[CANCER RESEARCH 57, 4608-4614. October 15. 1997)

Alterations of the Cyclin Dl/pl6-pRB Pathway in Mantle Cell Lymphoma

Martin H. Dreyling,1 Lars Bullinger, German Ott, Stephan Stilgenbauer, Hans K. Müller-Hermelink, Martin Bentz,

Wolfgang Hiddemann, and Hartmut DöhnerDepartment t>f Hematology/Oncology, University of Göttingen, D-37075 Göttingen, Germany [M. H. D., W. H.J: Department of Hemalology/Oneology and Rheumatology.University nf Heidelberg, 0-69115 Heidelberg. Germany ¡LB., S. S.. M. B.. H. D.]: and Department of Pathology, University of Würzburg.D-970XO Würzburg,Germany [G. Ó..H. K. M-H.l

ABSTRACT

Mantle cell lymphoma (MCL) has recently become generally acceptedas a subentity of malignant lymphomas that is characterized by thechromosomal translocation t(ll;14)(ql3;q32), resulting in the overexpres-sion of ryc/ínDI. Cyclin Dl forms a complex with cell cycle-dependent

kinase (cdk) 4, which inactivates the retinoblastoma protein (pRB) viaphosphorylation. However, in transgenic mice, the overexpression of cy-

din DI alone is not sufficient for the development of malignant lymphoma.To determine whether other members of the pRB pathway contribute tothe malignant transformation of MCL, we analyzed 37 cases of MCL thatwere well characterized by morphology, immunophenotype, and/or inter-

phase cytogenetics [detection of t(ll;14)(ql3;q32)|. Interphase fluorescence in situ hybridization was performed using a cosmid contig (250 kb)of the CDKN2/pl6 region (encoding an inhibitor of the cyclin Dl/cdk4complex) and a phage contig (200 kb) of the Rb region. CDKN2/pI6deletion was detected in 15 cases (41%), including 6 homozygous deletions; Rb was deleted in 15 cases (41%), all of which were hemizygousdeletions. Nine cases (24%) had deletions of both CDKN2/pl6 and Rb.Further analysis of a subset of 17 MCLs revealed a highly significantcorrelation between CDKN2/pl6 deletion and proliferation index, determined by the rate of KÌ67expression (/' = 0.014; / test). No significant

correlation was found between CDKN2/pl6 deletion and the blastoidvariant of MCL (P = 0.23; Fisher's test) or between proliferation indexand blastoid morphology i/' = 0.51; i test). Deletion iti Kb did not have any

impact on cell proliferation in addition to CDKN2/pl6 deletion (P = 0.76;

/ test). Additional analysis of 13ql4 deletions suggests that these deletionsmay target another gene telomeric to Rb. We conclude that deletion ofCDKN2/pl6 occurs in approximately one-half of MCLs and is a more

relevant indicator of the proliferative features as compared to morphological criteria. In contrast, although deletions of chromosomal band13ql4 are frequent in MCL, inactivation of Rb seems not to be involved inthe pathogenesis of MCL.

INTRODUCTION

The malignant transformation of the tumor cell is determined bya sequence of genetic alterations. Over the last few years, alterations of cell cycle-regulating genes have been evolving as an

important principle of promoting tumor development (1, 2). Inactivation of p53 promotes cell proliferation via down-regulation ofp21, an inhibitor of cdks (3, 4). The Kb2 gene, which is inactivated

in a variety of solid tumors, plays a central role in the transitionfrom late G, to S phase (checkpoint 1; Fig. 1; Ref. 5). Phosphorylation and concomitant inactivation of pRB is induced by acomplex of cdk 4 and cyclin Dl (6). As a result, the transcriptionfactor E2F is released, and transcription of multiple genes thatregulate cell proliferation is induced (c-myb. B-myb, cdc2, dihy-

drofolate reducíase, and thymidine kinase; Ref. 7). At this point.

Received 3/20/97; accepied 8/11/97.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section 1734 solely lo indicate this fact.

1To whom requests for reprints should he addressed, at Department of Hematology/Oncology. University of Göttingen. Robert-Koch-Strasse 40. D-37075 Göttingen.

Germany.- The abbreviations used are: Kb. relinoblastoma gene: cdk, cyclin-dependent kinase;

pRB. retinohlastoma protein; MCL. mantle cell lymphoma: FISH, fluorescence in situhybridization; YAC. yeast artificial chromosome: CLL. chronic lymphocytic leukemia.

the cell enters an irreversible state of S phase (5). Accordingly, incell culture experiments, overexpression of cyclin DI results in anabnormal proliferation of cells with shortened G, phase and lessdependence on growth factors (8). The kinase activity of the cyclinDl/cdk4 complex is inhibited by pl6 (MTS1, CDK4I, INK4A) andpl5, two members of a recently cloned family of inhibitors of cdks(9-11).

All of the genes involved in pRB regulation have been shown to befrequently altered in malignant disorders: cdk4 overexpression ormutation can be detected in various tumor types, including glioblas-

toma and melanoma (12, 13). Cyclin D l overexpression is a negativeprognostic factor in a variety of solid tumors (14). CDKN2/pl6deletion occurs in a broad spectrum of solid tumors and is the mostfrequently observed genetic alteration in adult T-cell acute lympho-

blastic leukemia (2, 10, 15, 16). Interestingly, in solid tumors, there isa reciprocal correlation between genetic alterations of single membersof the pRB pathway, namely Rb or CDKN2/pI6 inactivation andcyclin Dìor cdk4 overexpression (12, 13, 17. 18). Therefore, one hitat one position of the pRB pathway seems to be sufficient to destabilize cell cycle regulation.

Cyclin Dìoverexpression is also detected in the vast majority ofMCLs (19-22). This subentity of non-Hodgkin's lymphoma corre

sponds to the centrocytic lymphoma of the Kiel classification; classical MCL consists of small lymphoid cells with irregularly shaped,cleaved nuclei that coexpress CD5 and B-cell markers but lack CD23,which is typically strongly expressed in CLL (23-25). In addition, a

blastoid variant with either large pleomorphic or lymphoblastoid cellshas been recognized (23, 26-28). The clinical course of MCL is

aggressive and appears to be incurable with conventional therapy(29-32).

A characteristic feature of MCL is the chromosomal translocation t(l I;14)(ql3;q32), which leads to juxtaposition of the immu-noglobulin heavy chain promoter and the CTÕ-//HDìgene, resulting

in an overexpression of cyclin Dl in virtually all cases (19-22).

However, overexpression of cyclin Dl alone is not sufficient topromote the development of lymphoma in transgenic mice (33, 34).To determine whether secondary alterations of other members ofthe pRB pathway contribute to the malignant transformation ofMCL, we analyzed 37 cases of MCL. All cases were well characterized by morphology and immunophenotype or were analyzed forthe presence of the chromosomal translocation t(l I;14)(ql3;q32)(21, 26, 35).

Interphase FISH was performed to detect deletions of CDKN2/pI6and Rb (36, 37). This technique allows the analysis of genetic deletions on a single cell level, thereby allowing the reliable detection ofhemizygous deletions and the analysis of clinical samples with admixtures of normal cells. In previous studies, both CDKN2/p¡6 andRb probes provided the high hybridization efficiency that is necessary

for an accurate deletion analysis (36, 37). To further estimate thebiological effect of CDKN2/pi6 or Rb alterations, deletion state wascorrelated to proliferation index and morphological criteria in a subsetof 17 MCLs (26).

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PRB PATHWAY IN MANTLE CELL LYMPHOMA

CELLG2 CYCLE

E2FFig. 1.Cell cycle diagram and pRh pathway. Al the transition from G, to S phase, pRb

is phosphorylated hy the cyclin Dl/cdk4 complex. Consequently, the transcription factorE2F is released. The phosphorylation of pRb is inhibited by p!5 and pl6.

MATERIALS AND METHODS

Selection of Samples. Thirty-seven lymphomas were eligible on the baseof the characteristic histology of MCL (n = 24), immunophenotype (n = 34),and/or detection of the chromosomal translocation t( 11; I4)(q 13;q32) (n = 22;

Table 1; Rets. 26 and 35). Interphase cytogenetics were performed usingunique probes of the immunoxlohulin heavy chain region and the cyclin DIregion (38).

In 34 of 37 cases, immunophenotype was analyzed for CD5. CD23, and aB-cell marker (CD19. CD20. and/or CD22) by immunohistochemistry of

native lymphoma tissue or flow cytometry of peripheral blood (Table 1).Thirty-two of 34 cases (94%) showed the characteristic immunophenotype ofMCL: CD5 + . CD19/20/22 + , and CD23- (23. 24. 39). Two other cases were

found to be positive for CD23 by flow cytometry (case 7 and 9). However,

diagnosis of MCL was confirmed by the characteristic cleaved appearance ofcells, the presence of the chromosomal translocation t(l I:14)(ql3:q32), andthe clinical presentation and course.

In a subset of 18 cases. MCLs were classified according to cytomorpho-

logical features as classical, small cell MCL (13 cases) or blastoid variant (5cases), either lymphoblastoid or pleomorphic type, with a mixed population ofmedium and large cells (23, 26, 28). In this subset, cyclin DI rearrangement oroverexpression had been previously detected in 13 of 15 analyzed cases (87%;Table 1; Refs. 26. 35, and 40). In 17 of the cases, the proliferation index wasanalyzed using a KÌ67antibody (MIB1, DAKO, Hamburg. Germany) onparaffin sections (Fig. 2) as described (26). Briefly, paraffin sections of 4-6

p.m were mounted on slides, deparaffinized. and rehydraled in decreasing

ethanol concentrations. After microwave irradiation, immunohislochemicalstaining was performed using the peroxidase antiperoxidase method.

FISH. Mononuclear cells of patients samples had been grown in short-term

culture or processed directly, harvested using standard cell culture techniques,and stored in fixative for up to several years (41 ). Alternatively, cytospins wereprepared from frozen tissue blocks and processed in a similar manner (36).

FISH probes were generated from a cosmid contig containing CDKN2/pl6and the neighboring pl5 gene (8 cosmids, 250 kb) and a phage contig of theRB region (16 phages, 200 kb) using a sequence-independent amplification

technique (36, 37, 42). In previous studies, two hybridization signals weredetected in 92% (Rb) and 94% (CDKN2/pl6) of normal peripheral blood cells,respectively, indicating a high hybridization efficiency necessary for the reli-

Table 1 Characteristics of MCLs

Morphology, immunophenotype. genotypic characterization (or/in DI rearrangements and deletion status of CDKN2/plfi. Rh, and DI3S25), and proliferation index (KÌ67)of 37MCLs.

MCL pathology Immunophenotype

No.12345678910111213141516171819202122232425262728293031323334353637SamplePB"PBPBPBPBPBPBPBPBPBPBPBPBLNLNLNLNLNLNPBPBPBLNLNLNLNLNLNLNLNLNLNLNLNLNLNLNSourceCyloCytoCytoCytoCytoCytoCytoCytoCytoCytoCytoCytoCytoHistHisHisHisHisHisHisHisHisHisHisHisHistHisiHistHisHisHisHisHisHisHisHisHisSubtypeCDSND

+NDNDND+ND+ND+ND+ND+ND+ND+ND+ND+ND+NDNDND+ND+ND

+Classic+Classic+Classic

+ND+ND+ND

NDClassic+Classic+Classic

+Pleo+Classic+Classic+Classic

+Pleo+Classic+Classic+Blasloid+Blastoid

+Pleo+Classic+Classic

+B

cell CD23 Cyclin DI rearranged pl6Rb++ (FISH) +/+-/+ND

ND + (FISH) +/++/+++ (FISH) +/++/+++ (FISH) -/+-/++

+/- + (FISH) -/++/+++/- + (FISH) -/+*-/++

+ + (FISH) +/+-/+++ (FISH) -l-h-/++

+ + (FISH) +/++/+++ (FISH) +/++/+++ (FISH) +/+'+/+'+

+/- + (FISH) -/+''-/+NDND + (FISH) +/+-/++

+ (FISH) +/++/+-1- + (FISH) +/+r-/+''+

+ (FISH) +/+-/+++ (FISH) +/+-/+++ (FISH) +/++/++

+ (FISH, 1C) -/+-/++ND ND +/++/++

ND +/++/+NDND ND +/++/++

- (SB) -/--/+++ (SB. 1C) -/++/++

+ (1C) —¿�/-+/++-1-(SB, 1C) +/+c+/+'•+

ND +/++/++ND +/++/++ND +/++/++

-1-(1C) -/-'-l+c+

+ (FISH. SB) +/++/+++ (SB, 1C) +/++/+++ (SB, 1C) -/-+/++

+ (FISH, SB, 1C) -/+-/+++ (FISH. 1C) -/+r+/+''+

- (SB) -/++/++- + (1C) -/- -/+DI3S25-/++/++/+-/++/+-/+—

/+-/+—

/+—/—+/+c-/+-/++/+-/+'-/+—

/++/+—

/+NDNDNDNDNDNDNDNDNDNDNDNDNDNDNDNDNDNDKÌ67NDNDNDNI)NDNDNDNDNDNDNDNDNDNDNDND40%10%50%NDNDND60%20%40%20%20%40%ND40';20%20%80%50%20%60S40%

" PB. peripheral hlood; LN. lymph node; cyto. cytology; nisi, histology;+/+, wild type; —¿�/+. hemizygous deletion; —¿�/ —¿�. homozygous deletion.

'' Subclones with aberrant plfi deletion status.' Hyperdiploid.

pleo. pleomorphic; ND. not done; +/-. weakly positive; SB. Southern blot; 1C.immunohistochemislry (21);

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Fig. 2. Proliferation index (KÌ67)of MCLs. A, arepresentative MCL with a low proliferation index(10%; Table I, case 18). B, in case 23, 60% of thecells were positive for KÌ67.

able analysis of deletions (36. 37). In addition, cases 1-19 were hybridized

with a cosmid probe of DI3S25, which is located on chromosomal band 13ql4telomeric to Rb (43). In all cases with deletions, a YAC with similar hybridization efficiency, either YAC 284D6 (320 kb) from chromosomal band 8q22or YAC 166 from 18p, was used as a control probe (35, 44). FISH wasperformed as described previously (36, 37). Briefly, the hybridization solutioncontained approximately 0.1 /^.gof each probe, 1 ^ig of human Cotl-DNA. 0.6fig of human placenta! DNA. and 1 >xgof salmon sperm DNA/slide in a 10-filvolume. The probes were either directly labeled with Cy-3-dUTP or biotiny-lated and detected with FITC-conjugated avidin and amplified with antiavidin-FITC, if necessary. The slides were counterstained with 4',6'-diamidino-2-

phenylindole dihydrochloride. and were analyzed using a fluorescencemicroscope (Zeiss, Jena, Germany). For each case, at least 200 single, intactinterphase cells were analyzed. In some cases, two-color FISH with the unique

probes and the corresponding centromeric probes was performed to detectnumerical chromosomal aberrations (Fig. 3). For Figs. 3-5, separate gray-scaleimages of 4'.6'-diamidino-2-phenylindole dihydrochloride-stained cells and

fluorescence signals were captured using a charge-coupled device camera

(Xillix, Richmond, British Columbia, Canada) and were merged using AdobePhotoshop software (Adobe Systems, Mountain View, CA).

Statistical Analysis. The t test for independent samples and Fisher's exact

test were applied for analysis of correlations between deletion status, cytomor-

phological features, and proliferation index.

RESULTS

FISH Analysis. Fifteen of the 37 MCLs (41%) exhibited CDKN2/pl6 deletions, 9 cases (24%) had hemizygous deletions, and 6 cases(16%) had homozygous deletions (Figs. 3 and 4).

Rb was deleted in 15 cases (41%). All of these lymphomas hadhemizygous deletions (Fig. 4). DÌ3S25was deleted in all analyzedcases with Rb deletion. In addition, DI3S25 was hemizygously andhomozygously deleted in cases 9 and 10 (Table 1), respectively,whereas both copies of Rb were retained.

Nine cases (24%) were deleted for both CDKN2/pl6 and Rb (Fig.4; Table 2). The percentage of cells with CDKN2/p!6 or Rb deletionvaried between 26.0 and 99.0% and between 55.0 and 96.5%, respectively. Subclones of lymphoma cells with different CDKN2/pJ6 deletion pattern were detected in three cases (8%; Fig. 5; Table 1): in

cases 6, 8, and 12, CDKN2/pl6 was deleted in 27.5, 35, and 26% ofthe cells, respectively, whereas Rb deletions were detected in 55, 56.3,and 67.5% of the interphase nuclei. In contrast, two signals of thecontrol probe were detected in more than 95% of the cells.

In all diploid cases with deletions, two copies of the control probewere detected in 94.3 ±2.0%, proving the reliability of the method(Fig. 6). In most cases with CDKN2/pl6 or Rb deletion, a significantrate of cells with two hybridization signals was detected, especially inthe peripheral blood samples (5.0-72.5%; Fig. 5). These cells possi

bly reflect the admixture of normal cells, which would have complicated Southern blot analysis.

In the total group of 37 MCLs, there was a borderline correlationbetween Rb and CDKN2/pl6 deletion (Rb was deleted in 60% ofMCLs with CDKN2/pl6 deletion versus 27% in MCLs withoutCDKN2/pl6 deletion; P = 0.05; Fisher's test).

Correlations of CDKN2/pl6 Deletion Status. In a subset of 17histologically characterized MCLs, CDKN2/pl6 deletion was closelyrelated to proliferation index (46 ±18.4% in MCLs with CDKN2/pl6deletion versus 24.3 ±11.3% in MCLs without CDKN2/pl6 deletion;Fig. 7). This correlation was highly significant (P = 0.014; t test). The

proliferation index in cases with hemizygous and homozygousCDKN2/pl6 deletion was not significantly different (40 ± 18.7%versus 52 ±17.9%; P = 0.33; i test). There was a tendency toward

higher deletion frequency in the blastoid subtype of MCL (80% ofblastoid variant of MCL versus 46% of classic MCL), but this correlation was not significant (P = 0.23; Fisher's test).

Correlations of Rb Deletion Status. Rb deletion showed a borderline relation to the proliferation index as well (46.7 ±8.2% inMCL with Rb deletion versus 31.8 ±21.4% in MCL without Rbdeletion; P = 0.06; / test). However, in cases with CDKN2/pI6

deletion, Rb deletion did not have additional impact on the proliferation index (48 ±8.4% in MCL with Rb deletion versus 44 ±26.1%in MCL without Rb deletion; P = 0.76; t test; Fig. 7). Rb deletion did

not correlate to cytomorphological subtype of MCL (deletion in 60%of blastoid variant of MCL versus 40% of classic MCL; P = 0.56;Fisher's test).

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PRB PATHWAY IN MANTLE CELL LYMPHOMA

Fig. 3. Hypcrdiploid MCL wilh hemizygousCDKN2/pl6 deletion (Iwo-color FISH of case 35

(see Table 1)|. An interphase cell with four centromere 9 signals (small arrows} but only two CDKN2pl6 signals (large arrows) is shown.

Morphological Features and Proliferation Index. In the smallseries of 17 MCLs, there was a tendency toward a higher proliferationindex in the blastoid variant of MCL (42 ±24.9% in the blastoidvariant of MCL versus 35 ±16.8% in classical MCL). However, thisdifference was not significant due to a rather high SD (P = 0.51;Fisher's test).

DISCUSSION

Dysregulation of cell proliferation is one of the hallmarks of malignant neoplasias. Kb, a well-known tumor suppressor gene that is

inactivated in a variety of solid tumors, represents the central checkpoint at the transition from G, to S phase (Fig. 1; Ref. 5). In fact, allmembers of the pRb pathway are involved in malignant transformation (1,2, 5). Interestingly, in solid tumors, one hit at one position ofthe pRb pathway seems to be sufficient to destabilize cell cycleregulation: in glioblastoma, either cdk4 overexpression or CDKN2/pl6 deletion occurs (13). In lung cancer and melanoma, there is areciprocal correlation between RB inactivation and CDKN2/p!6 deletion or cyclin Dl overexpression (12, 17, 18). In contrast, not allcases follow this excluding alteration pattern in lymphoid neoplasias

(15). We investigated genomic alterations of the pRb pathway inMCL, which is characterized by deregulation of cell cycle control viaoverexpression of cyclin Dl. In our study, a second hit of the pRBpathway, namely the genomic deletion of CDKN2/pl6, was one of themost frequent genomic aberrations in MCL. Homozygous deletionwas detected in 6 of 37 cases (16%), and 9 of 37 cases (25%) hadhemizygous deletion of CDKN2/pI6. In the latter cases, the secondalíelemay be inactivated either by microdeletion/point mutation of theencoding region or methylation of the promoter region (45). In contrast, previous studies could rarely detect point mutations of CDKN2/p 16 in lymphoid malignancies (37,46). In addition, methylation of theCDKN2/pl6 promoter was not present in a series of acute lympho-

blastic leukemias (47). Alternatively, there may be a dosage effect ofnonhomozygous CDKN2/pI6 deletions, although the proliferationindex was not significantly different in cases with hemizygous andhomozygous CDKN2/pl6 deletion. However, our data clearly indicatea close correlation between CDKN2/pl6 deletion and cell cycle activity. The finding that deletion of CDKN2/pl6 has additional effectson cell cycle regulation in MCL implies that overexpression oícyclinDl does not completely destabilize the pRb pathway. This biological

Fig. 4. MCL wilh homozygous CDKN2/pl6 deletion and hcmi/ygous Rb deletion (two-color FISHof case 23 (see Table 1)J. A, no CDKN2/plf> signalsand two centromere 9 signals (small arrows) weredetected in interphase cells, fi. only one Rb signal(large arrows} and three centromere 13/21 signals(small arrows) were detectable. C. in contrast, twosignals of the control YAC (large arrows} and twocentromere 8 signals were detected (small arrows:compare Fig. 2fl and Fig. 6).

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PRB PATHWAY IN MANTLE CELL LYMPHOMA

Fig. 5. Different subclones of MCL with hemizy-gous Rb deletion and normal CDKN2/pl6 or hem-izygous deletion. (Two-color FISH with CDKN2/pl6

and Rb of case 12. Table 1). Three cells have one Rbsignal (small arrows); in addition, one of the cells,(top left) has only one CDKN2/pl6 signal (largearrows), indicating a hemizygous CDKN2/pl6 deletion in a subpopulation of cells. One cell with twohybridization signals of both CDKN2/pl6 and Rb(bottom right) is probably a normal lymphocyte.

Table 2 CDKN2/pI6 and Rb deletions in MCL

Rb normalRb deleted

TotalCDKN2/pl6

normal16(43%)

6(16%)22 (59%)CDKN2/pl6

deleted6(16%)

9 (25%)15(41%)Total22

(59%)15(41%)37(100%)

behavior may be a result of the physiological expression of othermembers of the cyclin D family in lymphoid tissue (Fig. 1; Ref. l).Therefore, overexpression of cyclin Dl, which is normally notexpressed in lymphocytes, may be partially compensated for by down-regulation of cyclin D2 and cyclin D3. In fact, in a previous study,cyclin D3 was completely down-regulated in all of 26 investigatedMCLs (40).

In accordance with other studies, there was a tendency toward ahigher deletion frequency of CDKN2/pl6 in the blastoid variant ofMCL, but this tendency was not significant, possibly due to thesmall number of analyzed cases (48, 49). It is not known whetherCDKN2/pl6 deletion is a negative prognostic factor in MCL. Intwo recent studies, the blastoid variant of MCL correlated with ashorter survival and higher rates of CDKN2/p!6 deletion (48, 49).In contrast, various studies in acute lymphoblastic leukemia couldnot confirm an independent prognostic value of CDKN2/pl6 deletion (50). Therefore, the poor prognosis of the blastoid variant ofMCL may be due to the higher rate of p53 inactivalions (51-53).Thus far, it is not known whether alterations of p53 correlate todeletions of CDKN2/pl6 in MCL. In diffuse large cell lymphoma,inactivation of both CDKN2/pl6 and p53 seems to be a late eventin lymphomagenesis, which correlates with progression or histo-logical transformation from low malignant to high malignant lymphoma but occurs rarely in de novo diffuse large cell lymphoma(54).3 In three cases of our study, subclones with different CDKN2/

pió deletion status were identified, suggesting that the CDKN2/pl6 deletion is a late event in MCL as well. On the other hand, all

' M. H. Dreyling, S. K. Bohlander, and O. I. Olopade. CDKN2 (^/6INK4A) deletion is

a frequent event in lymphoid tumor progression, submitted for publication, 1997.

variant MCLs with CDKN2/pl6 deletion were blastoid at primarypresentation. Therefore, deletion of CDKN2/pl6 is no specificindicator of histological transformation from classical to blastoidMCL. Thus, further studies are necessary to elucidate the biological role of CDKN2/pl6 deletion in MCL.

Similar to CLL, genomic deletion of Rb at 13ql4 was a frequentevent in MCL and occurred in 41% of our cases (36). However, thereare some hints that Rb is not the target of these genomic deletions.First of all, in contrast to solid tumors, there was no reciprocalcorrelation between CDKN2/pl6 and Rb deletion. Secondly, all deletions detected were hemizygous. Thus far, there are no data on pointmutations of Rb in malignant lymphoma, which may have inactivatedthe second alíele.However, two recent studies could not detect anyloss of pRB expression in MCL (55, 56). In contrast, an even higherexpression of pRB was correlated with the blastoid variant of MCL,suggesting that pRB expression is a secondary physiological effect of

control

RB0

signals/cellpl6

Fig. 6. Quantitative analysis of a MCL with homozygous CDKN2/pl6 and hemizygousRb deletion (Table 1, case 23). Interphase FISH with CDKN2/p!6 (•),Rb (D) and thecontrol YAC (D). Shown is the percentage of cells with zero, one, and two hybridizationsignals.

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PRB PATHWAY IN MANTLE CELL LYMPHOMA

70 T

60--

50 •¿�•

= 40--

30--

20--

10 -•

T 70

--60

-•50

--40

--30

-•20

•¿�•10

pionormal deleted

onlypio

deleted

pii.

and Rbdeleted

Fig. 7. Proliferation index (KÌ67)and deletion status. Proliferation index is defined asthe percentage of KÌ67positive cells (mean ±SD). *, significant difference between MCLwithout (n = 10) and with (n = 7) CDKN2/pl6 deletion (P = 0.014; / test). Among MCLswith CDKN2/p¡6 deletion, the proliferation index is similar in cases with (n = 5) andwithout (n = 5) Rb deletion (P = 0.76; I test).

cell cycle regulation. Accordingly, in our study, 13q deletions did nothave an effect on cell cycle proliferation in cases with CDKN2/pI6deletion. Finally, D13S25, another locus on chromosomal band13ql4, was more frequently deleted than Rb. Taken together, thesedata indicate that 13q deletions are frequent in MCL but do not targetcell cycle regulation via Rb deletion.

In summary, deletion of CDKN2/pl6 is a frequent event in MCL.Moreover, it is an important indicator of the proliferative featuresof MCL, superior to morphological criteria. In contrast, Rb deletion seems to be not involved in malignant transformation of MCL.In analogy to CLL, the 13ql4 deletions in MCL may reflect thefrequent deletion of a tumor suppressor locus more telomeric to Rb(43).

ACKNOWLEDGMENTS

We thank O. I. Olopade and S. K. Bohlander who contributed to thegeneration of the CDKN2/pI6 probe, T. P. Dryer for providing the Rb probe,and M. Unterhalt for assistance in statistical analysis.

REFERENCES

1

12.

16

17

18.

19.

20.

21.

22.

23.

24.

25.

26.

27.

Hunter. T.. and Pines. J. Cyclins and cancer II: cyclin D and CDK inhibitors come of 28.age. Cell, 79: 573-582, 1994

2. Hirama. T.. and Koeffler. H. P. Role of the cyclin dependent kinase inhibitors in thedevelopment of cancer. Blood. 86: 841-854, 1995. 29.

3. El-Deiry, W. S.. Tokino. T.. Velculescu. V. E.. Levy. D. B.. Parsons. R.. Trent. J. M..Lin, D.. Mercer. W. E., Kinzler. K. W.. and Vogelstein. B. WAFI. a potentialmediator of p53 lumor suppression. Cell, 75: 817-825. 1993.

4. Xiong, Y., Hannon. G. J., Zhang, H., Casso. D., Kobayashi, R., and Beach, D. p21 isa universal inhibitor of cyclin kinases. Nature (Lond.), 366: 701-704. 1993. 30.

5. Weinberg, R. B. The retinoblastoma protein and cell cycle control. Cell, 81: 323-330,1995.

6. Dowdy. S. F.. Hinds. P. W., Louie, K., Reed. S. I.. Arnold, A., and Weinberg. R. A.

4613

Physical interaction of the retinoblastoma protein with human cyclins. Cell, 73:499-511, 1993.Nevins, J. R. E2F: a link between the Rb tumor suppressor protein and viraloncoproteins. Science (Washington DC), 258: 424-429. 1992.Quelle. D. E.. Ashmun. R. A.. Shurtleff, S. A., Kalo. J.. Bar-Sagi. D.. Roussel. M. F.,and Sherr. C. J. Overexpression of mouse D-type cyclins accelerates G, phase inrodent fibroblasts. Genes Dev.. 7: 1559-1571, 1993.Hannon, G. J., and Beach. D. pl5'NK4B is a potential effector of TGF-ßinduced cell

cycle arrest. Nature (Lond.), 371: 257-261. 1994.Kamb, A.. Gruís,N. A., Weaver, F. J.. Liu. Q.. Harshman. K., Tavtigian, S. V.,Stocken, E., Day, R., HI, Johnson. B. E.. and Skolnick, M. H. A cell cycle regulatorpotentially involved in genesis of many tumor types. Science (Washington DC), 264:436-440, 1994.Serrano. M.. Hannon. G. J.. and Beach. D. A new regulatory motif in cell-cyclecontrol causing specific inhibition of cyclin D/CDK4. Nature (Lond.). 366: 704-707.

1993.Bartkova. J.. Lukas. J.. Guldberg. P., Aisner, J., Kirkin, A. F.. Zeuthen, J., and Bartek,J. The pl6-cyclin D/cdk4-pRb pathway as a functional unit frequently altered inmelanoma palhogenesis. Cancer Res.. 56: 5475-5483. 1996.He, J., Allen, J. R., Collins, V. P.. Allalunis-Turner. M. J., Godboul. R., Day, R. S.,III. and James. C. D. CDK4 amplification is an alternative mechanism to pl6homozygous deletion in glioma cell lines. Cancer Res.. 54: 5804-5807, 1994.

Michalides. R.. Van Veelen. N.. Hart, A.. Loftus. B.. Wientjens. E.. and Balm. A.Overexpression of cyclin Dl correlates with recurrence in a group of forty-sevenoperable squamous cell carcinomas of the head and neck. Cancer (Phila.). 55:975-978. 1995.Hangaishi. A., Ogawa, S.. Imamura. N., Miyawaki, S., Miura. Y.. Uike. N..Shimazaki. C.. Emi, N.. Kunihiko. T.. Hirosawa. S.. Kamada. N.. Kobayashi, Y.,Takemoto. Y.. Kitani. T.. Toyama. K.. Ohtake. S.. Yazaki. Y.. Ueda. R., and Hirai. H.Inactivation of multiple tumor suppressor genes involved in negative regulation of thecell cycle. MTSl/pl6!NK*A/CDKN2, MTS2/pl5<NK*K, p53, and Rb genes in primary

lymphoid malignancies. Blood. 87: 4949-4958. 1996.

Quesnel. B.. Preudhomme, C., Philippe. N.. Vanrumbeke. M.. Dervite, I.. Lai, J. L.,Bauters. F.. Wattel. E.. and Fenaux. P. piò homozygous deletions in acute lympho-blastic leukemia. Blood. 85: 657-663. 1994.Shapiro. G. I.. Edwards, C. D., Kobzik. L., Godleski, J.. Richards. W.. Sugarbaker.D. J.. and Rollins. B. J. Reciprocal RB inactivation and the pl6INK4A expression in

primary lung cancers and cell lines. Cancer Res.. 55: 505-509. 1995.

Schauer. I. E., Siriwardana. S.. Langan, T. A., and Sclafani, R. I. Cyclin DlOverexpression vs. retinoblastoma inactivation: implications for growth control evasion in non-small and small cell lung cancer. Proc. Nati. Acad. Sci. USA. 91:7827-7831, 1994.Bosch. F.. Jares, P., Campo. E.. Lopez-Guillermo. A.. Piris. M. A.. Villamor. N..Tassies. D.. Jaffe, E. S., Montserrat. E.. Rozman, C., and Cardesa. A. Prad-1/cyclin

DI gene Overexpression in chronic lymphoproliferative disorders: a highly specificmarker of mantle cell lymphoma. Blood. 84: 2726-2732, 1994.de Boer. C. J.. van Krieken. J. H. M.. Kluin-Nelemans, H. C.. Kluin. P. M.. andSchuuring. E. Cyclin Dl messenger RNA Overexpression as a marker for mantle celllymphoma. Oncogene. 10: 1833-1840. 1995.

Ott. M. M.. Helbing. A.. Ott. G.. Bartek. J., Fischer. L.. Dürr.A.. Kreipe, H., andMüller-Hermelink. H. K. bcl-l rearrangement and cyclin Dl protein expression inmantle cell lymphoma. J. Palhol., 179: 238-242, 1996.

Rosenberg, C., Wong. E.. Petty, E., Bale. A., Tsujimoto. Y., Harris, N.. and Arnold.A. Overexpression of PRADI, a candidate BCLI breakpoint region oncogene, incentrocytic lymphomas. Proc. Nati. Acad. Sci. USA. 88: 9638-9642. 1991.

Harris. N. L.. Jaffe. E. S., Stein. H., Banks. P. M., Chan, J. K. C.. Cleary. M. L.,Delsol. G.. Wolf-Peelers, C. D.. Falini, B., Gatter, K. C.. Grogan, T. M., Isaacson,P. G.. Knowles. D. M.. Mason, D. Y., Müller-Hermelink. H-K.. Pilen. S. A.. Piris.M. A., Ralfkiaer, E.. and Wamke. R. A. A revised European-American classificationof lymphoid neoplasms: a proposal from the international lymphoma study group.Blood. 84: 1361-1392. 1994.

Weisenburger, D. D.. and Armitage. J. O. Mantle cell lymphoma: an enlily comes ofage. Blood, 87: 4483-4494, 1996.

Tolksdorf. G., Stein. H.. and Lennert. K. Morphological and immunological definitionof a malignant lymphoma derived from germinal centre cells with cleaved nuclei(centrocytes). Br. J. Cancer. 41: 168-182. 1980.On, G., Kalla, J.. Ott, M.. Schryen, B.. Katzenberger. T.. Müller.G. J.. and Müller-Hermelink, H. K. Blastoid variant of mantle cell lymphoma: frequent BCL-1 rearrangements at the MTC locus and tetraploid chromosome clones. Blood. 89: 1421-1429, 1997.Ott, M. M., Ott, G., Kuse, R., Porowski, P., Gunzer, U., Feller. A. C.. and Müller-Hermelink, H. K. The anaplastic variant of centrocytic lymphoma is marked byfrequent rearrangements of the bel-1 gene and high proliferation indices. Histopa-thology. 24: 329-334. 1994.

Lardelli. P.. Bookman. M. A., Sundeen. J.. Longo, D. L., and Jaffe. E. S. Lymphocyticlymphoma of intermediate differentiation. Morphologic and immunophenotypic spectrum and clinical correlation. Am. J. Surg. Pathol.. 14: 752-763. 1990.Fisher. R. I.. Dahlberg. S.. Nathwani. B. N.. Banks. P. M.. Miller. T. P.. and Grogan.T. M. A clinical analysis of two indolent lymphoma entities: mantle cell lymphomaand marginal zone lymphoma (including the mucosa-associated lymphoid tissue andmoncytoid B-cell subcategories)—a southwest oncology group study. Blood. 85:1075-1082. 1995.

Hiddemann. W.. Brittinger. G., Tiemann. M.. Parwareseh, R.. Stein. H.. Lister. A. T.,Norton. A.. Piltaluga. S., de Wolf-Peeters. C.. van Hoof, A.. Coiffier, B.. Berger, F.,Kluin-Nelemanns. H.. van Krieken. H.. Kluin. P.. Cavalli. F.. Roggero, E., Pedrinis.

E.. Montserrat, E.. Piris, M. A., and Unterhalt. M. Clinical characteristics and

Research. on August 20, 2019. © 1997 American Association for Cancercancerres.aacrjournals.org Downloaded from

PRB PATHWAY IN MANTLE CELL LYMPHOMA

response to chemotherapy of mantle cell lymphomas: results of a European survey.Blood, SS (Sappi \): 674a, 1996.

31. Hiddemann. W.. Unterhalt, M.. Herrmann, R.. Wöltjen.H-H., Kreuser, E-D.. Triimper,L., Reuss. M.. Terhardt-Karsten. E.. Busch. M., Neubauer. A.. Kaiser, U.. Hanrath. R-D..

Middeke. H.. Helm. G.. Freund. M.. Stein. H.. Tiemann. M., and Parwaresch. R. Mantlecell lymphomas have a more widespread disease and a slower response to chemotherapyas compared to follicle center lymphomas. Results of a prospective comparative analysisof the German Low Grade Lymphoma Study Group. JCO. in press. 1997.

32. Zucca. E., Roggero. E., Pinolli. G., Pedrinis. E., Cappella. C.. Venco. A., and Cavalli.F. Pattern of survival in mantle cell lymphoma. Ann. Oncol.. 6: 257-262. 1995.

33. Lovée.H., Grzeschiczek. A.. Kowalski. M. B., and Möröy.T. Cyclin Dl/bcl-Icooperates with the tnvc genes in the generation of B-cell lymphoma in transgenicmice. EMBO J.. 13: 3487-3495. 1994.

34. Bodrug. S. E.. Warner. B. J.. Bath. M. L., Lindemann. G. J.. and Harris. A. W. CyclinDìtransgene impedes lymphocyte maturation and collaborates in lymphogenesis withthe m.vt-gene. EMBO J., 13: 2124-2130. 1994.

35. Bullinger. L.. Schroder. M.. Möller. P.. Barth. T.. Wilhelm. S.. Stilgenbauer, S.,Lichter, P., Bentz. M., and Döhner. H. Secondary chromosome abnormalities int( 11:14)(ql3;q32) positive non-Hodgkin's lymphomas detected by fluorescence in

situ hybridization. Ann. Hematol., 73 (Suppl. II): A124, 1996.36. Stilgenbauer, S.. Döhner,H.. Bulgay-Mörschel. M.. Weitz. S.. Bentz, M., and Lichter,

P. High frequency of monoallelic retinoblastoma gene deletion in B-cell chroniclymphoid leukemia shown by interphase cytogenetics. Blood, 81: 2118-2124. 1993.

37. Dreyling. M. H.. Bohlander. S. K.. Le Beau. M. M., and Olopade. O. I. Refinedmapping of genomic rearrangements involving the short arm of chromosome 9 inacute lymphoblastic leukemia and other hematological malignancies. Blood, 86:1931-1938, 1995.

38. Döhner. H.. Stilgenbauer. S.. James, M. R.. Benner. A.. Weilguni. T.. Bentz, M.,Fischer. K.. Hunstein. W., and Lichter. P. llq deletions identify a new subset ofB-cell chronic lymphocytic leukemia characterized by extensive nodal involvementand inferior prognosis. Blood. «9:2516-2522. 1997.

39. Zucca, E., Stein. H., and Coiffier, B. European Lymphoma Task Force (ELFT): reponof the workshop on Mantle Cell Lymphoma (MCL). Ann. Oncol.. 5: 507-522, 1994.

40. Ott. M. M.. Bartek. J.. Duerr. A., Ott. G.. Miiller-Hermelink. H. K., and Kreipe, H.

Mantle cell lymphomas with the U11;14) exhibit protein expression of cyclin DI anddownregulation of cyclin D3 in contrast to normal B-Iymphocytes. Ann. Hematol.. 73

(Suppl. II); AI3I. 1996.41. Le Beau. M. M. Cytogenetic analysis of hematological malignant diseases. In: M. 1.

Barch (ed.). The ACT Cytogenetics Laboratory Manual, pp. 395-445. New York:

Raven Press Ltd., 1994.42. Bohlander. S. K., Espinosa. R., Ill, Fernald, A. A., Rowley, J. D., Le Beau. M. M..

and Diaz. M. Sequence-independent amplification and labeling of yeast artificial

chromosomes for fluorescence in situ hybridization. Cytogenet. Cell Genet., 65:108-110, 1994.

43. Slilgenbauer, S.. Leupolt, E., Ohi, S.. Weiß,G., Schröder,M., Fischer, K., Bentz, M..Lichter. P.. and Döhner. H. Heterogeneity of deletions involving RB-I and theD13S25 locus in B-cell chronic lymphocytic leukemia revealed by fluorescence msitu hybridization. Cancer Res.. 55: 3475-3477. 1995.

44. Erickson, P.. Gao. J.. Chang. K. S.. Look. T.. Whisenant. E., Raimondi. S.. Lasher, R.,Trujillo, J., Rowley, J. D.. and Drabkin, H. Identification of breakpoints in t(8;21)acute myelogenous leukemia and isolation of a fusion transcript AML1/ETO, withsimilarity to drosophila segmentation gene. runt. Blood. 80: 1825-1831, 1992.

45. Merlo, A., Herman. J. G., Mao. L.. Lee. D. J.. Gabrielson, E.. Burger. P. C, Baylin.S. B., and Sidransky. D. 5' CpG island methylation is associated with transcriptional

silencing of the tumour suppressor ¡¡16/CDKN2/MTSIin human cancers. Nat. Med.,/: 686-692, 1995.

46. Ohnishi, H.. Kawamura. M., Ida, K.. Sheng. X. M., Hanada. R.. Nobori. T..Yamamori. S.. and Hayashi. Y. Homozygous deletions of ¡>16/MTS1gene are frequent but mutations are infrequent in childhood T-cell acute lymphoblastic leukemia.Blood. 86: 1269-1275, 1995.

47. Herman. J. G.. Jen. J.. Merlo. A., and Baylin. S. B. Hypermethylation-associatedinactivation indicates a tumor suppressor role for /;/51NK4B1. Cancer Res., 56:

722-727. 1996.

48. Pinyol. M.. Hernandez. L.. Carzola, M.. Balbin, M., Jares, P., Fernandez. P. L..Montserrat. E.. Cardesa. A.. Lopez-Otin, C.. and Campo, E. Deletions and loss ofexpression of/j/6!NK4A and/?2/VVAI*1genes are associated with aggressive variants of

mantle cell lymphomas. Blood. «9:272-280. 1997.49. Williams. M. E.. Finkelstein. S. D.. and Swerdlow. S. H. p53 mutations and cyclin-

dependent kinase inhibitor pi5 (MTS2) and piò (MTS1. CDKN2) deletions in mantlecell lymphoma: association with extranodal disease. Ann. Oncol.. 7 (Suppl. 3): 36,1996.

50. Fizzotti. M.. Cimino. G.. Pisegna. S.. Alimena. G.. Quartarone. C., Mandelli, F.,Pelicci, P. G., and Lo Coco, F. Detection of homozygous deletions of the cyclin-dependent kinase 4 inhibitor l/>/6) gene in acute lymphoblastic leukemia and association with adverse prognostic features. Blood. 85: 2685-2690, 1995.

51. Hernandez, L.. Fest, T., Carzola, M., Teruya-Feldstein, J., Bosch, F., Peinado, M. A.,

Pins, M. A., Montserrat, E., Cardesa, A., Jaffe, E. S., Campo, E., and Raffeid, M. p53gene mutations and protein overexpression are associated with aggressive variants ofmantle cell lymphomas. Blood, 86: 3351-3359, 1996.

52. Greiner. T. C., Moynihan. M. J.. Chan, W. C.. Lytle. D. M.. Pederson. A.. Anderson.J. R.. and Weisenburger. D. D. p53 mutations in mantle eel! lymphoma are associatedwith variant cytology and predict a poor prognosis. Blood. 87: 4302-4310. 1996.

53. Louie, D. C.. Offit. K.. Jaslow, R., Parza. N. Z.. Murty, V. V. V. S.. Schluger. A., andChaganti, R. S. K. p53 overexpression as a marker of poor prognosis in mantle celllymphomas with 1(11:l4)(ql3:q32). Blood. 86: 2892-2899. 1995.

54. Lo Coco, F.. Gaidano. G.. Louie. D. C.. Offit. K.. Chaganti. R. S. K.. and Dalla-

Favera, R. p53 mutations are associated with histologie transformation of follicularlymphoma. Blood. 82: 2289-2295, 1993.

55. Jares, P. E., Pinyol, M.. Bosch, F.. Miquel. R.. Fernandez. P. L.. Sánchez-Beato. M..Soler, F., Pérez-Losada,A.. Nayach, I., Mallofre, C.. Piris, M. A.. Montserrat, E., and

Cardesa. A. Expression of retinohlastoma gene product (pRB) in mantle cell lymphomas. Am. J. Pathol., 148: 1591-1600, 1996.

56. Zukerberg. L. R.. Benedict. W. F.. Arnold, A., Dyson. N.. Harlow. E., and Harris.N. L. Expression of the retinoblastoma protein in low-grade B-cell lymphoma:relationship to cyclin DI. Blood. 88: 268-276, 1996.

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