCL (n = 33), ALK(‐)ALCL (n = 25), AITL (n = 9) and PTCL‐

NOS (n = 11) were obtained from four institutions (University of Torino‐Italy; Memorial Sloan‐Kettering

Cancer Center, New York; Children’s Oncology Group (COG) and University of Nebraska Medical

Center, Omaha) with informed patient consent in accordance with the Declaration of Helsinki and

approval by the local Institutional Review Boards. The cases were diagnosed by expert

hematopathologists in accordance with the WHO classification1. The clinical and molecular data

including ALK‐translocation status and immunohistochemical profiles are listed in Table 1. The miRNA




profiles of B‐cell lymphomas were used for comparative analysis, and have been previously

described25.

The NPM‐ALK(+)ALCL cell lines (SUP‐M2/TS, Karpas299, L82, JB6 and SUDHL‐1) and ALK(‐) ALCL cell line

(MAC‐1) were cultured in RPMI‐1640 medium containing 10% fetal calf serum (FCS, Lonza, Rockland,

ME) and 2 mM L‐glutamine and 1% streptomycin. Normal T‐cell CD3(+) and B‐cell subsets25were

obtained from fresh tonsils as described previously26 using magnetic MACS® microbeads according to

the manufacturer’s instructions (Miltenyi Biotec, CA), and pooled RNA and miRNA from at least 6‐9

healthy individuals were profiled in three different experimental settings. The stromal cells were

isolated from minced human tonsils digested with 2 mg/ml collagenase type IV (Worthington

Biochemical, Freehold, NJ) and 0.1 mg/ml DNase I (Sigma‐Aldrich, St. Louis, MO ) and processed as

described previously27.

Knockdown of ALK in ALCL cell lines

ShRNA directed against the cytoplasmic domain of ALK was utilized to knockdown (KD) ALK in SUP‐

M2/TS cells, as done previously19,28. The expression plasmid for inducible NPM‐ALK silencing was

produced by subcloning the H1 promoter‐ALK‐shRNA cassette into the pLVTH vector as described

previously28. ShRNA expression was induced by the presence of doxycycline (1ug/ml). The KD efficiency

of the shRNA was estimated by qRT‐PCR method as described by Agnelli et.al.16. Briefly, 100ng of total

RNA extracted from transduced cells was reverse transcribed with the miScript Reverse Transcription

Kit (Qiagen, Valencia, CA), according to the manufacturer’s instructions. qRT‐PCR assays were

performed in triplicate in the Thermal iCycler (Bio‐Rad) and was normalized to GAPDH and relative

expression of ALK was calculated using the standard ΔCT method as described by Agnelli et.al.16. The




miRNA differential expression between control cell lines and KD cell lines was evaluated using standard

statistical tests as described below in data analysis section.

Conditional knockdown of miR‐17~92 in ALK(+)ALCL cell lines

For KD of miR‐17~92, Karpas299 and JB6 were transduced with lentivirus packaged with the pTRIPZ‐

sponge construct, pMD2G envelope vector and psPAX2 packaging plasmid in HEK293T‐cells as

described previously29. Transduced cells were selected with puromycin, and doxycycline‐induced GFP‐

expressing cells were isolated by flow cytometry (FACSCalibur; BD Biosciences). The cell‐cycle analysis

of transduced cells was conducted using Hoechst 33342 staining (H3570; Invitrogen, Carlsbad, CA).

Apoptosis of cells was determined by staining with Annexin V‐PE (Apoptosis detection Kit, BD

Pharmingen) according to the manufacturer’s instructions. Both cell cycle and apoptosis assays of

transduced cells (i.e. GFP(+)cells) were analysed by flow cytometry (FACSCalibur; BD Biosciences) as

described previously29.

Treatment of ALK(+)ALCL cell lines with STAT3 inhibitor

The viability of cell lines (Karpas299, JB6, L82) after treatment with the STAT3 small molecule inhibitor,

Stattic® (Calbiochem®, Inc; Billerica, MA) which blocks STAT3 phosphorylation at Tyr705 was

determined by using the CellTiter 96® Aqueous kit (Promega, Madison, WI) according to the

manufacturer’s instructions29 in time (12‐24 hours) and dose (1‐5uM) dependent manner. The

antibodies for immunoblotting in this study were as follows: anti‐PTEN, anti‐STAT3 (total), anti‐STAT3

(phosphorylated), anti‐p‐AKTser473 (Cell Signaling Biotechnology, Beverly, MA) and anti‐β‐actin (Santa

Cruz Biotechnology, Paso Robles, CA). The qRT‐PCR of miR‐17~92 cluster members (miR‐19a and miR92)

was performed as described by Agnelli et. al.16




RNA isolation and miRNA profiling

Total RNA for miRNA profiling was extracted from FFPE tissues utilizing 2 tissue cores (~ 1 mm2

diameter) –from an area with predominant tumor cells (>70%), using the RecoverAllTM total nucleic acid

isolation kit. RNA was obtained from cell lines and normal cells with mirVana™ miRNA isolation kit

according to the manufacturer’s instructions (Ambion, Austin, TX). Reverse‐transcription was carried out

using input amounts of 300ng of total RNA from cell lines and normal cells and 100ng of total RNA from

FFPE samples, with Megaplex™ RT Primers and enzyme kit, This was followed by a subsequent step of

pre‐amplification (12 cycles) using Megaplex™ PreAmp Primers to enhance assay sensitivity, as

recommended by the manufacturer (ABI, Foster City, CA). The pre‐amplified cDNAs were loaded onto

384‐well format miRNA assays plates (Taqman® Array Human MicoRNA A Card, V2.0, ABI, Foster City,

CA), and then qRT‐PCR was performed on a 7900HT Fast Real‐Time PCR System (ABI, Foster City, CA).

The threshold cycle was defined as the fractional cycle number at which the fluorescence exceeds the

fixed threshold of 0.1 with an automatic baseline using the RQ Manager‐1.2 software according to the

instruction from the manufacturer (ABI, Foster City, CA).

Immunohistochemical staining, fluorescence in‐situ hybridization (FISH), T‐cell receptor γ (TCR‐γ)

gene rearrangement analysis

Immunohistochemical (IHC) stains for T‐cell markers including CD3, CD2, CD4, CD5, CD8, CD43,

cytotoxic markers (TIA, granzyme B, and perforin) and CD30 were performed on FFPE tissue sections as

described previously30. For IHC staining of ALK, the rabbit monoclonal antibody SP8 against ALK1 was

used on a Ventana ES automated immunostainer (Ventana Biotek, Tucson, AZ) with a streptavidin‐


http://marketing.appliedbiosystems.com/mk/submit/TAQMAN_ARRAY_HMP_MS_RD?_JS=T&rd=bk



biotin peroxidase detection system. Positive signals were localized in nucleus, nucleolus, cytoplasm and

/or membrane with different staining pattern indicating different translocation variants. ALK

translocation was detected at initial cytogenetics review or by using a commercially available LSI ALK

dual color break apart probe (Vysis, Downers Grove, IL) according to the manufacturer's

instructions. Analysis of TCR‐γ gene rearrangement using PCR‐based methods was performed on a

subset of cases with adequate materials as reported previously31.

MiRNA profiling Data analysis

The raw data were uploaded into BRB‐ArrayTools (version 3.9.0)32 for analysis. Briefly, we performed

global median normalization for the entire data set from FFPE cases prior to any further analysis. To

select miRNAs for analysis, we used three approaches: (i) exclude miRNA showing minimal variation

across the arrays from analysis by including only miRNAs whose expression differed by at least 2 fold

from the median in at least 10% of the cases (ii) exclude miRNAs if the log intensity variation was not

significant (p>0.05) compared to the median of all the variances (iii) CT =30 or higher were used as

threshold for the minimum level of expression. For the CD3(+)T cell specific miRNA signature, we used

the average difference (>2 CT value difference) and t‐test (p<0.01) between CT values of CD3(+)T cells

and other normal cells examined and fold difference was expressed by converting the log scale (Δ CT)

to normal scale. The miRNA classifier for ALK(+)ALCLs and ALK(‐) ALCLs was constructed using a

Bayesian algorithm that estimated the probability of a case belonging to one subtype compared to

another subtype as described earlier33. In our series, differentially expressed miRNAs were selected at

a significance of p<0.05 and a mean fold‐difference of >4 between any two group comparisons. The

miRNA data from fresh frozen cell lines or normal cells was used for comparative analysis only.




Survival analysis

Event free survival (EFS; event = progression or death from any causes after the start of chemotherapy)

and overall survival (OS; event = death from any causes) were estimated using the Kaplan‐Meier method

and differences were assessed using the log rank test.

Results

Patient characteristics

The clinical and pathological characteristics of patients included in the study are summarized in Table

1. As expected, the majority of the ALK(+)ALCL patients were younger with a median age of 18 years (

range 3‐62 years) ,with a marginal male predominance (M:F = 1.31) and significantly better clinical

outcome (p < 0.05) compared with ALK(‐)ALCLs and other PTCLs (Supplemental Figure 1). The ALK(‐

)ALCL patients were older with a median age of 60 years (range 16‐84 years) at time of diagnosis and

prominent male predominance (M:F = 4.75). The IHC profile showed characteristic antigen profiles in

ALK(+)ALCL and ALK(‐)ALCL cases with abnormal expression of pan‐T‐cell markers (Table 1). The

expression of at least one cytotoxic marker (i.e. TIA‐1, granzyme B and perforin) was more frequently

observed in ALK(+)ALCLs (78%; 14 of 18) than ALK(‐)ALCL cases(52.63%; 10 of 19). There was strong

and uniform expression of CD30 in all cases of ALCL. The t(2;5) was observed by FISH in 91.67% (11 of

12) of ALK(+)ALCLs, while the remaining case was evaluated by IHC alone and was positive for ALK

expression. Clonal TCR gene rearrangements were observed in the majority of evaluable cases (Table

1). The immunophenotypic profiles of other PTCLs were consistent with their diagnosis and were

distinct from the ALCLs (Table 1).




Identification of normal CD3(+)T‐cell specific miRNA signature and its expression in ALCLs and PTCLs

We identified a pan T‐cell miRNA signature by comparing the miRNA profiles of normal CD3(+)T‐cells

with normal B‐cell subsets (centroblast, centrocyte, naïve B cell and peripheral B cell) and stromal cells.

We identified 6 miRNAs that showed significantly higher expression (p < 0.01 and > 4 fold change) in

CD3(+)T‐cells (Figure 1a). While 5 of 6 miRNAs (miR‐135a, miR‐146a, miR‐146b‐3p, miR‐146b‐5p and

miR‐21) have been associated with T‐cells in previous studies34,35, we identified one novel miRNA (miR‐

642) expressed in T‐cell that has not been previously reported. When the expression of these miRNAs

was correlated with ALCL and other PTCLs and further compared with B cell lymphoma entities (DLBCL,

BL and MCL), we observed that this miRNA signature was enriched in ALCL, PTCLs, and some cases of

DLBCL that correlated with the T‐cell and stromal cell content of the tumour, probably representing T‐

cell rich DLBCLs (Figure 1b). Although expression of many T‐cell antigens is lost in ALCL 36, our T‐cell

miRNA signature was consistently expressed in the majority of the ALCLs at a level comparable to other

PTCLs. However, of special interest was the consistent low expression of miR‐146a in the majority of

ALK(+)ALCLs and in a subset of ALK(‐) ALCLs. In contrast, miR‐135a expression was more often low in

the other PTCLs and unexpectedly high in MCL.

In further analysis, we ranked miRNAs in the order of their expression level in normal T‐cells. Of the top

10% highly expressed (38), miRNA a substantial proportion (29 of 38; 76.3%) was also represented in

the top 10% highly expressed miRNA in both ALCL subgroups. This suggests that ALCL maintains a

substantial T‐cell related miRNA profile (Figure 1c). Seven other miRNAs, including miR‐7b, miR‐7d,

miR‐155, miR‐126, miR‐145, miR‐34a and miR‐886‐5p were shared by both ALCL subgroups. When we

examined the enriched miRNAs in normal T‐cells (upper half by expression level), we observed seven

miRNAs (Supplemental Table 1) that were significantly expressed at lower level (p < 0.05 and fold > 4)




in ALCL indicating loss of function of these miRNAs. In contrast, 18 miRNAs enriched in ALCL entities,

were not expressed in normal T‐cells or stromal cells, with miR‐10b and miR‐512‐3p showing >100 fold

change (Supplemental Table 1), suggesting an abnormal gain of function of these miRNAs. A small

subset of seven miRNAs were specifically enriched in only ALK(+)ALCLs, and three miRNAs were

specifically enriched in ALK(‐)ALCL compared to normal T‐cells (Supplemental Table 1).

In vitro analysis of ALK associated miRNA signature

We determined the ALK associated miRNA signature by knockdown (KD) of ALK by specific shRNAs in

an ALK(+)ALCL cell line (SUP‐M2/TS cells) resulting in at least 90% decrease in ALK mRNA as shown in

supplemental Figure 2. The differentially expressed miRNAs (Supplemental Table 2) after ALK KD

compared to control included 22 down‐regulated miRNAs mainly associated with cell proliferation such

as let‐7d, miR101, miR‐17~92 cluster and its paralogues (miR‐93, miR‐363, miR‐106a) consistent with

the role of ALK in inducing cell proliferation. We correlated this miRNA signature from the KD

experiments with the miRNA profiles of ALK(+)ALCLs vs. ALK(‐)ALCLs and PTCL entities and only a

fraction of the miRNAs demonstrated the expected changes in ALCL patient samples. These include

miR‐629, miR‐886, miR‐93, members of miR17~92 cluster and miR‐106 on ALK KD (Supplemental Table

2).

To further validate the functional effects of miR‐17~92 in vitro, we analysed the functional changes of

reducing miR‐17~92 in Karpas 299 by a sponge construct that sequesters the corresponding miRNAs29

(Figure 2a). Lentiviral transduction of a miR‐17~92 sponge resulted in an increased percentage of cells

in G2/M phase (2 fold) with a concomitant reduction in the percentage of cells in Go/G1 and S‐phases,

suggesting impaired cell cycle progression (Figure 2b). In addition, we observed that expression of miR‐




17~92 sponge increased apoptosis as compared to control vector in Karpas299 cell line (Figure 2c).

Similar results were obtained with JB6 cell line (data not shown). Since STAT3 is a major downstream

substrate for ALK, we further examined the miR‐17 ~ 92 expression level, upon inhibition of STAT3

phosphorylation by the small molecule inhibitor (Stattic® Calbiochem, Inc; Billerica, MA). The two

representative members of this cluster, miR19a and miR92 were measured by qRT‐PCR in Karpas299

cells and decreased expression was observed at 2µM Stattic® compared to untreated cells (Figure 2d

upper panel). However, there was little effect on STAT3 phosphorylation at concentrations up to 2μM

(Figure 2d, lower panel) when marked toxicity was observed. Interestingly, miR‐17~92 inhibition (by

sponge construct) in Karpas299 cell line combined with Stattic® at sub‐STAT3 inhibitory doses (1.5 µM

concentration) showed enhanced cytotoxicity (Figure 2e), suggesting that miR‐17~92 is crucial for cell

survival particularly in the presence of toxic agents. Even though PTEN is a canonical miR‐17~92 target,

we did not observe a consistent increase of PTEN protein expression nor changes in the activation of

AKT pathway in sponge transduced cells (Figure 2f). The ALK associated miRNA signature included

several other miRNAs that could target PTEN such as miR‐22, miR‐106b‐25, miR‐221 and miR‐22237 and

miR‐17~92 may act mainly through some other targets.

Diagnostic miRNA expression signature for ALCL

Unsupervised hierarchical clustering (HC) of all PTCL cases (n = 78) showed that ALCL samples formed

distinct clusters with respect to PTCL‐NOS and AITL, with two subgroups of ALCL interspersed among

each other (Figure 3a). ALCL cases (n = 58) formed two distinct clusters, a major (n = 44) and a minor (n

= 14) cluster, with the latter clustering closer to other PTCLs. Of the 178 differentially expressed

miRNAs the majority showed lower expression in ALCL cases compared to other PTCLs. However a




subset of miRNAs including miR‐517c, miR‐517a, miR‐512‐3p, miR‐135b, miR‐135a, miR‐708, miR‐886‐

5p and miR‐886‐3p, were more highly expressed in ALCL than in other PTCL (Figure 3a, red bar). There

is also a cluster of miRNAs that are highly expressed in some ALCL cases (blue bar). This cluster is also

highly expressed in stromal cells and their expression levels may be related to the stromal content. To

identify the miRNA signature specific for ALCL cases, we applied a Bayesian algorithm using ALCL as a

group versus other PTCLs, and identified twenty‐eight miRNAs including 13 up‐regulated and 15 down‐

regulated miRNAs to distinguish ALCLs from other PTCLs (Figure 3b). Note that none of the very highly

expressed miRNAs are part of the stromal signature indicating that the stromal component does not

have a significant impact in the diagnostic signature. This signature was predictive of most cases in the

major ALCL HC (40/44, 90%) with > 90% probability, whereas a small subset of nine ALCL cases showed

probability of >70%‐90% [6 ALK(+)ALCL and 3 ALK(‐)ALCL], and seven ALK(‐)ALCL were below 70%. Two

ALK(‐)ALCL cases were classified as PTCLs (probability >90%). The majority of ALCL cases, which were

not classified as ALCL by the Bayesian algorithm, were part of the minor ALCL cluster (12/14; 86%) in

unsupervised hierarchical clustering analysis. This subset of ALCL cases shared many of the 15 miRNA

more highly expressed in PTCL‐NOS and AITL.

Diagnostic miRNA expression signature for ALK(+)ALCL

According to the 2008 WHO classification1, ALK(+)ALCL and ALK(‐)ALCL were designated as different

entities. We observed seven miRNAs including five up‐regulated miRNAs (miR‐512‐3p, miR‐886‐5p,

miR‐886‐3p, miR‐708 and miR‐135b) and two down‐regulated miRNAs (miR‐155 and miR‐146a)

differentially expressed in ALK(+)ALCLs compared with ALK(‐)ALCLs and other PTCLs (Figure 3c).

Interestingly two ALK(‐)ALCL cases were classified as ALK(+)ALCLs with this miRNA signature. The class




precision analysis indicated that 25 of 33 ALK(+)ALCLs were classified with > 90% probability, and a

subset of ALK(+)ALCL cases (n = 7) was unclassifiable with probability of 60%‐90% (n=3), and <50% (n =

4). One ALK (+)ALCL case was classified as other PTCL. This unclassifiable subset of cases showed no

major difference in morphology, tumor content (% neoplastic cells) or T‐cell and stromal cell miRNA

signature, but patients in this unclassifiable group were relatively older (median age 38 years, range

21‐71) compared with the molecularly classified cases (median age 15 years, range 2‐62), though the

difference was not statistically significant.

Other than the miRNAs in the classifier, we analysed the differential expression of miRNAs between

molecularly defined ALK(+)ALCL cases and other PTCLs (p < 0.05 and FDR<0.1), and observed that 22

miRNAs, including the chr19q13.41‐42 miRNA (C19MC) cluster: miR‐519a, miR‐517c, miR‐517d, were

upregulated in ALK(+)ALCL. A small subset of these miRNAs may be contributed by the stromal cells

(e.g. miR‐221, miR‐376a, miR‐134, miR‐495, miR‐379 (Supplemental Figure 3a). Of the 17 down‐

regulated miRNAs, aside from the miRNA‐146a and miR‐155, several other interesting miRNAs are

noted including miR‐150, miR‐101, miR‐342‐3p, miR‐342‐5p, miR‐142‐3p and miR‐142‐5p. This group of

miRNAs was also highly expressed in normal T‐cells.

MiRNA Signature for differentiating ALK(‐)ALCLs from PTCLs

Using a similar approach as above, we obtained a 11 miRNA signature including 4 up‐regulated ( miR‐

210,‐miR‐197,‐miR‐191, miR‐512‐3p) and 7 down‐regulated (miR‐451, miR‐146a, miR‐22, miR‐455‐3p,

miR‐455‐5p, miR‐143, miR‐494) miRNAs, which distinguished ALK(‐)ALCL cases ( 16 of 25) with > 90%

probability (Figure 3d). However of the remaining nine cases, 3 cases had probability <50% and one

case (<10% probability) was classified as PTCLs. We observed no significant differences in tumour




content or morphology between these two subsets. Interestingly, the 7 downregulated miRNAs in the

classifier were highly expressed in T‐cell or stromal cell.

We further compared the differential miRNA expression between molecularly defined ALK(‐)ALCLs and

other PTCLs (p < 0.05, FDR<0.1), and observed 47 miRNAs differentially expressed, including up‐

regulation of miR‐210, miR‐512‐3p, miR‐135b, miR‐191 and miR‐886‐3p and many down‐regulated

miRNAs including miR‐451, miR‐22, miR‐146a and miR‐101 in ALK(‐)ALCLs (Supplemental Figure 3b).

We also compared the miRNA profiles of ALK(‐)ALCLs and ALK(+)ALCLs, and observed 43 differentially

expressed miRNAs, which included 27 highly expressed miRNAs in ALK(+)ALCLs and 16 miRNA more

highly expressed by ALK(‐)ALCLs (Supplemental Figure 3c). Many of these miRNAs were common to

those observed to be differentially expressed between ALK(+) ALCL and other T‐cell lymphomas.

Discussion

We included a relatively large series of well characterized PTCL cases with the aim of determining

distinct patterns of miRNA expression for the ALCLs and to further subdivide ALK(‐)ALCL from AITL and

PTCL‐NOS. We were able to construct miRNA classifiers that can distinguish the majority of ALCL

entities, and also identified several miRNAs that may play a role in the pathogenesis of ALCL.

One interesting observation is that while many T‐cell associated genes are downregulated in ALCLs,

many T‐cell associated miRNAs were still expressed indicating the preservation of the cell of origin

miRNA expression profile.

The known molecular mechanism of the pathogenesis ALK(+)ALCL involves activation of STAT3, a major

substrate of ALK, which is required for the survival and growth of ALK(+)ALCL cells in vivo and in vitro3,6.

We performed in vitro experiments to identify miRNAs that are associated with ALK activation, and




observed that the miR‐17~92 cluster and its paralogues were downregulated on ALK KD. It is known

that miR‐17~92 cluster is transcriptionally regulated by STAT3, suggesting a role of this miRNA cluster

in the pathogenesis of ALK(+)ALCL. Consistent with this possibility, we observed that miR‐17~92 is

generally highly expressed in ALK(+) ALCL and KD of this miRNA cluster did have deleterious effects on

cell survival and proliferation. We were not able to demonstrate the regulation of miR‐17‐92

expression by pYSTAT3 using the small molecular inhibitor Stattic because it was highly toxic to the

ALCL cells before clear STAT3 inhibition was observed. Interestingly, inhibition of miR17~92 by the

sponge construct enhanced the toxicity of Stattic. Thus, the miRNA cluster is probably important in

promoting survival of ALCL cells especially when they are under stress. However, one critical target of

miR‐17~92, PTEN, show only mild upregulation on reducing the level of the miRNA cluster37 and there

was little changes in pAKT suggesting that the effect of miR17~92 may be exerted through other

targets. There were only a limited number of additional miRNAs identified by the ALK KD experiments

that have the expected pattern in tumor samples. These included miR‐93, miR‐100, miR‐629 and miR‐

886‐5p. The lack of positive correlation between experiments performed in cell lines and the

observations in ALK(+)ALCL cases suggested either (i) complex regulation of the miRNAs in‐vivo or (ii)

presence of various stromal components in tissue specimens that contributed to the variability of the

miRNA expression profile or (iii) alterations specific to cell lines.

The pathognomonic feature of ALK(+)ALCL can be determined readily by IHC and gene rearrangement

of ALK. However, we identified a seven miRNA [five upregulated miRNAs (miR512‐3p, miR‐886‐5p, miR‐

886‐3p, miR‐708, and miR‐135b) and two down‐regulated miRNAs (miR‐146a and miR‐155) signature

characteristic of ALK(+)ALCL cases, that also defines some important aspects of ALCL tumor biology.

This miRNA signature includes a high expression miR‐135b (>15 fold) which, has been shown to




mediate the NPM‐ALK induced IL‐17 producing immunophenotype in ALK(+)ALCL23, an observation

consistent with our GEP study13. The high expression of miR‐886‐5p and miR‐886‐3p may also be

related to ALK expression, as ALK KD was associated with significant downregulation of these miRNAs.

The miRNA‐886 has been shown to target Bax38, a pro‐apoptotic gene, thus may deregulate apoptosis.

The high expression of miR‐708 and miR‐512‐3p in ALK(+)ALCL compared to other PTCLs may be

relevant, as miR‐708 have been shown to target TMEM88, a negative regulator of Wnt signaling39 and

miR‐512‐3p targets the cell cycle inhibitor p21(Waf1/Cip1)40. Thus the miRNAs in the signature may

contribute to the expression of an IL17 associated phenotype, and inhibition of apoptosis as well as

promoting cell cycle progression. However, the function of miRNA on gene expression is cell and

microenvironment dependant, and therefore must be investigated in a context specific manner.

MiRNA‐155, a known oncomir in DLBCL41 is expressed at a significantly lower level in ALK(+)ALCL

compared to ALK(‐)ALCLs and PTCLs. This suggests the use of a different oncogenic pathway. It is

noteworthy that the miR17~92 cluster is highly expressed in ALK(+) ALCL and possibly may serves as an

alternative to miR‐155. The low expression of miR‐146a is a striking feature of ALK(+)ALCLs . MiR‐146a

is known to regulate innate immunity and is repressed by MYC while upregulated by NF‐κB and TCR

signalling. Its role in ALCL pathogenesis is unclear but its low level may be related to the lack of TCR

signalling. miR‐146a has been shown to target STAT1 and its low expression may lead to increased

STAT1 expression 42. Our miRNA signature is distinct from the one derived previously from ALCL cell

lines21. This may be partially attributed to its derivation from tissue specimen and evaluation against

PTCL entities. In our experience, cell lines show distinct miRNA profile from primary tumors with loss of

many miRNAs. While this may be partially due to the lack of stromal components, there are probably

changes associated with immortalization in cell culture as well as culture associated changes. However,




a number of our observations are consistent with Merkel’s report21 such as the high expression of the

miR‐17~92 cluster and miR‐886‐3p, and low expression of miR‐155. The miR‐17~92 cluster is located at

13q31.3 that is frequently amplified in lymphomas and other cancers and are highly expressed in

cancer cells20. It is also induced by MYC and cooperates with MYC in lymphomagenesis43,44. An auto‐

regulatory loop has been shown between STAT3 and miR‐17~9245 that may be the major mechanism of

its upregulation in ALCL.

The distinction of ALK(‐)ALCLs from PTCLs‐NOS can be challenging with conventional diagnostics. The

11 miRNA signature that could distinguish ALK(‐)ALCL from PTCL‐NOS and AITL including the

upregulation of miR‐512‐3p and downregulation of miR‐146a was also noted in ALK(+)ALCL, suggesting

a shared role of these miRNAs in ALCL biology. The other three upregulated miRNA, include (i) miR‐

210, a regulator of hypoxic responses and has been associated with tumor progression46 (ii) miR‐197, a

negative regulator of a tumor suppressor gene FUS1 in lung cancer 47 and (iii) miR‐191 that is

associated with hepatocellular carcinoma48. The down regulated miRNAs including miR‐451, miR‐146a,

miR‐22, miR‐455‐3p, and miR146 as noted above. miR‐2249 and miR‐45150 have reported association

with tumor suppressor activities.

Other than miRNAs in the classifier, we observed 35 miRNAs differentially expressed between ALK(‐)

ALCLs and PTCLs, with 21 up‐regulated and 14 down‐regulated miRNAs including miR‐886‐3p/miR‐886‐

5p, whose expression was observed in both types of ALCL although higher in ALK positive cases. Two

miRNAs, miR‐29b1 and miR‐29a may be de‐regulated by recurrent t(6;7)(p25.3;q32.3) translocation in

ALK(‐)ALCLs18, but only miR‐29b was upregulated in ALK(‐)ALCL compared to other PTCLs and

ALK(+)ALCL.




Another major aim of this study was to compare ALK(+)ALCLs and ALK(‐)ALCLs; however, analysis of

unsupervised HC had shown that ALCL entities were interspersed regardless of ALK expression status,

and the top 10% of expressed miRNA in ALK(‐)ALCLs and ALK(+)ALCLs showed a significant overlap

(36/38; 94.7%), suggesting that ALK(+)ALCL and ALK(‐)ALCL share common miRNA profiles. There were

a set of miRNAs expressed by ALCL cases regardless of their ALK expression, including miR‐517c, miR‐

517a, miR‐518e, miR‐519d, miR‐519a, miR‐135a, miR‐152 when compared with other PTCLs. However,

there were also significant differences between the two entities (Supplemental Figure 3c). Consistent

with previous reports, miR‐29b, miR‐29a24, and miR‐1622, which have been shown to be

downregulated by ALK were also expressed at lower levels in ALK(+)ALCL cases in our series.

One notable observation was that there was a minor HC cluster including both subtypes of ALCL that

accounted for most of the molecularly not classifiable cases. For the minor ALK(+)ALCL subset, there

tended to be lower expression of miRNA associated with ALCL but at the same time, the expression of

miRNAs associated with PTCLs was also low. It is possible that these cases just had low tumor cell

content but the stromal signature was not high and review of the sections did not show obviously

lower number of neoplastic cells or distinct morphological features. The minor subset of the ALK(‐)ALCL

cases actually showed a higher expression of the ALCL signature compared with the major subset but it

also had a stronger expression of the PTCL associated miRNAs. It is possible that this reflect a higher

infiltration of T‐cells but one would anticipate a decrease in the ALCL signature and an increase in the

stromal signature that was, however, not the case. Review of the section showed consistent

morphological features of ALCL and also substantial number of neoplastic cells. In fact these minor

groups of ALCL cases in general expressed a set of miRNAs common to all ALCL.




Overall, our study confirmed that miRNA signatures can be used as a diagnostic tool for ALCL and play

a significant role in their pathogenesis. We have also shown that ALK (‐) ALCL has a distinct miRNA

profile compared to PTCL‐NOS, and shares partially the miRNAs expression profile of ALK(+)ALCL.

However a larger series of cases would be required to further refine the diagnostic signatures and to

elucidate the biologic differences between the major and minor subset of ALCL cases.




Author Contributions

CL, JI, GI and WCC: designed the experiments, analyzed the data and wrote the manuscript.

YL, MJD performed experiments. KD provided fellowship to MJD, and assisted in paper writing

MSL and GI, SKB, ML, DDW, TG, PA, SLP, WCC, JTF provide materials and perform pathology review.

CK, XH, CCL, RP performed in vitro experiments.

JOA and JMV provided the clinical data and other support.

Competing Interests: The authors have declared that no competing interests exist.

Acknowledgments

The authors would like to thank Martin Bast and Xin Huang for coordinating clinical outcome data and

analysis. This work was supported in part by a NCI grant to WCC (5U01/CA114778).




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Table 1: Immunophenotype, ALK status and basic clinical information of cases in this study

ALK(+)ALCL (n = 33)

ALK(‐)ALCL (n = 25)

AITL (n = 9)

PTCL‐NOS (n = 11)

Age (median, range) 18 (3‐62) 60 (16‐84) 63 (30‐83) 72 (24‐97) Gender Male : Female 1.31 4.75 1.25 2.67 Stage I/II 3/18 (16.67%) 3/12 (25%) NA 2/4 (50%)

III/IV 15/18 (83.33%) 9/12 (75%) NA 2/4 (50%) OS (median, range)(months) 40.9 (2‐206) 19.8 (8‐163.2) 26.6 (6.7‐113.9) 27.8 (0.5‐67.4) EFS (median, range)(months) 28.7 (1.1‐206) 10.32 (3.2‐47) 6.9 (1.1‐92.4) 6.15 (0.5‐34.8)

Immun

ophe

notype

ALK 33/33 (100%) 0/25 (0%) 0/3 (0%) 0/5 (0%) CD30 33/33 (100%) 25/25 (100%) 5/7 (71.43%) 1/7 (14.29%) CD3 5/30 (16.67%) 14/24 (58.33%) 9/9 (100%) 11/11 (100%) CD2 3/6 (50%) 6/6 (100%) NA 3/3 (100%) CD4 6/14 (42.86%) 9/15 (60%) 4/4 (100%) 5/5 (100%) CD5 5/13 (38.46%) 5/11 (45.45%) 7/7 (100%) 9/9 (100%) CD8 2/18 (11.11%) 3/20 (15%) 1/3 (33.33%) 0/4 (0%) CD43 6/7 (85.71%) 4/7 (57.14%) 5/5 (100%) 5/5 (100%)

Granzyme B/Perforin/TIA‐1

14/18 (77.78%) 10/19 (52.63%) NA NA

PAX‐5 0/15 (0%) 0/17 (0%) NA NA CD20 0/20 (0%) 0/13 (0%) 1/8 (12.5%) 0/9 (0%)

Cytogenetic abnormalities

ALK translocation

11/12 (91.67%) 0/9 (0%) NA 1/1 (100%)

TCR rearrangement

4/6 (66.67%) 13/14 (92.86%) 3/3 (100%) 3/4 (75%)




Figures legends Figure 1: Identification of T‐cell associated miRNA signature. (a) The 6 miRNA signature differentially

expressed (p < 0.01, fold change > 4) in normal T‐ cells compared with B‐cell subsets and stromal cells.

(b) Expression of T‐cell miRNA signature in different subtypes of T‐cell and B‐cell lymphomas Majority

of PTCL cases (including ALK(+)ALCL, ALK(‐)ALCL, AITL and PTCL‐NOS) and a few DLBCL cases showed

enrichment of T‐cell miRNA as well as stromal cell miRNA signature. Abbreviations: ALK, anaplastic

lymphoma kinase; ALCL, anaplastic large cell lymphoma; AITL, angioimmunoblastic T‐cell lymphoma;

PTCL‐NOS, peripheral T‐cell lymphoma ‐ not otherwise specified; DLBCL, diffuse large B‐cell lymphoma;

BL, Burkitt lymphoma; MCL, mantle cell lymphoma. (c) Overlap in the expression of the top 10% of

miRNAs from CD3(+)T‐cells, ALK(+)ALCLs and ALK(‐)ALCLs.

Figures 2: Knockdown of miR‐17~92 in ALK (+)ALCL cell lines (a) Diagram of the lentivirus construct

expressing GFP and a sponge which has 7 tandem binding sites for each of the four miRNA families

present in the miR‐17~92 cluster (targeting, from 5’ to 3’, miR‐17/20, ‐18, ‐19, and ‐92). (b) Cell cycle

analysis (c) Apoptosis analysis in Karpas299 post miR‐17~92 KD (d) Relative expression of two

representative miR17~92 cluster members (miR‐19a and miR‐92) after Stattic® treatment for 12 hours

(upper panel). pYSTAT3 levels after treatment with various concentrations of Stattic® in ALCL cell lines

(Karpass 299, JB6, L82) (e) IC50 for the STAT3 inhibitor, Stattic (Calbiochem®, Inc; Billerica, MA) in

Karpas 299 cell (right panel) and combined effect of Stattic and miR‐17‐92 inhibition in Kapras 299 (left

panel) (f) Immunoblotting of PTEN, pYSTAT3, STAT3(total), pAKT and pRPS6 with or without miR‐17~92

knockdown.

Figure 3: (a) Unsupervised Hierarchical clustering (HC) of PTCL cases. ALCL cases formed two distinct

clusters (major and minor HC clusters) with respect to AITLs and PTCL‐NOS. ALK(+)ALCL and ALK(‐)ALCL

cases were interspersed with each other. A group of miRNAs highly expressed in ALCL cases are

highlighted (yellow box) and listed on the right side (red bar). A strong stromal signature is represented

by the blue bar. (b) miRNA signature associated with ALCL (vs AITL/PTCL‐NOS). The Bayesian classifier

included 28‐miRNAs. Most of the cases in the ALCL minor hierarchical cluster were in the intermediate

group. Yellow boxes highlighted minor ALCL group. (c) miRNA signature associated with ALK(+) ALCL. A




7‐miRNA classifier was derived using Bayesian algorithm, included 5 upregulated and 2 downregulated

miRNAs. Orange box highlighted the expression of this signature among CD3(+) normal T‐cells, stromal

cells and ALCL cell lines. (d) miRNA signature associated with ALK(‐) ALCL. A 11‐miRNA classifier

included 4 upregulated and 7 downregulated miRNAs. Orange box highlighted the comparative

analysis of this signature in CD3(+) normal T‐cells, stromal cells and ALCL cell lines.




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Median expression (n = 3)

6 miRNAs

2 miRNAs5 miRNAs

64 miRNAs

miR-135amiR-642miR-146amiR-146b-3pmiR-146b-5pmiR-21

Figure 1a

Figure 1b

-miR-135a-miR-642-miR-146a-miR-146b-3p-miR-146b-5p-miR-21

-CD3(+)T cell-Stromal cell

ALK(+)ALCL ALK(-)ALCL AITL PTCL-NOS DLBCL BL MCL

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Figure 1c : Common top 10% miRNAs between CD3(+)T cells, ALK(+)ALCLs and ALK(-)ALCLs.

miR-708miR-886-3p

miR-193b

miR-140-5p

let‐7elet‐7gmiR‐17miR‐19bmiR‐92amiR‐20amiR‐20bmiR‐106amiR‐146amiR‐150miR‐21miR‐16miR‐24miR‐26amiR‐29amiR‐30bmiR‐30cmiR‐93miR‐142‐3pmiR‐146b‐5pmiR‐186miR‐191miR‐222miR‐223miR‐320miR‐331‐3pmiR‐342‐3pmiR‐374bmiR‐484

let‐7blet‐7dmiR‐155miR‐126miR‐145miR‐34amiR‐886‐5p

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Figure 2 : Knockdown of miR-17~92 in ALK (+) ALCL cell lines

Cha

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Karpas 299control sponge

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DM

SO

DMSO

DM

SO

μM μM

(d)

(f)

ControlSponge

1.5uM1uM

Nor

mal

ized

(%)

cell

num

ber

(e)

Karpas-299

Concentration (μm)

Nor

mal

ized

(%)c

ell

num

ber

F




From



Figure 3a: Unsupervised Hierarchical clustering (HC) of PTCL cases

-4Lo

g2 I

nten

sity

4

ALK(+)ALCLALK(-)ALCL

AITLPTCL-NOS

Tumor specimen (n = 78)

178

diffe

rent

ly e

xpre

ssed

miR

NAs

-miR-517c-miR-517a-miR-512-3p-miR-518e-miR-519d-miR-519a-miR-135b-miR-135a-miR-708-miR-152-miR-886-5p-miR-886-3p

Major ALCL Minor ALCL

T ce

llSt

rom

al

cell

-miR-204-miR-133a-miR-337-5p-miR-495-miR-379-miR-134-miR-376a-miR-376c-miR-411-miR-539-miR-127-3p-miR-214

For personal use only.

at Università di T

orino on June 26, 2013. bloodjournal.hem

atologylibrary.orgF

rom



Figure 3b : miRNA signature associated with ALCL

-miR-155-miR-455-3p-miR-15a-miR-22-miR-455-5p-miR-660-miR-194-miR-101-miR-125b-miR-100-miR-99a-miR-143-miR-146a-miR-342-5p-miR-150-miR-149-miR-210-miR-517c-miR-517a-miR-512-3p-miR-339-5p-miR-339-3p-miR-197-miR-886-5p-miR-886-3p-miR-708-miR-135b-miR-223

LOO

CV

prob

abilit

y1

0.80.60.40.2

0

ALCLAITL/PTCL-NOS

Median signature expression

-T-cell (normal)

-Stromal cells

AITLPTCL-NOS

ALCL groupMajor NC

Median expression

ALK(+)/ALK(‐)ALCL AITL/PTCL‐NOS

Pathology diagnosis

F




From



Figure 3c: miRNA signature associated with ALK(+) ALCL

Others (ALK(-)ALCL/ AITL /PTCL-NOS)ALK(+)ALCL

Others PTCLsALK(+)ALCL

LOO

CV

prob

abili

ty 10.80.60.40.2

0

-miR-512-3p-miR-886-5p-miR-886-3p-miR-708-miR-135b-miR-155-miR-146a

ALK

(+)A

LCL

(NC

)

ALK

(+)A

LCL

cell

line

-CD3(+)T cells

-Stromal cells

Median expression

ALK

(+)A

LCL

ALK

(-)A

LCL

cell

line

AIT

L

PTC

L-N

OS

CD

3(+)

T ce

lls

Stro

mal

cel

ls


ALK

(-) A

LCL

Pathology diagnosis

F




From



-miR-210-miR-197-miR-191-miR-512-3p-miR-451-miR-146a-miR-22-miR-455-3p-miR-455-5p-miR-143-miR-494

10.80.60.40.2

0

ALK(-)ALCLAITL / PTCL-NOSLO

OC

Vpr

obab

ility

AIT

L

PTC

L-N

OS

ALK

(-)A

LCL

(NC

)

ALK

(-)A

LCL

CD

3(+)

T c

ells

Stro

mal

cel

ls

ALK

(+)A

LCL

cell

line

ALK

(-)A

LCL

cell

line


-CD3(+)T cells-Stromal cells

Median expression

Pathology diagnosis

Figure 3d: miRNA signature associated with ALK(-) ALCL

ALK(-)ALCLAITL/ PTCL-NOS

F




From



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MicroRNA expression profiling identifies molecular signatures associated with anaplastic large cell...

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