Circulating lymphoma cells in patients with B & T non-hodgkin's lymphoma detected by immunoglobulin and T-cell receptor gene

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1 Br. J. Cncer Br. J. Cncer (1987). c Thc Mcmilln Press 1987 (1987). 56, The Mcmilln Press Ltd., , Circulting lymphom cells in ptients with B & T non-hodgkin's lymphom detected by immunoglobulin nd T-cell receptor gene rerrngement M. Brd', S. Mizutni2, H. Molgrd2, J.P. Slone', J. Treleven', A. Horwich' & M.J. Peckhm' 1Institutc of Cnccr Reserch nd TIie Rovl Mcsdcez Hospitl, Downs Rod, Sutton, Surrey; nd 2LRF Centre, Clester Bcttui Lbortories, Institute of Cncer Reserlch, Fullimn Rod, London SW3 6JJ, UK. Summry We studied peripherl blood mononucler c6lls from 5 ptients with ctive B- nd T-cell non- Hodgkin's lymphom by DNA hybridistion. Nineteen ptients (38%) hd circulting clones of cells detected by immunoglobulin gene rerrngement (17 ptients) or T-cell receptor gene rerrngement (2 ptients) with J,, nd J¾2 probes. Lymphom tissue nd peripherl blood were studied simultneously in 22 ptients, 9 of which hd circulting clone of cells in peripherl blood. In 7 ptients the gene rerrngement in lymphom tissue nd peripherl blood mononucler cells ws identicl. However, in 2 ptients both hevy chin nd light chin gene rerrngements were different in tissue nd peripherl blood. The incidence of peripherl blood involvement ws commonest in dvnced CSIII & IV disese (5%) compred to CSI & II disese (18%) (P<.5), nd in low grde (5%) compred to intermedite nd high grde lymphom (31%) (difference not sttisticlly significnt). Only ptients hd definite lymphom cells seen on peripherl blood smer. The presence of circulting lymphom cells correlted with conventionl ssessment of bone mrrow involvement lthough circulting clones were detected in 3% (12/) of ptients with pprently norml bone mrrow. Lymphom cells hve been detected in peripherl blood by routine morphologicl exmintion of the blood smer (Come et l., 198; Dick et l., 197; Foucr et l., 1982; Grrett et l., 1979; McKenn et l., 1975). Their presence in B-cell lymphom hs lso been implied with k nd. light chin stining nd the demonstrtion of bnorml k/. rtio (Sobol et l., 1985; Johnson et l., 1985), clonl excess (Ligler et l., 198; Weinberg et l., 198) or by cytofluorimetric studies (Smith et l., 198). With the dvent of clonl nlysis by immunoglobulin (Ig) nd T-cell receptor (TCR) gene rerrngement studies of B- nd T-ccll lymphoms (Arnold et l., 1983; Clery et l., 198; Bertness et l., 1985; O'Connor et l., 1985), it hs become possible to detect clonl rerrngement with up to I % sensitivity (Arnold et l. 1983). This llows for the detection of clones of cells in peripherl blood nd hs lredy been successfully pplied to ptients with low grde B-cell lymphom who hve high frequency of bone mrrow nd peripherl blood involvement (Hu et l., 1985). We hve set out to estblish whether the clones of cells in peripherl blood represent circulting lymphom cells nd to ssesses the frequency of peripherl blood involvement in ll histologicl types nd stges of non-hodgkin's lymphom (NHL). Where possible we lso compred the ssessment of bone mrrow involvement by conventionl nd DNA hybridiztion techniques. Ptients nd methods Ptients We studied 32 consecutive untreted ptients with non- Hodgkin's lymphom (NHL) referred to the Lymphom Unit t the Royl Mrsden Hospitl nd 18 ptients with recurrent lymphom undergoing tissue biopsy. All ptients hd full stging investigtions which included full blood count, differentil white count, routine biochemistry, bone mrrow spirte nd trephine biopsy, chest X-ry nd CT scn of chest nd bdomen. Selected ptients hd bipedl Correspondence: M. Brd. Received 2 Jnury 1987; nd in revised form. 5 My lymphogrphy. Their clinicl stge ws ssigned ccording to Ann Arbor clssifiction (Crbone et l., 1971). The histology ws reviewed by single pthologist (JPS) nd clssified ccording to the Working Formultion (The Non- Hodgkin's Lymphom Pthologic Clssifiction Project (1982)). In 5 ptients clssifiction ws bsed on the referring hospitl's report. Peripherl blood cytology ws exmined independently of the DNA nlysis by single hemtologist (JT). Peripherl blood involvement by lymphom ws defined s the presence of more thn four cells resembling lymphom cells on microscopic exmintion of 15 high power fields (x ). Detection of 2- bnorml cells ws defined s 'suspicious of involvement'. DNA hybridistion of peripherl blood mononucleur cells ws performed on ll 5 specimens. We lso exmined the DNA from bone mrrow spirtes of 12 nd from tissue biopsy of 22 of these ptients. Peripherl blood ws lso obtined from 15 controls - 7 ptients with Hodgkin's disese, 3 norml volunteers, 2 ptients with chronic myeloid leukemi (CML) in chronic phse nd 3 with other conditions (1 undifferentited tumour nd 2 rective lymphdenopthy). In ddition we studied 9 control lymph node biopsies from 6 ptients with Hodgkin's disese t presenttion nd 3 with other conditions (s bove). Methods Forty ml of venous blood nticogulted with preservtivefree heprin were seprted on Ficoll/Isopque density grdient to obtin mononucleur cell frction. Cells were wshed twice in buffered tissue culture medium nd frozen until further nlysis. Where vilble, bone mrrow spirte ws treted in similr mnner. Tissue biopsy mteril ws frozen nd kept t -9 C. Before digestion the tissue ws disrupted by grinding in liquid nitrogen. DNA ws prepred by stndrd methods (Ford et l., 1983). DNA (IOpg) ws digested with restriction enzymes- EcoRI, XbI nd in selected cses with HindII, EcoRV or BnmHI. Frgments were seprted by electrophoresis on.7% grose gel, trnsferred onto nitrocellulose filter (Southern, 1975) nd hybridized with immunogiobulin gene or T-cell receptor gene probes. These were rdio-lbelled with, 32P-CTP by rndom primer extension method. We

2 18 M. BRADA et l. initilly used J. DNA probe (Bgl If - Bgl II frgment excised from CH28-6; Rvetch et l., 1981) nd J.2 probe (.2kb EcoRI restriction frgment of J,2 region) kindly provided by Dr P. Leder nd Dr B. Toyong. BmHI digests were hybridized with CK probe. DNA from ll specimens ws initilly digested with EcoRI nd XbI restriction enzymes nd hybridised with J,, nd J.2 probes. If rerrngement ws detected with only one enzyme, DNA ws further digested with either HindlII or EcoRV restriction enzymes nd hybridised with JH or JP2 respectively. A circulting clone of cells ws considered to be present if one or two rerrnged bnds in ddition to germline bnd were present on t lest two seprte enzyme digests. One ptient hd rerrngement detected on CK probing of BmHI digest lone. Results The clinicl stge nd histologicl grde of 32 untreted nd 18 relpsed ptients with non-hodgkin's lymphom re shown in Tble I. Twenty-four ptients hd low grde (A = 3, B = 15, C = 6, 1 uncertin), nd 25 intermedite nd high grde lymphoms (D=1, E=3, F=, G=O, H=, Tble I Fifty ptients with non-hodgkin's lymphom (The Royl Mrsden Hospitl, 1986) Previously untreted Recurrent disese Histology' CSI & II CSII & IV CSI & ilb CSIJI & I Vb Low grde Intermedite nd high grdec Clssified ccording to Working Formultion; bclinicl stge t the time of relpse; cincludes 1 ptient with ggressive cutneous T- cell lymphom. I= 1, 1 uncertin). One ptient with ggressive cutneous T- cell lymphom ws included in the ltter ctegory. On immunohistochemicl nd in some cses on dditionl gene rerrngement criteri 6 ptients hd B- nd T-cell lymphom. One ptient hd coexistent folliculr smll cleved cell lymphom with cutneous T-cell lymphom. The men ge (rnge; s.d.) t the time of study ws 53 (19-73; 1.5) yers. Nineteen of 5 ptients studied (38%) hd Ig or TCR gene rerrngement detected in peripherl blood. Two hd T-cell nd 17 B-cell lymphom. Exmples re shown in Figure 1. Peripherl blood from 15 control subjects ws norml without detectble rerrngement. Gene rerrngement in peripherl blood nd lymphom tissue We were ble to study biopsy tissue nd peripherl blood simultneously in 22 ptients. All 22 biopsy specimens showed gene rerrngement; 2 on JH nd on JP2 probing. One ptient with T-cell lymphoblstic lymphom on immunohistochemicl criteri hd both TCR gene nd Ig gene rerrngement. The ltter ws only detected by hevy chin probing (JH) with light chin gene (CK nd ca) in germline configurtion (dt not shown). One ptient hd coexistent B- nd T-cell lymphom (see bove). We detected gene rerrngement in mononucler cells from peripherl blood from 9 of these ptients. In 7 the rerrngement in peripherl blood nd tissue biopsy mteril ws identicl on JH (5 ptients) or JB2 (2 ptients) probing (e.g. Figure 2). Two ptients hd different rerrngement in the 2 specimens. One ptient with recurrent diffuse lrge cell lymphom confined to single nodl site (Figure 3) nd one with extensive recurrence of diffuse smll cleved cell lymphom which initilly presented in nodulr form. On further nlysis of light chin gene with BmHI digestion nd C, probing we lso detected different rerrngement in lymphom tissue nd peripherl blood in both ptients. Peripherl blood JH probe Pts. X 28 (GY) H E 29 (AE) X x 33 (KG) H E.. EcoRI X.. Xbl H.. Hindill Figure 1 Exmples of Southern blot nlyses of DNA extrcted from mononucler cell lyer of peripherl blood from 3 ptients with ctive non-hodgkin's lymphom showing immunoglobulin gene rerrngement. Ech DNA ws digested with t lest 2 enzymes nd hybridised with the JH probe. Open tringle denotes the position of germline bnd nd closed tringle shows position of fint rerrnged bnd(s). (.. represents rtefcts due to prtil digestion or contmintion.)

3 CIRCULATING LYMPHOMA CELLS 19 Frequency ofperipherl blood involvement Of 19 ptients with detectble clonl rerrngement in peripherl blood seven hd recurrent disese nd 12 were untreted. Fifteen hd Ig nd 2 TCR gene rerrngement in ssocition with B- nd T-cell lymphoms respectively. The distribution of peripherl blood involvement in reltion to stge nd histology is shown in Tble II. It ws more common in dvnced compred to loclised disese (CSI & II vs. CSIII & IV; 5% vs. 18%) nd in low grde compred to high nd intermedite grde lymphom (6% vs. 31%) lthough the ltter did not rech sttisticl significnce. The difference between erly nd dvnced disese ws mintined when the extent of disese ws corrected for histologicl grde. Gene rerrngement nd conventionl ssessment ofperipherl blood Of 19 ptients with Ig nd TCR gene rerrngement detectble in peripherl blood only hd suspected lymphom cells on routine peripherl blood film stined with My- Grunwld-Giems stin (Tble III). In 1 ptient with positive smer there ws no detectble bnormlity on DNA hybridistion. Three of 9 ptients with suspicious peripherl blood smer nd 12 of 35 with pprently norml smer hd rerrngement detected in peripherl mononucler cells by DNA hybridistion. The totl white count nd mononucler cell counts did not differ significntly between ptients with nd without rerrngement (Figure ). Bone mrrow involvement All ptients hd conventionl bone mrrow ssessment by spirte nd trephine biopsy. Bone mrrow ws considered to be involved by lymphom when the histology nd/or cytology were bnorml. The frequency of peripherl blood involvement in reltion to bone mrrow cytology nd histology is shown in Tble IV. Seventy per cent of ptients with bone mrrow disese hd clonl rerrngement detected in circulting mononucler cell frction compred to 3% of ptients with norml bone mrrow. The incidence ws not relted to the histologicl grde of lymphom. DNA nlysis ws performed on bone mrrow spirte from 12 ptients. Four hd detectble rerrngement which in 3 cses ws identicl to peripherl blood (exmple in Figure 1). Comprison of DNA hybridistion nd conventionl bone mrrow ssessment (Tble V) shows flse negtive rte for histology of 2% (2/1). Discussion As shown previously (Hu et l., 1985; Berliner et l., 1986), we were ble to demonstrte the presence of circulting clones of mononucler cells in ptients with non-hodgkin's lymphom s specific immunoglobulin or T-cell receptor gene rerrngements. Seven ptients hd identicl gene rerrngement in peripherl blood nd lymphom tissue, suggesting tht circulting clones of cells represent lymphom cells. In 2 ptients, both with recurrent disese, the pttern of hevy nd light chin rerrngement differed between peripherl blood nd lymphom tissue nd this could be scribed to biclonlity (Sklr et l., 198). Preservtion of specific trnsloction detected by bcl-2 probe (pfl- 2; Clery et l., 1985) nd identicl VDJ joining sequences in cses described by Sklr et l. (198) suggest tht the pprent biclonlity my lso be due to somtic muttion (Clery, personl communiction). In our cses this would hve to be explined by two muttions. Permnent cell lines of B-cell linege cn undergo further rerrngement by exchnge with n upstrem V segment (Reth et l., 1986; Kleinfield et l., 1986). Although such ltertions hve not been demonstrted in vivo they my lso be cuse of pprent biclonlity bsed on Ig gene rerrngement studies lone. Tble II Frequency of detection of Ig nd TCR gene rerrngement in peripherl blood in 5 ptients with NHL (The Royl Mrsden Hospitl, 1986) CsI & II CSIII & IV All stges Number Number Number Histology of of of of lymphom ptientsb % ptients % ptients % Low grde 2/8 25 9/ /2 6 Intermedite nd high grde 2/1 1 6/12 5 8/26 31 All histologies / / /5 38 Grdes ccording to Working Formultion; bexpressed s number of ptients with detectble rerrngement/number of ptients tested. Tble III Detection of circulting lymphom cells: Comprison of conventionl cytology with DNA nlysis Gene rerrngement in peripherl blood mononucler cells Cytology ofperipherl blood smer Detected Not detected Totl Positive 1 5 Suspicious Negtive Not vilble - 1 Totl Tble IV Frequency of detection of circulting lymphom cells in reltion to conventionl bone mrrow involvement (The Royl Mrsden Hospitl, 1986) Involved' Bone mrrow Not involved' Histology Number of Number of of lymphom ptientsb % ptients % Low grde 6/7 86 5/17 29 Intermedite nd high grde 1/3 33 7/23 3 All histologies 7/1 7 12/ 3 Assessed by cytology of bone mrrow spirte nd histology of trephine biopsy; bexpressed s number of ptients with detectble rerrngement in peripherl blood/number of ptients studied. Tble V Bone mrrow involvement by lymphom: Comprison of cytology nd histology with DNA nlysis in 12 ptients (The Royl Mrsden Hospitl, 1986) Conventionl histology nd cytology of bone mrrow Gene rerrngement in bone mrrow* Involved Not involved Detected 2 2 Not detected 8 Number of ptients.

4 15 M. BRADA et l. In pb bm JH probe In pb bm EcoRI Xbl Pt.15 (RG) Figure 2 Autordiogrphs of DNA nlyses obtined from lymphom tissue (In), peripherl blood mononucler cells (pb) nd mononucler cells from bone mrrow (bm). The pttern of immunoglobulin gene rerrngement ws obtined by digestion with two seprte enzymes (EcoRI nd XbI) nd hybridistion of Southern blots with JH probe. Open tringles indicte the position of germline bnd nd closed tringles the position of rerrnged bnds. JH probe In pb In pb b ck probe In pb 1... BmHI EcoRI Pt.1 (EM) Pt.1 (EM) Figure 3 Anlysis of immunoglobulin gene rerrngements in lymphom tissue (ln) nd peripherl blood mononuclel cells (pb) from ptient with recurrent diffuse lrge cell lymphom. () Autordiogrph of Southern blot nlysis of EcoRl nd XhI DNA digests probed with JI, probe. (b) BmHI digest probed with ck probe. [Open tringle indictes the position of germline bnd nd closed tringle the position of rerrnged bnd(s).] Xbl

5 - 12 ) x ) 9 Q C. 6 x U3 ( C) L. I lb 3 v b 5 r I v-, ES IB Undetected Go eo e OB Undetected o m No Detected (19.1) (1.3) (8.8) Detected Circulting lymphom cells Figure Totl white count () nd mononucler cell count (b) in ptients with nd without circulting lymphom cells s defined by immunoglobulin or T-cell receptor gene rerrngement in peripherl blood. Filled symbols represent ptients with lymphom cells seen on peripherl blood smer. Numbers in brckets indicte the individul cell counts outside the rnge in figure. Hu et l. (1985) reported identicl Ig gene rerrngement in lymphom tissue nd circulting cells in 7 ptients with folliculr B-cell lymphom. Identicl light chins on circulting mononucler cells nd lymphom tissue lso suggest tht the circulting clonl popultion represents lymphom cells (Smith ct l., 198). We detected circulting clones of cells in 19/5 (38%) ptients with ctive lymphom nd the incidence of blood involvement ws relted to the extent of disese nd possibly to histologicl grde (Tble II). Lymphom cells hve been detected on peripherl blood smer in 8-2% of ptients with NHL (Come et l., 198; Dick et l., 197; Foucr et l., 1982; McKenn et l., 1975; Morr et l., 1985). With the use of immunocytochemicl stining of light chins with nti k nd A. ntibodies the presence of circulting lymphom cells hs been implied in lrger proportion of NHL ptients. Abnorml k/2 rtio or Iclonl excess' suggested lymphom cells in peripherl blood of 36-55% of ptients (Grrett et l., 1979; Johnson et l., 1985: igler et l.. 198; Lindemlm et l., 1985; Sobol et l., 1985). With cytofluorimetric nlysis of k nd A stined mononucler cell popultions the peripherl blood involvement hs been reported in up to 78% of the ptients studied (Smith et l., 198). Although, to some extent, these results reflect incresed sensitivity of the more sophisticted techniques (Smith et l., 198; Berliner et l., 1986), they re lso dependent on ptient selection prticulrly s ll studies hd shown correltion with stge nd histologicl grde similr to our findings. The presence of circulting tumour cells, which is conventionlly considered feture of high grde mlignncy is commoner in the more benign lymphoms of low grde. It my reflect wht Jffe describes s 'benign' nture of low grde lymphom (Jffe, 1983), where tumour cells my retin some of the recircultion properties of norml lymphocytes (de Sous, 1981). The site of origin of circulting lymphom cells is, however, not certin. The mjority of ptients with these cells hve morphologicl bone mrrow involvement; only 3% with norml bone nrrow hve peripherl blood lymphom cells. As the flse negtive rte of 2% for conventionl bone mrrow cytology nd histology is similr, circulting lymphom cells my reflect bone mrrow involvement. This view is supported by studies where bnorml peripherl blood cytology ws detected only in ssocition with positive bone mrrow lthough Smith et l. (198), using cytofluorimetry, detected circulting clones of cells in 8% of ptients with norml bone mrrow. The possibility of peripherl lymphom cells originting in lymphom tissue cnnot be excluded prticulrly if bone mrrow is considered trnsient stop in the recircultion of lymphocytes or if we dhere to the trditionl view of spred from primry to metsttic sites vi the blood strem. To nswer the question of tumour cell origin it will be necessry to perform longitudinl studies, prticulrly in ptients receiving only locl therpy. Clinicl relevnce nd conclusion It remins to be shown if the detection of lymphom cells in peripherl blood with such high sensitivity is of prognostic or therpeutic importnce. Our findings support the view of low grde lymphom s systemic disese (Jffe, 1983). The detection of lymphom cells in blood is unlikely to lter current tretment strtegies except in erly disese where locl rdiotherpy is the tretment of choice (Pryni et l., 1983; Sutcliffe et l., 1985). Our findings of 25% of peripherl blood involvement in CSI nd II NHL my indicte the source of filure in proportion of these ptients. If peripherl blood lymphom cells represent bone mrrow disese we my lso speculte tht in low grde lymphom their detection will be of no prognostic significnce (Brtl et l., 1982; Lindemlm et l., 1985; Bennett et l., 1986). Similr considertions pply to intermedite nd high grde lymphoms lthough bone mrrow involvement in these tumours is considered poor prognostic fctor (Fisher et l., 1981; Gms et l., 1985; Stewrd et l., 198; Bennett et l., 1986). With the incresingly successful use of chemotherpy in erly-stge disese (Miller et l., 1983; Cbnills, 1983) there is less need for exct delinetion of tumour sites. However, if we consider locl rdiotherpy less toxic tretment, the detection of circulting lymphom cells my help in choosing the pproprite therpy, with systemic tretment reserved for truly systemic disese. In ddition sensitive method of detection of miniml disese in peripherl blood nd bone mrrow my hve n importnt role in bone mrrow trnsplnttion, prticulrly if utologous mrrow is used. The serch for circulting lymphom cells by DNA hybridistion cnnot t present be considered prt of the routinie ssessment of lymphom ptients. The exct role of this investigtion remins to be defined nd so fr it is too unwieldy for regulr clinicl ppliction. However, the prospect of observing tumour cells with such precision nd CIRCULATING LYMPHOMA CELLS 151

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