Genotype and phenotype: A practical approach to the immunogenetic analysis of lymphoproliferative disorders

Genotype and Phenotype: A Practical Approach to the lmmunogenetic Analysis of Lymphoproliferative Disorders ANNA M. WU, PHD, CARL D. WINBERG, MD, KHALIL SHEIBANI, MD, ANNE M. COLOMBERO, BA R. BRUCE WALLACE, PHD, AND HENRY RAPPAPORT, MD Determination of cell lineage and clonality in lymphoproliferative disorders (LPD) is greatly enhanced by molecular genetic analysis in conjunction with morphologic and immunologic techniques. We now report on a technique in which we used cryostatcut, fresh-frozen sections (CCFFS) prepared from tissues in a manner that allows DNA hybridization studies to be coordinated readily with routine morphologic and immunohistologic studies. Thirty-seven cases representing a broad spectrum of reactive and malignant LPD were examined with this method. Samples of DNA were extracted from frozen sections, subjected to Southern blot hybridization, and probed for rearrangements of the immunoglobulin (Ig) heavy-chain and the K and A light-chain genes, as well as for the T-cell receptor P-chain gene. We also evaluated the effects of (1) diagnostic category of LPD, (2) volume of the tissue sample, and (3) fibrosis, necrosis, and ice crystal artifacts in the sample on the recovery of DNA. Ice artifact and sample size had the greatest negative impacts on the quantity and condition of DNA recovered. Of 19 samples involved by B-cell LPD, the results of immunogenetic studies were consistent with the immunophenotypes in all but one case. Of the T-cell lymphomas from which sufficient DNA was available (three out of five of the T-cell cases), all showed rearrangements of the T-cell P-chain gene. In order to reduce sample processing time, we evaluated alternate blot hybridization methods, rapid alkaline transfers, and direct hybridization of synthetic oligonucleotides in dried agarose gels, and found that they decreased the time required for hybridization studies. In summary, the use of CCFFS as the source of DNA allows study of gene rearrangements and, at the same time, preserves frozen-tissue blocks in tumor banks for further immunologic studies. The development of time-effective methods will make the routine use of molecular-genetic analysis more practical in the diagnostic hematopathology laboratory. HUM PATHOL 21:1132-1141. 0 1990 by W.B. Saunders Company.

From thejames Irvine Center for the Study of Leukemia and Lymphoma. Dlviaion of Anatomic Pathology. and the Department of Molecular Biochemistry. City of’ Hope National Medical Center, Duarte. CA. Accepted for publication January 23. 1990. Supported bk (it-ants No. <:A-‘Ltitix and (:,4-09%)8 awarded by the National (iancer Institute, Department of IIealth and HIIman Services. Bethesda, MD: by Hematopathology Tutorials. Inc. Duarte. CA; by the Mobil Foundation, New York, NY; and by the James Irvine Foundation, San Francisco, CA. Drs Wu, Sheibani. Wallace. and Kappaport are members of the City of Hope <:ancer Kesearch (:enter which is supported by Grant No. CA-33572 awarded by the National Cancer Institute. This work is also made possible in part by support from the James H. Hat-less Kesearch Fund. Duarte, (:A. Dr Winberg is presently with the Department of Pathology. Saint Joseph Medical Center. Burbank, (3. Key ulor&: Ipmphomas, frozen sections. oligonucleotide hybridization, alkaline transfer. gene rearrangements. Address correspondence and reprint requests to Anna Wu. PhD. Division of Pathology, (:ity of Hope National Medical <:enter. 1500 East Duarte Kd, Duarte, C.4 Y1010. 0 1990 by W.B. Saunders Company. 0046-8 17719oi’L 1 1 1-0008$5.00/0

1132

Multidisciplinary studies of human lymphoproliferative disorders (LPD) have included morphologic, cytochemical, immunologic, cytogenetic, cellkinetic, and electron-microscopic analyses. These analyses frequently provide information about the LPD which could not be obtained from morphologic evaluation alone. Recently, molecular hybridization techniques have made possible the identification of gene rearrangements in leukemias and lymphomas. K’ These techniques have proven useful for both diagnosis and research.‘-“J,“‘-” In most reported studies on LPD that involved molecular hybridization techniques, investigations have used either cell suspensions prepared from fresh tissues or large fragments of frozen tissues that had been stored in frozen-tissue tumor banks, I.:4.-l.i,7.1~).1”-17 We now report on a practical method for immunogenetic analysis of LPD which is ideally suited for use in the diagnostic hematopathology laboratory. This method, in which cryostat-cut, fresh-frozen sections (CCFFS) are used for molecular hybridization, can be adapted to multidisciplinary studies in which morphologic, immunologic, and molecular hybridization results are evaluated simultaneously. We describe and evaluate two alternative hybridization methods, alkaline transferlH and dried-gel hybridization.“‘~“” which reduce the total time required for molecular hybridization studies. The results of our evaluation of the usefulness of our methods in a study of 37 tissue samples involved by a variety of LPD are presented. MATERIALS AND METHODS

Patientsand Samples The 37 tissue samples analyzed as part of this study, were selected f’rom 340 samples evaluated at the City ot Hope National Medical (Ienter, Duarte, CA. between March lYH.5 and July 1985. The cases were chosen with the following objectives: C1) the series would have a representative distribution of’ samples involved by reactive and neoplastic LPD, as well as by nonhematopoletic tumors; (2) the histologic findings in each case were characteristic of the morphologic diagnosis; (3) the immmlologic characterization of the disease process was complete; and (4) f’rozen tissue was available in our Hematopathology Frozen-Tissue Tumor Bank. Under ideal circumstances, the specimens were received in the f’resh state in physiologic saline from the op crating room and immediately placed in a Petri dish containing Hanks’ balanced salt solution with 1% bovine serum albumin, 17r penicillin-streptomycin, and 0.17( gentamicin.

IMMUNOGENETIC ANALYSIS OF LPD [wu et al)

Chnditions leading to drying of the specimens were avoided. .Thc fresh tissue samples waere di\:ided for tnultidiscipliii;rr~ laboratory studies as previously described.“-“’ Spectmens were cut into parallel sections !?-mm thick, and then alternate sections were processed as (1) iixed tissue for morphologic studies. (2) frozen tissue fat the tumor bank, and (3) fresh tissue for cell suspensions. Tissue samples were prepared for our Hematopathologv FroLrn-‘I‘issue Tumor Bank as described previousIv.“.~” Briefly, embedding medium (OCT compound, Lab ‘l‘rch Products. Naperville, II.) was applied to a disposable composite cork disk. The specimen was set into the embedding medium anti oriented f’or a good cross sectional cut. After the specimrtt was covered with OCT compound so that it wa.r protected front freezer desiccation. the sample was snap-f’rozrtt either in a freezing chamber of‘ A cryobath unil or in isopentane precooled by liquid nitrttgett. The satnple was lhrti wrapped in precooied aluminum f-oil, placed in a small f’reezer bag. and stored in a freerer at 8oY:.

c.otnpietion of the molecular hybt-idiLation studies wa:i not a prerequisite for inclusion of a case in this study* since we wanted to evaluate the efftct of processing artifacts on the results of the molecular hybridization studirs. ILfost s;1t1ilh3 were ittitialiv prepared in our laboratory, but some were snapfrozen I& referring pathologists and sent to our taboratorv bv courier service. Succe:;st'~~l

lmmunohistochemical

Proteinase K (Boehringer-Mannheitn, Indianapolis, IN) to 0.5 mg/mL. Proteinase digestion was accomplished overnight at 55°C. On the following day, the samples were extracted twice with phenol-chlorofortn (?: 1, equilibrated against 10 mmol/L Tris-HCI, pH 8). followed b) one extraction with chloroform. Nucleic acids were precipitated by addition of ‘/I,, volume of 3 tnol!L sodium acetate (NaAc), pH 7. and two volumes of cold ethanol. A fibrous DNA precipitate was always visible: it was spooled onto a glass capillary and redissolved in -150 ~1 10 mmol/L TrisHCI, 1 mmoi/L EDTA overnight at 4°C:. The DNA samples were stored at 4°C. Concentrations were determined b? measurement of LTV absorbance at 260 nm. Absorbance was also measured at 280 nm, providing an index of the purity of the DNA. Control DNA was prepared by the method of Km et al” from peripheral bloocl from a normal human volunteer.

Calculation of Recovery and Yield of DNA The total amount of DNA recovered f’rom each sample is given in micrograms (kg). The yield of DNA was defined as the quantity of DNA (pg) recovered per volume of tissue satnple digested. ‘I-he volume was determined by the measurement of the surface area (mm”) of the tissue in an itnmunostained CUFFS, and multiplication of this area by the total thickness of’ the sections (2 X IO ’ ttttn). l‘he final vield of DNA was obtained by the equarion:

Studies

For intntttttohistocIlemica1 study of C(:FFS, the direct techniclue was used in the evaluation of surface membraneassociated ilnirrttrnoglobulin light chains.” In testing the remaining ivnrphocyte surface antigens. we used a modification of’the avidin-hiotin complex technique.“’ !!’ ‘I’he resulting stained f’rc,-/en tissue sections wet-e evairtared according IO ;I prc\iouslv tiescribed protocol.” l’et.oxiclase-cott,jugated F(ab’), goat antihutnatt antisrr‘a for lg(;, Ig.4, igM, and IgD heavy chains, and for K attd A light chains. were used f'Ot'the detection 01 surface ittit~iutioglol~ulitts (‘I‘AGO. Burlingame. CA). Thr samples were also !*tudied with a panel of‘ tnonocional antibodies, inc!uding (;1)5 (t-ractive with -1‘ cells and some B-ceil lymphoproliftt.;ttivr disordersj, CD8 (reactive with suppressor/ c) totoxic ‘I‘ cells), CD1 (reactive with helper/inducer .r cells and some histioc.ytes), and HLA-DR (reactive with B ceils. and activated ‘1’ cells), obtained from Bectontnonocytes, Dickinsott Laboratories, Sunnyvale, CA. We also used the tt~onociottal antibodies CD20 (reactive with B ceils of pcripheral blood and lymphoid organs), CD2 1 (reactive with surface itnir~unogtot)ulin-positive B lymphocytes md dettdritic c-ells). and (:D2 (reactive with T cells), ali f’rotn (kntiter Itnmrtnologv, Hialeah. FL.

Isolation and Purification of DNA I‘he DN.4 was prepared from a sample of snap-frozen tissue obtained from IO CCFFS cut at 20 pm thickness. In a preiitninarv studv (unpublished data) we found that the quantity antd’condi&on of the DNA recovered were affected adversely when the CCFFS were cut at less than 20 pm. wttereas
Yield = DN,4 (pg)/Volume

(mrn’r

To determine the causes of inadequate or low DNA vieid. we examined the corresponding histologic sections and determined whether fibrosis, necrosis, and/or iymphocyte depletion were present. The degree of fibrosis and the extent of necrosis were each graded according to the percentage of the lymph node surface involved: +, less than 34%; + + , 34’;: to tX%.; and + + + 1more than 67% of the surface area. We defined lymphocyte depletion as reduction in the number of iytnphoid ceils per unit surface area, assessed at 125X magnification; this was a :subjective evaiuation, and no grading was used. Frozemtissue samples were also evaluated for the presence of’ ice crvstal artifacts.

Molecular Hybridization Techniques Restrictwn mdonuclease digestion of DNA. Restriction digests were done in a loo-p1 reaction under buffer conditions recommended by the supplier (New England BioLabs. Beverly, MA). Twenty to 30 units of enzyme were used for digestion of 8 pg of DNA, and incubation was performed at 37°C for 2 to 3 hours so that c.omplete digestion was ensured. The extent of digestion could also be monitored by the appearance of “satellite bands” in the gel lanes after eiectrophoresis, due to the presence of reiterated sequences in the genomic DNA. The products of restriction endonuclease digestion were separated by electrophoresis on 0.7% agarose gels in 10 mmol/L Tris-acetate, pH X.0. 1 tnmol/L EDTA. HindHI-digested phage A DN.4 was used as a size standard: digested control human DNA was also included on each gel as a hybridization control and size marker. Eiectrophoresis was performed overnight at 2 V/c,m. Gels were stained with ethidium bromide (1 &ml,) and photographed under ultraviolet light. Southrm trunsfer. Gels were denatured in 03 mol/L sodium chloride (NaCl), 0.2 N sodium hydroxide (NaOH) for 30 minutes and neutralized in 1.5 moi/L N&l, I mol/L Tris-HCI. pH 7.5, for 30 minutes. The DNA was transferred to Biotrans activated nylon membranes (ICN Biochemicals, Costa Mesa. CA) in 10 X SS(: bv capillary blot-

1133

HUMAN PATHOLOGY

Volume 21, No. 11 [November 1990)

ting (1 X SSC is 0.15 mol/L NaCl, 0.015 mol/L sodium citrate. pH 7). Following overnight transfer, the membranes were baked at 80°C under vacuum for 2 hours.‘” Alkaline transfer. DNA was transferred from agarose gels to nylon membranes (Genetran 45, Plasco, Inc, Woburn, MA) according to the method of Reed and Mann.‘s Following pretreatment of gels in 0.25 N HCl for 15 minutes, capillary transfer was done in 0.4 N NaOH for 2 hours. Filters were then rinsed briefly in 2X SSC and air-dried. Hybridization

of nick-translated

probes

to filter-bound

DNA.

Supercoiled plasmids were purified by the alkaline-SDS lysis method of Birnboim and D01y.‘~ Gene inserts were isolated by digestion of plasmid DNA with the appropriate restriction enzymes (see “Hybridization Strategy,” below), followed by separation of insert from vector by agarose gel electrophoresis, and electrophoretic elution of the isolated restriction fragment. Two hundred nanograms of purified restriction fragment were routinely labeled in a 25-FL reaction; we used a commercial nick-translation kit (Bethesda Research Laboratories, Bethesda, MD) and a-32phosphorusdeoxycytidine triphosphate (3,000 Ciimmol, New England Nuclear, Boston, MA). The radiolabeled restriction fragment was separated from unincorporated label by chromatography on Sephadex G-100 (Pharmacia, Piscataway. NJ) in 0.3 mol/L NaCl, 10 mmol/L Tris-HCl, pH 8, 1 mmol/L EDTA. Specific activities of 2 to 5 x 10H cpmipg were generally obtained. Probes were stored frozen and were boiled just before use. Prehybridization of Biotrans membranes were carried out in 50% formamide (deionized), 3X SSC, 1X Denhardt’s solution, 0.1 mg/mL denatured salmon sperm DNA, 0.1% SDS, overnight at 42°C in an airtight storage bag. The hybridization buffer was the same with the addition of 5% dextran sulfate and 10” cpm/mL denatured probe. Hybridizations were performed at 42°C overnight. Filters were washed briefly twice at room temperature in 0.1X SSC, 0.1 CTSDS, followed by two washes for 45 minutes at 5 1“C in 0.1X SSC, 0.1%’ SDS. Blots were wrapped in plastic wrap and autoradiographed on Kodak XAR film (Eastman Kodak, Rochester, NY) with two intensifying screens at -70°C for 1 to 3 days. Prehybridization of Genetran 45 nylon membranes was done in 50% formamide, 5X Denhardt’s solution, 3X SSC, 0.5 mg/mL denatured salmon sperm DNA, and 1 ?G SDS in an airtight storage bag for 2 to 24 hours at 42°C. Hybridizations were conducted for 18 hours at 42°C in the same buffer, with the addition of 5% dextran sulfate and 10” cpm/mL of denatured probe. Washing conditions and autoradiography were the same as used for the Biotrans membranes. Hybridizution qf labeled oligonucleotide dried gels. We have used the procedure

probes

to DNA

in

of Tsao et al’” for direct hybridization of dried gels. Following staining and photography, gels were denatured and neutralized in low salt solutions (30 minutes in 0.5 mol/L NaOH, 0.15 moVL NaCl, followed by 30 minutes in 0.5 mol/L Tris, pH 8, 0.15 mol/L NaCl) and then dried onto Whatman 3MM paper (Clifton, NJ) under vacuum. Following rehydration in distilled H,O, excess liquid was blotted away, and the gels were placed in bags for hybridization. The A light-chain oligonucleotide (see below) was synthesized by the phosphoramidate method at the City of Hope DNA Synthesis Facility.“7 The oligonucleotide was end-labeled with T4 polynucleotide kinase (Bethesda Research Laboratories) and y-s’phosphorus-adenosine triphosphate” (P-ATP, 3,000 Ci/mmol, ICN Biochemicals). The labeled oligonucleotide 1134

was separated from unincorporated label by chromatography on diethylaminoethyl cellulose (Whatman). No prehybridization is necessary when oligonucleotides are hybridized to DNA in dried gels. Hybridizations were accomplished in heat-sealed storage bags in 5X SSPE. 0.1% SDS, 10 IJ-g/mL carrier DNA, and 2 x lOti cpm/mL kinased oligonucleotide. (SSPE is 0.15 mol/L NaCl, 10 mmol/L monosodium acid phosphate (NaH,PO,), I mol/L EDTA, pH 7.4). Based on the length and composition of the 23-base A light chain constant region probe, we calculated a Td of 74’Cz7 We have found that hybridization at 60°C followed by stringent washes at 67°C. gives excellent results with this probe. Hybridizations were done for 2 hours. Gels were then washed at room temperature in 6X SSC for 15 minutes, 15 minutes, and 2 hours. Finally, two l-minute washes in 6X SSC at the stringent wash temperature (67°C) were done. For autoradiography we used Kodak XAR film with two intensifying screens at a temperature of -70°C for 1 to 3 days.

Hybridization Strategy Immunoglobulin heavy-chain gene rearrangements were analyzed by hybridization of RamHI. EcoRI or HirLdIII blots with a 6 kilobasepair (kb) HamHI-Hind111 joining region fragment from the plasmid PJH.‘~ K Light-chain rearrangements were analyzed in BarnHI digests using a 2.5 kb EcoRI fragment from pHuK,:,“’ and h light-chain rearrangements were analyzed with EcoRI digests using a .5 kb Ecol-Hind111 fragment from pHuXC,.“” The configuration of the T-cell receptor P-chain locus was analyzed in BumHI, EcoRI, and HilLdIII digests using a 0.44 kb PstI-H&II fragment of pJurkat-2 as a pr0be.s’ Based on the published sequence of the A light-chain gene, we designed a mixed synthetic oligonucleotide probe 23 based long, with the following sequence: 5’CCTTCATG(A/C)GTGACCTGGCAGCT

3’.

This sequence is complementary to the human and mouse messenger RNA sequences corresponding to amino acids 197 900,s” . . to L

RESULTS Yield and Recovery of DNA In one spleen and five lymph node samples involved by reactive follicular hyperplasia (Table 1, cases no. 1 to 6). 227 to 481 pg of DNA were recovered (mean, 380 pg). The yield of DNA ranged from 2.8 to 23.1 kg/mms (Table 1). with a mean of 14.4 pg/mm”. In the lymph node sample with the lowest yield (2.8 pg/mm”), moderate fibrosis was identified. None of the six samples involved by reactive follicular hyperplasia was affected by ice crystal artifacts. In our four cases of Hodgkin’s disease (Table 1. cases no. 7 to IO), 136 to 460 kg of DNA were recovered, with a mean of 287 kg. The yields of DNA ranged from 3.2 to 18.1 pg/mm” (mean, 10.8 kg/ mm3). In two samples (cases no. 7 and lo), the yield of DNA was less than 10 pgimm”. Prominent ice crystal artifacts were identified in the sample with the lowest DNA yield (3.2 pg/mm”. case no. 7). whereas moderate ice crystal artifacts were identified in the frozen sections in case no. 10, which yielded 9.8 pgirnrn:’ of DNA. The total quantity of DNA recovered from the 23 samples involved by non-Hodgkin’s lymphomas (Table 2, cases no. 11 to 33). ranged from 11 to 1.161

IMMUNOGENETIC ANALYSIS OF LPD (wu et al)

TABLE 1.

Lymphoid Hyperplasia and Hodgkin’s Disease: DNA Recovery and Yield, Lymph Node Morphologic Diagnosis, and Immunologic Characterization

p_g,with ;i mean of.482 pg. ‘I‘hr yield of DN.4 ranget! from 5.8 to :%‘i.j pg/mm:‘, with a mean of’ 18.j ~g/mm”. Although too f’ew samples were available for statistical ;mal\sls, low DNA yields appeared to be correlated mc,‘;t closely lvith thd presence of‘ice crystal ;irtifkts in the frozen tissues. Of the four samples producing k+ldc of less than IO ~girnm (Grses no.

! 2, 17, 28. arid 3 I ). two containe4 prominent ice crystal artifacts, and one exhibited fibrosis. In these f’our cases. the low yields did not appear to h;-lvc an adverse effect on the &al DNA recovered. In the two specimens fl-om which iess than t 00 pg of DIVA were recovered (cases no. 19 and 23), the +Ads werr satisf’xtor) (1 I.2 ;~nti 27.5 pg/rn~n’~~, but

Non-Hodgkin’s Lymphomas, Mycosis Fungoides, and Nonhematopoietic Tumors: DNA Recovery and Yield, Lymph Node Morphology, and Immunologic Characterization --_-____ __-.-Uorphotogic Featllrrc I)NX

TABLE I!. _ ( ..*sc’ AC,. _____-

-____

l’i>.\clc, ,Scwl~c~

hIorpholoylc Diagnosi<

1.h 1.L

NPDI.

I.1 I.1

NI’DI.

1.h 1.h 1.h I.1 OK L\ SI’ (;I I.\ sti LX LX 1.N (;I I_!\ Sl (;I LX I.‘\ LX L r\ I.?\ L:i

Nl’l)l. N PDI. NSl

R.!XOXW\ (CLRJ

l’dti

ICY (:l\llt.ll

(pg/rrlm”)

,\rtil;lr

1.) nqh,c\tt

t

Fit,rosl\

Net rosia

Ikptcticm

--_

1mmunologic (.har-at ter-izalion

NPDI. N1’1)l.

sts: I)\VDL DM’DL Db’DI. I)1 Dhl D1.C :

Ills, 1Dl.C : 1)l.c: D1.C: D1.C. 111. DL I.13 LB 11F \l(:A MC::2 Stminoma

NCXI‘F.: See Iwt Illr grading system of morphotogir feature\. A&l-c_viatwrla: LS. t\-mph node; OR. orbit; SP. spleen; (;I. differentiated lymphocy:tic: NM. nodular mixed; N1.C:. nodular Inlet-mediate l~mphoc~tlc: DM, diffuse mixed tell: DL(:. diffuse Lullg-oidw: MC .4. Ineta\r;ttic carcinoma: B. U-cell tvmphoma: 7,

_ + _

_ _. +

i

_

++

_ _ _ _

_

+ +

Ii’

I’ t+

__---

poort$ gaslrointestinal tract; SK. skin; ST. soft lissue; NPDI.. nodular well-differentiated tcmphoc~ric : DI. diffuse tat-ge cell: DWDL. diftuse. large cell: DC‘. diffuse undifferentiated: LB. t!mphohtastic: MF. mycosis Tcell t\-mphomC~: P. pol!clonat Iymphoid poputariow

1135

Volume 21, No. 11 (November 1990)

HUMAN PATHOLOGY

TABLE 3.

Non-Hodgkin’s

Lymphomas,

Mycosis Fungoides, and Nonhematopoietic Tumors: lmmunophenotypic and Genotypic Characterization (;enr [g-Light

lrumunc~iogic (:haracterizatioll Case NO.

hlorpholo~qic Diagnosis NPDL NPDL NPDL NPDL NPDL KPDL Nhl x1.<; D!L’DL DWDL DWDL Dl Dhl DLC: DLC DLC DLC: DLC D1.C: DU DL‘ DI_T

t.n Ln hlF

Designation

n n n n n n n 13

n n n

1% T ‘I

n n .r n n n n n

Light <:hainli

IgG IgG IgM IgG

lgbl lghl-D IgM-L) IgM l#M lgrb-11

lghl lghl

lghl (i I)$; Ighl lghl lgM

-

Chain

lg-Ileav)

K

A

B

E:

B

(; K R R R R R R R R R (i/D (; NS R (i/D (; (C/D (i/D R R R R (; G

c;

R

-Ht%V\ Chains

Rearrangement

R (: (: C; (; (: R (; R (; R CG NS (; R (; R R (; R R (; C; (;

‘I‘-cell Keceptor p Chain

(Ihain

E

H

R R R R R R R R R (; (; R (; R R R R R

n

E

H (k G (i G K

(; (; NS (; (; (G G/D NS (; R ( iiD (; (; C; (; (; K

(;

(i (i (i

0 R c; R (;

R (; G C; (;

(; (;

Abbreviations: NPDL, noddar powly difterentiated Ivmphocytic: NM, nodular mixed: NLC, nodular large cell: DWDL, tiiff,tse. \vell-differentiated lpmphocytic; D1. diffuse intermediate lymphocytic: DM. diffuse mixed cell; DL(I. diffllse large cell; DLI, t\ifftlse undifferentiated; Ld. Iymphoblastic; MF. mycosis tungoides; B. B-cell lymphoma; T, T-cell Iymphoma; (;, germine; K. rearranged; NS, not sufficient sample. (Xl indicate5 that the grrmline hand was grratl:, reduced in intensitv. prerttmabl\ due to deletioll.

the total quantities of DNA recovered were only 37 pg and 11 Kg, indicating that the low DNA4 recovery was probably, related to the small tissue sample. Both specimens with low Dh’A recovery consisted of extranodal tissue; however, other samples from extranodal sites (gastrointestinal tract [cases no. 27 and 311) had adequate DNA recovery. but low yields. In the single case of lymph node involvement by mvcosis fungoides (case no. 34), the yield of DNA (421.5 kg/mm”) was the highest among our lymphoma cases, but the total quantity of DNA recovered was only 51 pg. The DNA yields of nonhematopoietic tumors involving tymph nodes (cases no. 35 to 37) were low (range, 6.0 to 9.0 pg/mm!‘), but the total quantity of DNA recovered was satisfactory (108 to 3 15 kg). Condition

of DNA

In all 37 cases. we were able to spool out high molecular weight DNA following the ethanol precipitation step. In other cases (not reported here) in which no fibrous precipitate was visible, the precipitated nucleic acids were collected by. centrifugation. However, following restriction d.igestlon and agarose gel electrophoresis, examination of stained geIs showed that the DNA obtained by centrifugation was badly degraded. In short. we found that the presence of spoolable DNA was correlated with high molecular 1136

weight and with suitability of the material tion digestion and hybridization studies.

for restric-

Analysis of Gene Rearrangements

Specimens from patients with reactive follicular hyperplasia, Hodgkin’s disease, and nonlymphoid neoplasms (cases no. 1 to 10, and 35 to 37) carried only the germline configuration at the immunoglobulln and T-cell receptor P-chain loci. In nonHodgkin’s lymphomas (Table 2), the 19 cases classified as B-cell lineage neoplasms on the basis of morphology and immunology (cases no. 11 to 22, 25. 26, and 28 to 32) all exhibited rearrapgement of the immunoglobulin heavy-chain gene. Light-chain gene rearrangements corresponded with the observed phenotype in agreement with the findings of Korsmeyer, et alzS2,“”with one exception. In case no. 1 1, which was marked immunologically as positive for h light chains, we were unable to detect any immunoglobulin lightchain gene rearrangements. In addition, four of 19 B-lineage LPDs showed rearrangements of the T-cell receptor P-chain genes. The five T-cell lineage LPDs in this series (cases no. 23, 24, 27, 33, and 34) did not show rearrangements of the immunoglobulin genes. Three of the cases (cases no. 23, 27, and 33) displayed rearrangement of the T-cell receptor P-chain gene. The re-

IMMUNOGENETIC ANALYSIS OF LPD (wu et al]

rtiaitiitig t\vo spw‘inirtis (cases no. 24 and 31) vieldcd iticuf.fic-ient I)S:\ fi)t- ;I complete anal\,sis.

Dried GelOligonucleotide Hybridization

Evaluation of Time-Saving Methods :I ccIttip;tt-inon of the alkaline transftt methotl \\ ith the ~~lattdai-d Southet-ti high salt transf’et- method is illustratrd in Fit 1. ~I‘he aisnals obtained after otilb L’hour5 c,f tratisf’tG time by t’&e alkaline method were as intense as those obtained f’olhnving overnight highs,tlt transfer. In ;tddition, the bands obtained af’ter the shot-t trarthf’er time ttanded to be sharprr. An es;tniplc of‘ the results of‘dri&f-q1 h!,bt-idizatton \vith the A olieonu~leotide is showti’in Fit 2. ‘I‘his c~ligonu~l~~otitlc hvbt-idires to and identifies the same bands that art’ de’tectecl with a nick-translatv(t h corlstatic-region probe. l)t-icd-,~el/oligotiuc~leoticlr hvhridi/;ttioti is ;I>’ svti5iti\e as the s;andard fillet-jnicktt~anslated t,rotw h\,t)t-idizatioti technicrue; similar aw tot-adioSr,;iI)ti~ tint& at-e required for detection of‘ the same signals. ~urthertnore, dried gels cm he stripped and t.rh\,itt.icii/etl.‘” ‘l‘hr chief advantage of(lt-ied-get I

I

I! Z s

A

B

C


Alkaline Transfer

High Salt Transfer

2 hr

18 hr

FIGURE 2. Dried gel-oligonucleotide hybridization. Control and patient DNA was digested with Ecd4. and electrophoresis on 0.7% agarose was performed. The gel was dried and hybridized to the human A light-chain oligonucleotide as described in the text. Arrows labeled “G” identify the germline position of A light-chain genes in individuals carrying type I polymorphism at this IOCUS.~~ Arrows labeled “R” point to rearranged bands In two of the specimens.

hybridization5 is that two overnight nated: the overnight transf’et-, and bridi~ation.

steps arr elimio\,erttight prehy-

DISCUSSION A Perspective on DNA Hybridization and Lymphoproliferative Disorders

FIGURE 1. Comparison of high-salt transfer with alkaline transfer Duplicate samples of patient DNA were digested with HindIll. and electrophores*ls on a 0.7% agarose gel was done. The gel was divided in half, and transfers were performed by the alkaline method or the standard high-salt Southern method, as described in the text. The two filters were hybridized in the same bag with the T-cell receptor p-chain probe, washed, and exposed to x-ray film slmultaneouslv

Kearrangements of’ the irnmuno~lobulin and the T-c-e11 receptor genes are objective markers for the lineage and clonality of‘ B and ‘I cells. t-espectiveDuring the differentiatiott of’ precut-sotly. ’ -++J’v~:‘L’~~5:’ cells into mature B or -1‘ lymphocytes, recombination of inactive gene segments (variable, diversity. joining. or constant regions) must occur fix acti1.e imtnunoglobulin or T-cell receptor genes to be generated. As a result. the identification of gene rearrangements provides an early marker of‘ commitment to thts B- ot T-cell lineage. Moreover, because the rearrangements carried b> ai\ one cell are essentially unique, rearran~ged genes provide clonal markers of’ ;I lvm-

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Volume 21, No. 11 [November 1990)

phoid cell should undergo neoplastic transformation. Because of these findings, molecular hybridization is assuming an increasingly important role in diagnostic and research hematopathology, and it is at a stase of development and application similar to that of the development of immunology and its application to hematopathology approximately a decade ago. The interpretation of the lmmunogenetic data. however, is complicated by the fact that rearrangements at the immunoglobulin heavy-chain locus have been identified in as many as 10% of cases of T-cell leukemias.“” Conversely, rearranged ‘T-cell receptor genes have been encountered in B-cell lymphomas as well as in B-cell lymphocytic leukemias.:‘-‘,?” In addition, we recently reported on three cases of nonHodgkin’s lymphoma in which there were rearrangements of both the T-cell-receptor P-chain genes and the immunoglobulin light-chain genes.36 A Practical Approach to Molecular Hybridization Studies of Lymphoproliferative Disorders In this report, we have described a practical method for incorporating DNA hybridization studies into a multidisciplinary laboratory approach to the study of tissues involved by LPD. For such a multidisciplinary approach to be successful, it is essential that the results of morphologic, immunologic, and immunogenetic studies be derived from tissue samples that are representative of the LPD. Moreover, for the efficient operation of a clinical-diagnostic hematopathology laboratory, it is desirable for the tissue samples to be subjected to minimal handling and to be stored easily. It is also important that the tissue samples can be preserved, in the event that additional studies are required. The method which we describe in this report accomplishes all of these objectives. The results presented here demonstrate that DNA hybridization studies by the Southern blot method can be performed easily and reliably on CCFFS prepared from tissue samples that have been maintained in a frozen-tissue tumor bank. The method for freezing and storing the tissue samples is identical to that used for immunologic studies. thus eliminating separate procedures for the preparation of frozen tissues for immunologic and immunogenetic analysis. The only difference between the methods for immunologic studies and those for immunogenetic studies is the thickness of the CCFFS: we found CCFFS cut at ‘LOkm provided satisfactory results for DNA hybridization studies. In the past, some laboratories have used fragments or “chunks” of frozen tissue for molecular hybridization studies. This method requires the maintenance of frozen-tissue tumor banks separate from those used for immunolit results in damage to the frozenogy; otherwise, tissue samples available for immunologic studies. With the use of CCFFS for hybridization, both of these problems are avoided. When a tissue sample is cut into thin, parallel sections (“breadloafed”) and alternating sections are 1138

submitted for morphology (fixation) and immunology/DNA hybridization (frozen-tissue tumor bank), greater knowledge of the nature of the lymphoproliferative process involving the various areas of the tissue is made available. Our method should provide for adequate evaluation of those cases in which a lymph node is only partially involved by an LPD, or is involved by multiple types of LPDs which are separate geographically. Moreover, the morphologic, immunologic, and immunogenetic studies are performed on tissue samples that are within millimeters of each other in size and, thus, the results can be correlated reliably. We recommend that the quantity, yield, and condition of the DNA be routinely evaluated on a case-by-case basis in new diagnostic/clinical molecular hybridization laboratories during the startup phase, as a means for evaluating the adequacy of the laboratory technique. The 37 cases analyzed in our study represent a broad spectrum of benign and malignant LPD. Highmolecular-weight DNA suitable for molecular hybridization was prepared from frozen sections of all of the specimens, although the recovery of DNA was low (less than 100 pg) in three cases. The cell lineage and clonality as determined by molecular hybridization were in agreement with the classification based on conventional morphologic and immunologic techniques. Four of the 19 neoplasms of the B-cell type were bigenotypic (ie, they contained both immunoglobulin and T-cell receptor gene rearrangements), in accordance with findings from other laboratories.‘“’ None of our T-cell lineage LPD showed any rearrangement of immunoglobulin genes; however, the sample size was small (five cases). The presence of ice crystal artifacts resulting from improper freezing and storage of the frozentissue samples appeared to be the factor which correlated best with low DNA yields. This is an important finding because ice crystal artifacts can easily be eliminated when proper procedures for freezing, transportation, and storage of frozen tissues are followed. The most common cause for ice crystal artifact is the overnight storage of frozen tissue samples in selfdefrosting cryostats and freezers.“’ Our findings indicate that cell lineage can be determined reliably from DNA hybridization studies when the tissue sample is large enough to produce sufficient DNA. From a practical standpoint, however, low yields did not necessarily have an adverse effect on the total DNA recovered. The results of Southern blot analysis were not shown to be affected adversely by the nature (ie, the morphologic subclassification) of the LPD, or by the presence of fibrosis and/or necrosis in the specimen. Small sample size, however, had an obvious negative affect on the recovery of DNA in some of the cases. We and others have reported on the increased occurrence of surface immunoglobulin (SIg)-negative B-cell lymphomas in extranodal sites, even when the sample is large and has been fixed optimally.21J7 The SIg negativity may be attributed to a technical problem related to increased background staining of stro-

IMMUNOGENETIC ANALYSIS OF LPD pVu et al)

ma1 tissue, or the lymphoma may not express SIg.” Even though the number of cases of LPD involving extranodlal sites in this series were small. our findings suggest that DNA hvbridi/.ation studies more readily enable the identific&on of cell lineage of extranodal 121’1>than immunologic studies. We ihave also described the incorporation of alternati\,e hvhridization techniques into gene rearI angement itnalvsis that result in a reduction in the time required t(; conduct the studies. If our standard Southern blot technique is used, an autoradiogram is available fi)r i-e\-iew on the eighth working day after submission of the sample. Use of’ the alternative t ransf’er or hvbridization methods reduc.es the required time to 7 or even ti days. A further advantage of dried-gel hvbridi~ation is the elimination of the rleed to ptirc.hase nylon membranes. Potential Indications for Diagnostic Application of DNA Hybridization At presetIt. DN.4 hybridization studies are laborilltensive, time-consuming, and expensive. hloreover, most LI’D can he diagnosed and classified on the basis of routine morphologic and immunologic analyses. Xs a result. DNA hybridization studies ma!’ he necess,iry anti f.ol- resolution of specific diagnostic problems. Rec,ent reports have summarized major areC+sin dia,ynostic hematopathology in which DNA hvbridiLation studies may be particularlv helpf ,,,, I~‘~.i~7.‘l.,:1.I1.il~.I7.:~x Tl lese include ( 1) the ‘identif‘ication 01’ mol~oclonal l~rnphoproliferatice disorders which wet-v not clearly found to be neoplastic based on the results of morphologic and immunologic studivs, (2) the detection of “minimal disease” in patients Lvith non-Ilodgkin’s lvmphomas, and (3) the diagnosis of B-cell lymphomas showing a predominance of ‘1 cells ‘(ao-called “pseLldo-periphei.~lt J.-cell 1) mphom,t\“). DNA hybridization studies ma) also be useful in the investigation of SIg-negative lymphomas, extranodal lymphomas. and lymphomas provisionallv diagnosed as “true histiocytic.” In’ ;I recent study of specimens from 509 consecutive c’ase:? of’ I.-cell-rich LPI) that were subjected to morphologic and immunologic study in our laboratorv. we encorrntered a number of diagnostic dilemIU& related to the diagnosis of peripheral I‘-cell lymphomas (P’IX:L) which may be resolved by DNA h?hridization studies.:‘!’ ‘I‘hese include (1) Y’I‘LC: of the small-cell it\ pe having an unremarkable immunophenotype; (2j P’I‘(:L with an immunophenotype lackin expression of‘ the usual pan-‘I‘ cell-associated antigens: (3) differentiation between PTCL and pseudoP’I‘CL; and (G) polymorphous large-cell proliferations in which the differential diagnosis includes PI‘CL, SIg-negative larce-cell lymptioma of the B-cell disease, tymphocyte depletion type, and Hodgkin’s DNA hybridization studies may be type. In addition, required fi)r demonstration of the presence of cutancous T-cell l\mphoma in patients who have known skin disease a;id Lvhose lymph nodes exhibit the features of‘ tlt~rrna~opathic lvmphadenitis.“’ DNA hy-

bridization techniques have become important research tools for the study of atypical imrnune reaction disorders and angioimmunoblastic lymphadenopaand L.PD suspected thy, ‘.*.w Hodgkin’s disease. 11’.12.1’~ of ~nulticlonalitv.‘~~.:~~~~’ tlcknoz~llr~,~mul1/. The authors gratef’ullv acknowledge technical assistance hy William Swartz. and preparation of the manuscript hy Janet Cahalin, Millie Dettloff, and Toni Martinez. The plasmids pHuJH. pHuCK., and pHuhC! were the generous gift of Dr P. Leder. Harvard Medical School, Boston, MA; and pJurkat-2 was the generous gift of’ Dr ‘I-. Mak. Ontario Cancer Institute (Ontario, Canada). APPENDIX 1 Preparation of DNA From Cryostat-cut Fresh-frozen Tissue 1. (Iut ten L’O-km froren sections and place in 1.5 nit. microfuge tube. 2. Add WO ~.LL0. 1 mol/L E:D’1‘.4, 0.05 mol/L ‘l‘ris-HCI. pH 7.5: add 10 ~.LL208 (w/v) sodium dodecyl sulf’ate; add 40 FL 5 mgiml. Proteinase I(; close and flick tubr to mix. Place tube in 35°C: water bath and incubate overnight. Shake tube occasionally to mix. 3. l’hen(,l-chlorofol-m extraction: add +3) +L phenolchloroform (2: I, equilibrated against 10 mmol/L 1 t-is-HCl, pH 8). Shake for .5 minutes. Spin fi)r :! minutes in microfuge to separate phases. Remove upper aqueous phase to fresh microfuge tube, using a P- 1.000 pipetman with the end of the tip cut of‘f (solutions containing high-molecular rveight DNA GUI he quite viscous). 110 not vc~rtex: vortexing tan shear DNA. 3. Repeat phenol-chlorofoI.1~~ extraction .fransfrr up per phase to tresh tube. .5. Extract for final time with chloroform only. T‘I-anster upper phase to fresh tube. 6. Ethanol precipitation: add 4.5 FL 3 rnol/L sodium acetate. pH 7: add 1 mL cold IOW ethanol; invert tube se\:eral times to mix. Should see fibrous whitr prrcipitate. Spool DNA onto glass capillary tube ((:lay-.4dams 5 ~1 pipettes work well, Recton Dickinson. Mountain View, (Z.4). Blot off excess ethanol in a fresh microfuge tube. 7. Redissolve DNA in 200 to 800 ~.LLTris-EDTA (10 tnmolil Tris-HCI, pH 7.6, 1 mmol/L EDT.4) in a fresh microfuge tubt- by twirling and stirring the capillarv tube in the buffer until the DNA slides off. Seal tube and rotate (Labquake rotator, Lab Industries, Berkeley, (:A) in cold room overnight to aid complete dissolution of’ the DNA. 8. Determine concentration and recover-y of DNA bl measuring I’V absorption. Dilute an aliquot of the DN.4 stock I :50 and measure absorbance at 26~) .h:~tl 280 nm. A, ,.,,, of 1 = .?A)pg/mL DNA. .%i,l/Aw,, ratio should be > 1.6. Lower- ratio indicates cotitamina~ion with protein or phenol. Sotr I. Frozen-tissue tumor samples should not be stored in a cryostat or freezer which has a self-defrosting cycle because thawing and refreezing af‘f’wts rhr sample b\ creating ice crystal at-M&t. .‘v’n/p2. Proteinase Ii (Boehrirlger-Mannheim, IndianI‘ris-HCI. pH apolis, IN) stock is 5 mg/mL in 25 mmol/L 7.5, 10 mmol/L EDTA. 100 mmol/L Na(:l. Stored frozen in aliquots at - 7CK. ,Yotr 3. One can use the same procedure tor prepav itlg DNA from a chunh of fro/.en tissue hv mincing the

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HUMAN PATHOLOGY

Volume 21, No. 11 [November 1990)

tissue quickly, placing tissue in microfrige tube. and adding Tris-EDTA, SDS, and Proteinase as above. Note. 4 DNA is prepared from cell suspensions by washing cells in phosphate-buffered saline and gently pelleting cells in a microfuge tuhe. Phosphate-buffered saline is removed. Cells are resuspended in Tris-EDTA before the addition of detergent and protease.

APPENDIX 2 Alkaline Transfer of Agarose Gels to Nylon Membranes

Materials Transttr is performed in snap-lid food storage coiitainer. Box should be 2 to 3 inches larger than gel length 45 menland width, and about 2 inches in height. (ienetran hranes are available from Plasco Inc, W&urn. hlA. Procedure 1. After staining and photographing gel as usual, soak gel in 0.25 mol/L HCI for 15 minutes. (Bromophenol blue marker will turn vellow.) 2. While gel is soaking, invert gel casting tray and place in bottom of elastic box to he used as blottine nlatlinm. 3. Cut &netran tnemhrane to dimens&d of. gel and place in distilled H,O l’or 5 minutes. 4. Cut eight sheets of M’hatman 3Mhl filter paper (Whatman) to dimensions of gel and set aside. 5. Cut four sheets of Whatman 3MM paper to be used as wick (same width as gel, but several inches longer, to be draped over platform and into bottom of blotting box). 6. Place wick on inverted gel tray. Pour 0.3 N NaOH to saturate wick. 7. Place gel on wick, making sure no air huhbles are trapped. Pour 0.4 mol/L NaOH on gel. 8. Place Genetran membrane on gel, laying down rniddle first, and again pour 0.4 mol/L NaOH on top. Remove all air bubbles. 9. Wet precut sheet of 3MM paper in water and place on filter. Repeat with second sheet. Remove all air bubbles. 10. Place remaining dry sheets of YMM onto stack. Trim paper towels and add until the stack reaches the height of the box rim. Check whether NaOH is sufficient for the transfer and that no “short circuits” are present. Place lid on box and seal. 11, Let transfer proceed for 2 hour-s. 12. Open box; carefully remove paper towels ancl 3hIbI uaDer. Peel off membrane and nel. mark lanes. number and ha;e membrane with black hal$o~nt pen. 13. Rinse membrane briefly in 2X SSC and blot dry. The membrane is now ready for prehybridization, No&. SSC is 0.15 m&L NaCl, O.Ol:i m&L sodium citrate, pH 7.

5. Place in gel dryer (HioRad, Inc, Richmond, CA). Dr\ with vacuum, only until gel is almost flat (about 30 minutes): then turn on heat to 60°C and continue drving for 30 minutes more. 6. Remove gel from clryer. Relr)drate ,tnd remove from paper hp placing in tray with dIstilled H,O and agitating gently. 7. Place gel in Seal-:t-meal bag and removr excess wdter. H. Preh~t)ridizatio!l is not necessary. Hybridize gel in 55 SSPE:, 0.1% sodium dodecyl sulfate, IO ~g/mL~ denatured salmon sperm DNA, and 2 X IO” cpni/niL endlabeled oliaonucleotitle. H~bridizauona are done overnight at a temperature that i> determined by the length and composition of the oligonucleotide probe being used. I‘he A lighr chain oligonucleotide is hvbridiLed at 60°C. 9. Ali washes are in 6X SSC, \vith shakirlg. Pl’ash: 13 minutes at room teniwxature. 15 minutc5 at room temperature, 2 hours at t&n temperature. I .Finiirland I minute at stringent utes at stringent temperature, teniperaMre. ‘The stringent \vash temperature is tletel-mined b\ thy length and composition of the oligoriucl~otide. The A ‘lightchain oligonucleoticle is washed at 67°C:. IO. Blot dry. wrap in plastic wrap. and ;urtor;ltliograph. Solr.~. S!X: is 0.15 niol/L Sa(:l, 0.01.5 niol/I. sodium citrate, pH 7. SSPE i\ 0.13 molil. NaCl, IO mmolil. monosodium acid phosphate. I mmol/I, EDTA. pH 7.4. To rehyhridiLe, repeat steps 2 and 3 to remove old probe. Timex can be reduced to 15 minutes.

REFERENCES

.5. Knowles DELI. Peticci I’-(;. Datla-Fa\er-a K: Inlrnullogl(,t,lllill and I‘-cdl receptor beta chain gene DNA probec in Ihc diagnosi? anti cta5sification of human I) mph&t neopta5ia. Mel (:ell Prot,es 1: 15-SI, 19x7

p subunit of the antigenihlH(: during inrrathvmic ontog!en\

APPENDIX 3 Preparation and Hybridization of Dried Agarose Gels With Synthetic Oligonucleotides 1. Stain and photograph gel as usual. 2. Denature gel in 0.5 mol/L NaOHIO. 15 mol/I, NaCl for 30 minutes at room temperature, shaking gently. 3. Neutralize in 0.5 m&L Tris-HCI. pH 8/O. 15 mot/L NaCl for 30 minutes at room temperature, with shaking. 4. Place gel on two sheets of Whatman 3MM paper and overlay with plastic wrap.

1140

receptor under-go 1.c’al-rdngemenI prior to sur-face I‘?-Ti exl,ression.

IMMUNOGENETIC ANALYSIS OF LPD (wu et al)

.,

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irnmttr~oglobulin gcnc reart anqmrnt\ in human Iruk~mic ur-r-H-tell<. f’roc Nat1 Ac ad Sci ITSA 7X:7006-7 100, 19X I 34. Tn~a .A. Hozttmi N. Slitrtlen 11. ct dl: Kcarrattgement of the I -ccl1 tectpt8)r l3-chain gtmc’ in rum-‘I‘-rrll. non-I\-cell ac-lttr I\ mphohlnsticleukemta of chil(lhoo
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