Immunoglobulin and T cell receptor beta chain gene DNA probes in the diagnosis and classification of human lymphoid neoplasia

Molecular and Cellular Probes (1987) 1, 1 5 -31

REVIEW

Immunoglobulin and T cell receptor beta chain gene DNA probes in the diagnosis and classification of human lymphoid neoplasia

Daniel M . Knowles II,'t Pier-Giuseppe Peliccit and Riccardo Dalla-Faverat *Immunopathology Laboratory, Department of Pathology, and the tKaplan Cancer Center, New York University School of Medicine, New York, NY 10016, USA (Received 7 November 1986, Accepted 17 December 1986)

KEYWORDS : immunoglobulin gene rearrangements, T cell receptor gene rearrangements, non-Hodgkin's lymphoma, Hodgkin's disease, acute lymphoblastic leukaemia, T,-lymphoproliferative disease, lymphomatoid papulosis, hairy cell leukaemia .

INTRODUCTION The human lymphoproliferative disorders are a heterogeneous collection of benign

and malignant lymphoid neoplasms exhibiting marked clinical and histopathologic diversity' . Distinguishing between benign and malignant lymphoid proliferations, between non-Hodgkin's lymphoma (NHL) and Hodgkin's disease (HD), and appropriately categorizing the various subtypes of NHL and lymphoid leukaemia (LL) and distinguishing them from one another carries clinical significance with respect to management, therapy and prognosis' . The more than occasional difficulties encountered with morphologic discrimination between the various lymphoprolifer-

ative disorders have been partially remedied by immunophenotypic analysis' . The operational basis for this approach is that the majority of NHLs and many LLs represent homogeneous proliferations of surface immunoglobulin (Slg) positive B cells which express a single immunoglobulin light chain isotype, either kappa or lambda . However, immunophenotypic analysis based upon the demonstration of immunoglobulin light chain isotypic exclusion also has shortcomings . First, correctly demonstrating the presence of a single immunoglobulin light chain class on the cell Correspondence and reprint requests : Dr Daniel M . Knowles, New York University Medical Center, University Hospital-Department of Pathology, 550 First Avenue, New York, NY 10016, USA .

0890-8508/87/010015 + 17 $03 .00/0

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surface, using either cell suspension or tissue section techniques, may be difficult due to poor reagents or Fc receptor binding . In the case of tissue section techniques, the presence of serum immunoglobulin in the interstitium may also prevent accurate demonstration of the cell surface immunoglobulin light chain class . Second, many B cell lineage-derived neoplasms lack surface and cytoplasmic immunoglobulin ; therefore, their clonality cannot be determined at all . Third, benign polyclonal B lymphocytes may be intermixed with a malignant monoclonal B cell proliferation resulting in only small deviations in the normal kappa to lambda light chain ratio which causes difficulty in demonstrating the monoclonal B cell population'. ' . In addition, a significant proportion of NHLs and LLs are derived from the T cell lineage' , ', ' . Unfortunately, T cells lack a phenotypically demonstrable cell surface marker comparable to immunoglobulin in B cells that can be routinely employed to determine the clonality of a T cell proliferation . Therefore, the malignant nature of a T cell proliferation usually must be inferred from the lesion's clinical aggressiveness, the cytomorphology, homogeneous and/or anomalous cell surface antigen expression, karyotypic abnormalities or a combination of these characteristics . None the less, the lack of a suitable marker for T cell clonality often causes difficulty in determining the benign or malignant nature of a particular T cell proliferation . For example, it may be difficult to determine whether a peripheral blood T4 or T8 lymphocytosis merely represents a quantitative alteration in the normal T cell subsets or a clonal neoplastic expansion of one subpopulation . The antigen recognition molecules of B and T cells, immunoglobulin and T cell receptor(s), respectively, are encoded by genetic loci that undergo somatic recombinations (rearrangements) to become functionally active in mature lymphocytes 5 '8 . We and other investigators have demonstrated that clonal rearrangements of the immunoglobulin and the T cell receptor beta chain (T ß) gene loci represent accurate and objective molecular genetic markers for the lineage and clonality of B and T cells, respectively"' . Southern blot hybridization analysis for clonal immunoglobulin and Tß gene rearrangements, so-called immunogenotypic analysis, has provided investigators with a uniquely sensitive assay with which to successfully determine the lineage and the clonality of the lymphoproliferative disorders . Current technology permits investigators to detect a clonal population whose DNA comprises only about 2% of the total DNA present in a pathologic sample" 12 Immunogenotypic analysis has, therefore, become an important tool in the investigation of lymphoid neoplasia l3, ' 4 . In this review, we shall discuss the rationale for immunogenotypic analysis and briefly summarize selected contributions already made to the diagnosis and classification of lymphoid neoplasia by our and other laboratories employing immunogenotypic analysis .

REVIEW Immunoglobulin gene rearrangements Immunoglobulin genes coding for the immunoglobulin molecules which serve as cell surface antigen receptors are organized as discontinuous segments of DNA in the germline configuration . An early event in B cell differentiation is the somatic



DNA probes in human lymphoid neoplasia

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recombination of these discontinuously arranged DNA segments to place variable, constant, joining and diversity segments into an appropriate juxtaposition to allow the formation of an active and functional immunoglobulin gene . This process of immunoglobulin gene rearrangement occurs consistently as an early event in B cell differentiation but occurs only uncommonly in other cell populations . Therefore, the rearrangement of immunoglobulin genes can be used as a useful indicator of a cell's commitment to the B cell lineage . Second, the considerable degree of variability of recombination of immunoglobulin gene DNA segments and the fact that each individual B cell expresses only a single antibody idiotype makes the immunoglobulin rearrangement configuration a specific marker for a given B cell and its clonal progeny . The immunoglobulin gene rearrangement pattern is unique for each B cell clone, its diversity representing the genetic basis for antibody diversity . In other words, each immunoglobulin gene rearrangement pattern serves as a 'fingerprint' for a particular B cell clone and its further proliferation (Fig . 1) . Third, immunoglobulin gene rearrangements proceed according to a developmental hierarchy . The heavy chain gene locus rearranges first and this is followed by rearrangement of the kappa and then the lambda light chain gene loci . Thus, the hierarchical rearrangement of the immunoglobulin genes provides a genetic basis for defining discrete stages of B cell differentiation (reviewed by Korsmeyer' S ) . In

C

I

2

3

4

Kb 17-

Fig. 1 . Southern blot hybridization analysis of the immunogobulin heavy chain (IgH) gene locus in DNAs extracted from monoclonal B cell neoplasms . Lane C contains human fibroblast DNA . Lanes 1-4 contain different B cell neoplasms . The DNAs were digested with the EcoRl restriction endonuclease and hybridized to a joining region heavy chain (J©) gene DNA probe . Rearrangement bands are indicated by arrows . Lane C displays the IgH germline configuration without new hybridizing bands indicating the absence of clonal B populations . Lane 1 displays a prominent germline band consistent with the presence of large numbers of residual benign, polyclonal lymphoid cells and a single new rearrangement band demonstrating the presence of a clonal B cell population . Lane 2 exhibits two rearrangement bands and a germline band which is diminished in intensity and probably represents small numbers of residual polyclonal lymphoid cells . Lane 3 shows one new hybridizing band consistent with rearrangement of one allele and deletion of the other allele . Lane 4 shows two rearrangement bands consistent with biallelic rearrangement.



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summary, the identification of immunoglobulin gene rearrangements provides a useful genetic marker for the B cell lineage, serves as a genetic indicator of the clonal nature of a B cell population, and defines a discrete stage of B cell differentiation . Investigators have utilized these fundamental properties of immunoglobulin gene rearrangements to determine the lineage, clonality, and stage of differentiation of surface immunoglobulin negative B cell neoplasms 16-19 and to clarify the nature of hairy cell leukaemia" .

Non-B, Non-T acute lymphoblastic leukaemia and non-Hodgkin's lymphomas The large proportion of acute lymphoblastic leukaemias (ALL) which lack surface immunoglobulin, sheep erythrocyte rosette receptors, and T cell associated differentiation antigens, have been designated non-B, non-T or null ALL . Nadler et al." showed that some non-B, non-T ALLs express B cell associated antigen 131, and suggested that they, therefore, represent immature B cell neoplasms . Korsmeyer and his colleagues 16 exploited the fundamental properties of immunoglobulin gene rearrangements to demonstrate that the non-B, non-T ALLs are clonal proliferations of neoplastic B cells representative of various points in B cell differentiation prior to the synthesis of surface immunoglobulin . Korsmeyer et al ." further demonstrated that non-B, non-T ALLs undergo immunoglobulin heavy chain gene rearrangement and express HLA-DR antigens prior to the phenotypic expression of cALLa and BA-1 . We confirmed these observations and extended their studies . We demonstrated that non-B, non-T ALL cells exhibit the co-ordinate hierarchical expression of immunoglobulin heavy and light chain gene rearrangements and several B cell associated differentiation antigens18 . These findings suggest that the subcategories of non-B, non-T ALL, broadly defined phenotypically as cALLa - , Cu - , cALLa + Cu - and cALLa + Cu + , represent stages of B cell differentiation of increasing maturity which correlate with the developmental hierarchy of immunoglobulin gene rearrangements . We exploited the same fundamental properties of immunoglobulin gene rearrangements to show that the majority of non-B, non-T NHLs also are clonal proliferations of neoplastic B cells representative of stages in B cell differentiation prior to the synthesis of surface immunoglobulin18,1s We also investigated the correlative expression of B cell associated differentiation antigens and immunoglobulin gene rearrangements in the so-called non-B, non-T NHLs . We found that non-B, non-T NHLs express a constellation of B cell associated differentiation antigens distinct from those expressed by non-B, non-T ALLs . Specifically, the vast majority of non-B, non-T ALLs are TdT + BA2 + B1 } BL7 - while the vast majority of non-B, non-T NHLs are TdT - BA2 - B1 + BL7 + . Furthermore, non-B, non-T NHLs more commonly express the mature B cell associated differentiation antigens BL3, OKB1, OKB4 and OKB7 (Table 1) 18 . In addition, their phenotypic expression does not appear to correlate with the developmental hierarchy of immunoglobulin gene rearrangements . The results of our studies suggest that non-B, non-T ALL and NHL originate from distinctive lymphoprogenitor cells which differentiate along separate developmental pathways" .



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DNA probes in human lymphoid neoplasia

Table 1 . Comparative expression of B cell associated differentiation antigens by so-called non-B, non-T acute lymphoblastic leukaemia (ALL) and non-Hodgkin's lymphoma (NHL) B Cell associated antigen

ALL (n = 37)

TdT la

100 100

LEU12 BA2 BL2 OKB2 BL1

100 82 78 89

BAl B1 BL3 BU OKB1 OKB7 OKB4

NHL (n =14) 0 86 82 18 79

89 88 34 46 3 0

92 86 82 92 79 77 14

0 0

7 25

Numbers indicate the percentage of cases expressing each marker. Note that most ALLs are TdT'BA2'B1'BL7- while most NHLs are TdT - BA2 - Ba'BL7' .

Hairy cell leukaemia

Considerable controversy has surrounded the cellular origin and stage of differentiation of the neoplastic cells comprising hairy cell leukaemia (HCL) for several years . These neoplastic cells characteristically display cytoplasmic tartrate-resistant acid phosphatase (TRAP) 22 . They also display some monocytoid characteristics such as hair-like cytoplasmic projections22 and reactivity with monoclonal antibody OKM1 20 . Many investigators have demonstrated that HCLs express B cell associated antigens 23-24 and some have described T cell associated antigen s 25 . The majority of HCLs also exhibit Slg23 '24 but reports of multiple isotypes and the presence of avid Fc receptors has often raised the question of whether this represents intrinsic surface membrane or merely cytophilic immunoglobulin . These reports describing conflicting phenotypic characteristics have left the lineage and the stage of differentiation of the cell of origin of HCL unclear . Korsmeyer and his colleagues 20 demonstrated that HCL, including those with and those without surface immunoglobulin, consistently exhibit clonal immunoglobulin heavy and light chain gene rearrangements, characteristic of mature stages of B cell differentiation . They also detected mRNA responsible for immunoglobulin production in some instances . They further demonstrated that HCL cells consistently react with monoclonal antibody anti-Tac which detects the membrane receptor for Interleukin-2 (IL-2) . Their results strongly suggest that HCL is a clonal B cell malignancy which may be representative of a unique stage of B cell differentiation and/or activation .



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D. M. Knowles et al.

T cell receptor beta chain gene rearrangements DNA clones corresponding to the human T cell receptor genes also have been isolated and characterized 26 . The T cell receptor genes share structural and functional properties with immunoglobulin genes 7 '8 '27. Structural analyses of the gene coding for the beta chain of the T cell receptor (Tß) has identified variable, diversity, joining and constant regions which are similar to the corresponding immunoglobulin gene segments . These regions similarly represent discontinuously arranged DNA segments in the germline configuration . They are brought together during T cell differentiation by mechanisms which may be analogous to those responsible for immunoglobulin gene rearrangements in B cells" 27-31 . The obvious analogy between immunoglobulin and Tß gene rearrangements, both with respect to the antigen molecules that they encode and the mechanisms that regulate their expression, suggests that they may be used to identify the lineage and the clonality of T cell populations similar to the use of immunoglobulin gene rearrangements in B cell populations .

T beta (Tß) gene rearrangement analysis distinguishes non-T cells, polyclonal T cells and monoclonal T cells We investigated the organization of the T ß gene locus by Southern blot hybridization analysis employing a Tß gene DNA probe provided by Dr Tak Mak (Fig . 2) . The Tß gene probe was derived from the human cDNA Tß clone YT)-2 isolated from the Jurkat-2 lymphoma cell line 26 . We used either the intact probe or fragments corresponding to the constant and variable regions of the Tß gene generated by Hinc II digestion of the intact Tß clone ) " . DNAs extracted from multiple cell and tissue samples were analysed following digestion with each of three restriction enzymes, EcoRl, Barn HI and Hind Ill . EcoRl digestion has been routinely used to identify Tß gene rearrangements since this restriction endonuclease does not display genetic polymorphism at the Tß gene locus . First, we analysed an extensive panel of DNAs extracted from normal, non-T haematopoietic cells and from benign and malignant non-haematopoietic tissues in order to determine the germline configuration of the Tß gene . We demonstrated that four EcoRl hybridization bands, two derived from the constant region (12 . 0 and 4 . 2 kb) and two derived from the variable region (5 . 2 and 2 . 1 kb), characterize the germline T ß gene locus in non-T cells" (Fig . 2) . Second, we investigated the organization of the Tß gene locus in benign, polyclonal thymocytes and mature peripheral blood and lymphoid tissue E rosette forming (T) cells . We demonstrated that all of these benign, normal T cell populations consistently exhibit a biallelic deletion of the EcoRl 12 . 0 kb DNA fragment, which corresponds to a portion of the C B1 region . This consistent deletion represents a specific molecular genetic marker of the T cell lineage . We also demonstrated that benign polyclonal T cells lack new hybridizing bands, consistent with their lack of clonal rearrangements of the Tß gene locus" (Fig . 2) . Third, we investigated the organization of the T ß gene locus in a large panel of malignant T cell neoplasms",13,32 . We demonstrated that malignant T cell populations also exhibit the biallelic deletion of the EcoRl 12 . 0 kb band, consistent with their T cell lineage derivation . However, in contrast with benign polyclonal T

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Kb

12.0-

12.0

21

neoplasia

5

6

7

8

5*24*2-

4.2

Fig. 2. Southern blot hybridization analysrs of the T cell receptor beta charn CT,J gene locus in DNAs extracted from non-haematopoietic cells (Lane I), B cells (Lane 21, benign T cells (Lane 3) and T cell neoplasms (Lanes 5-8). The DNAs in Lanes I-3 were digested with EcoRl and hybridized to a Til gene DNA probe which detects rearrangements of the constant and variable regions of the TJ gene locus. Non-T cells (Lanes 1 and 2) exhibit four hybridization bands representative of the germline configuration of the TP gene locus. Benign T cells (Lane 3) display biallelic deletion of the EcoRl 12.0 kb band, a consistent and characteristic marker of the T cell lineage, but lack new hybridizing bands. DNAs in Lanes 4-8 were digested with EcoRl and hybridized to a T,, gene probe which detects the constant region of the TJ gene locus (T&L Human fibroblast DNA (Lane 4) exhibits the two hybridization bands characteristic of the germline constant region of the TP gene locus. T cell neoplasm DNAs (Lanes 5-8) display the biallelic deletion of the EcoRl 12.0 kb band consistent with their T ceil lineage derivation and new hybridizing bands (arrows) indicating the presence of clonal T cell populations. Case 8 is an example of T3 antigen-positive T;lymphoproliferative disease.

cells, malignant T cells consistently display one or more new unique bands upon hybridization with EcoRI, Hind III or Barn HI digested DNAs, consistent with the presence of clonal TP gene rearrangements”,13 (Fig. 2). In summary, we found that non-T cells, polyclonal T cells and monoclonal T cells may be readily distinguished by Southern blot hybridization analysis using a T, gene DNA probe. Non-T cells exhibit the germline configuration. Benign polyclonal T cells exhibit biallelic deletion of the EcoRl 12.0 kb DNA fragment in the absence of new hybridizing bands. Monoclonal T cells exhibit biallelic deletion of the EcoRl 12.0 kb DNA fragment and one or more new hybridizing bands upon hybridization of the Tg gene DNA probe to EcoRI, Barn HI or Hind Ill digested DNAs. These fundamental properties of TP gene rearrangements have been widely exploited by us and by other investigators to determine the T cell lineage and clonality of a spectrum of NHLs and LLs, to establish the clonality of most cases of T,-lymphoproliferative disease, and to investigate the lineage derivation and clonality of presumed histiocytic malignancies, lymphomatoid papulosis and Hodgkin’s disease, and to help demonstrate the existence of bigenotypic lymphoid neoplasm5



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Tß gene rearrangements in T cell neoplasia

We investigated approximately 60 T cell derived NHLs and LLs representative of almost the entire clinical and morphologic spectrum of T cell neoplasia 11,13,3 z These T cell neoplasms expressed a multiplicity of immunophenotypes representative of various intra-thymic and post-thymic stages of T cell differentiation . Some of the neoplasms expressed anomalous and atypical phenotypes which made their placement in the hierarchy of T cell differentiation difficult (Table 2) . None the less, we found that every T cell NHL and LL which we investigated exhibited the EcoRl 12 . 0 kg DNA fragment deletion characteristic of cells belonging to the T cell lineage and a clonal T ß gene rearrangement consistent with a clonal T cell proliferation (Fig . 2) . Each T cell neoplasm exhibited a unique pattern of new hybridizing bands consistent with an origin from a distinctive T cell clone . In some instances, EcoRl digestion did not permit detection of a new hybridizing band and an additional enzyme had to be used to detect the clonal Tß gene rearrangement . The results of our studies suggest that the T ß gene locus is rearranged in essentially all clinicopathologically distinct subcategories of T cell neoplasia . This appears to be true regardless of the immunophenotypic or functional stage of

Table 2. Immunophenotypic characteristics of 60 T cell lymphomas and leukaemias which exhibit clonal Tß gene rearrangements Immunophenotype E/T11 •

T3

ALL ALL ALL PLL PLL PLL CLL CLL CLL CLL SS

+ + + + + + + +

+ + + + +

LBL LBL LBL CTCL CTCL CTCL NHL NHL

+ + + + + +

No . of cases

Diagnosis

T-leukaemia 4 3 2 2 2 1 3 1 1 1 8

T-lymphoma 2 2 1 8 1 1 15 2

+

+

T4

+ + + + + + + + +

+ + + + +

+ + + +

T6

+ + -

T8

+

TdT

Tß gene

+ + + -

R R R R R R R R R R R

+

-

+

-

+ + -

+ +

-

-

+ + + -

-

R R R R R R R R

ALL, acute lymphoblastic leukaemia; CLL, chronic lymphocytic leukaemia ; CTCL, cutaneous T cell lymphoma ; LBL, lymphoblastic lymphoma; NHL, peripheral T cell non-Hodgkin's lymphoma; PLL, prolymphocytic leukaemia ; R, rearranged; SS, Sezary syndrome .



DNA probes in human lymphoid neoplasia

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differentiation . Therefore, Tß gene rearrangements appear to represent an extremely early marker of T cell differentiation and in this regard are analogous to immunoglobulin gene rearrangements in B cells . Our results have been confirmed by a number of other investigators' Z,33-36 Our observation that all T3 antigen positive T cell neoplasms are T ß rearranged and that Tß gene rearrangements also occur in T3 antigen negative neoplasms suggests that the T ß gene locus rearranges prior to T3 antigen expression . Pittaluga et al." confirmed our observations in an analysis of 16 terminal deoxynucleotidyl transferase (TdT) positive T-lymphoblastic neoplasms, tumours representative of the earliest stages of intra-thymic T cell differentiation . However, they extended our observations by demonstrating that T cell associated antigen 3A1 (CD7) was expressed prior to rearrangement of the T ß gene locus in two of these 16 very immature T cell neoplasms . These findings suggest that 3A1 (CD7) cell surface antigen expression occurs as an initial event in T cell differentiation and that this is followed by rearrangement of the T ß gene locus and subsequent expression of cell surface antigen T3 .

T y -Iymphoproliferative disease Approximately 10% of circulating peripheral blood lymphocytes belong to the large granular lymphocyte (LGL) population, so-named because of their large size and abundant azurophilic granular-rich cytoplasm 38 . The LGL population is phenotypically and functionally heterogeneous, containing cells which share T cell characteristics, e .g ., T11, T10, T8, T3 antigens and IL-2 responsiveness, and monocyte characteristics, e .g ., Mol expression, IL-1 production and broad cytotoxic activity 38 . This phenotypic and functional heterogeneity has left the lineage derivation of LGLs uncertain and controversial . Occasionally, patients develop a peripheral blood lymphocytosis composed of cells exhibiting the features of LGLs 39 . The phenotypic and functional characteristics of these LGL proliferations vary from patient to patient, reflecting the heterogeneity of the normal LGL population 39. Most commonly, the patients present with an asymptomatic lymphocytosis and have an indolent clinical course characterized by fluctuating numbers of peripheral blood LGLs 39. For this reason, and also because of the uncertainty surrounding the cellular origin and nature of normal peripheral blood LGLs, the clonal nature and malignant potential of these pathologic LGL proliferations have remained unclear . This uncertainty has caused the LGL proliferations to be variously referred to as T-cell chronic lymphocytic leukaemia, chronic T,r cell leukaemia, TY lymphocytosis or T 7-Iymphoproliferative disease (T,- LPD) . The last term is most commonly employed and is a non-committal designation conveying the uncertainty of the clonal and malignant nature of this disease . We investigated the organization of the Tß gene locus in 12 well-documented examples of T 7 -LPD (Table 3) . These 12 cases exhibited a variety of immunophenotypic and functional properties 40. Ten of the 12 cases were T3 antigen positive and the two remaining cases were T3 antigen negative . Nine of the 10 cases of T3 antigen positive T 7 -LPD exhibited the biallelic deletion of the EcoRl 12 . 0 kb DNA fragment characteristic of the T cell lineage . These 10 cases also exhibited clonal T ß gene rearrangements upon digestion with EcoRl, Barn HI or Hind III and hybridization to a



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D . M. Knowles et al .

Table 3. Results of immunophenotypic, functional and immunogenotypic analysis of 12 cases of T lymphoproliferative disease Immonophenotype No . of cases

Fc,

T11

1 1 5 2 2 1

+ + + + + +

+ + + + + +

T3

+ + + +

Function

T8

Leu7

OKM1

NK

ADCC

Tß gene

+ + + +

+

ND + + +

ND +

ND + + + + +

G G R R R R

+ + + +

+ -

ADCC, antibody-dependent cell mediated cytotoxicity ; G, germline ; ND, not done ; NK, natural killer; R, rearranged .

Tß gene DNA probe . The two T3 antigen negative cases of T,-LPD exhibited the T ß gene germline configuration upon digestion with EcoRl, Barn HI, Hind III and subsequent hybridization to the same T ß gene DNA probe" . Our results demonstrate that these pathologic LGL proliferations, commonly referred to as T,-LPD, can be broadly dividedd into two subcategories based upon T3 antigen expression and rearrangement of the T ß gene locus . T3 antigen positive T },LPD exhibits rearrangement of the T ß gene locus while T3 antigen negative T,-LPD retains the T ß gene locus in the germline configuration . Furthermore, our results demonstrate that the majority of cases of T,-LPD, despite their benign and indolent clinical course, do represent clonal proliferations . These results have been con12,33,41 firmed by other investigators

Bigenotypic lymphoid malignancies Rearrangement of the immunoglobulin and Tß gene loci were originally believed to be highly specific for the B and T cell lineages, respectively . However, occasional examples of lineage infidelity were noted by several investigators' 6,42 These observations led us to formally determine the frequency of this lineage infidelity by investigating the organization of the immunoglobulin and T ß gene loci in a collection of 33 B cell and 30 T cell neoplasms of considerable immunophenotypic diversity representative of the major clinical and pathologic subtypes of NHL and LL43 . As expected, each of the 33 B cell neoplasms displayed clonal rearrangements of the immunoglobulin heavy chain gene locus . Some of these B cell neoplasms also displayed rearrangements of the immunoglobulin light chain gene loci, kappa or kappa plus lambda . Conversely, each of the 30 T cell neoplasms displayed clonal rearrangements of the Tß gene locus . However, seven of the 63 cases (11%) displayed dual rearrangements, i .e ., five of the 33 B cell tumours (15%) displayed T ß gene rearrangements and two of the 30 T cell neoplasms (7%) displayed immunoglobulin heavy chain gene rearrangements . Therefore, in our experience, approximately 10% of lymphoid malignancies are bigenotypic, i .e ., display both immunoglobulin and Tß gene rearrange ments 43 (Fig . 3) . However, we and others' 0' 17) have found that immunoglobulin gene rearrangements occurring in non-B cell neoplasms

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Fig. 3. bigenotyplc extracted

Southern blot hybridization analysts of the T,$ and IgH gene loci In DNAs extracted from three T cell neoplasms. Lane C contains human fibroblast DNA. Lanes IV3 contain DNAs from human T cell neoplasms which were digested with EcoRl and hybridized to a Tr constant

region gene DNA probe and a joining region heavy chain (J,,) DNA probe. Rearrangement bands are Indicated by arrows. Lanes C demonstrate the germline configuratIon of the Tfl and IgH gene loci. Lanes 1-3 exhlblt the characteristic biallelic deletion of the EcoRl 12.0 kb fragment indicative of the T cell lineage. Lanes 1 and 3 also exhibit a new hybridizing band consistent with a clonal rearrangement of the T, gene locus. Each lane also exhibits one or more new hybridizing bands consistent with rearrangements of the IgH chain gene locus.

involve the heavy chain gene loci but do not involve the light chain gene loci. Possible explanations for these occurrences have been suggested and discussed elsewhere”“. These results suggested to us the possibility that clonal immunoglobulin and l/, gene rearrangements may not always be useful in determining the B or T cell lineage of a lymphoid neoplasm. Therefore, we further investigated the seven bigenotypic neoplasms to determine whether they expressed a corresponding biphenotype, i.e., expressed both B and T cell lineage associated antigens. We found that, despite their bigenotypic nature, these neoplastic cell populations express only B or T cell lineage restricted antigens, suggesting that they carry a completely fidelious phenotype4’ (Table 4). Therefore, in our experience, nearly all lymphoid neoplasms can be conclusively assigned to either the B or T lineage based upon the results of combined immunogenotypic and immunophenotypic analysis.

Histiocytic

malignancies

The use of immunophenotypic markers during the past decade classification of most NHLs and LLs as either B or T cell neoplasms.

has led to the However, some



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DNA probes in human lymphoid neoplasia

27

morphologic, ultrastructural and immunohistochemical studies have led to the suggestion that occasional malignancies are derived from the histiocyte/monocyte lineage . For example, Turner et al .

44 combined plastic section enzyme histochemical

and immunohistochemical studies to demonstrate the marked phenotypic heterogeneity of 20 such histiocytic malignancies . However, Weiss et al.

45

investigated the

organization of the immunoglobulin and T ß gene loci in six of these 20 histiocytic malignancies . They found that one of these neoplasms exhibited clonal rearrangements of the immunoglobulin heavy chain and kappa light chain genes while four of the five remaining neoplasms contained clonal T & gene rearrangements . Isaacson et al . 46 performed an analogous examination of four neoplasms which previously had

been classified as malignant histiocytosis of the intestine" . They demonstrated that cases of so-called intestinal malignant histiocytosis consistently exhibit clonal Tß gene rearrangements, suggesting that these lesions are more likely T cell neoplasms than histiocytic malignancies . The results of these studies strongly suggest that the majority of malignancies previously presumed to be of histiocytic/monocytic origin may actually represent additional examples of B or T cell derived lymphoid neoplasms, some of which merely express atypical and anomalous phenotypes .

Lymphomatoid papulosis Lymphomatoid papulosis is a clinically benign cutaneous disorder which has a chronic course punctuated by the occurrence of papular eruptions which often undergo spontaneous regression 48 . Despite its benign clinical behaviour, lympho-

matoid papulosis displays histopathologic features suggestive of malignancy . The lesions consist of epidermal and dermal infiltration by large, atypical mononuclear cells which often resemble the Reed-Sternberg cells of Hodgkin's disease 48. These

cells express T cell associated antigens and they may express the LeuM1 antigen, a granulocyte-associated antigen consistently expressed by Reed-Sternberg cell S41 . Lymphomatoid papulosis has always represented a controversial disease entity because of histopathologic difficulties in distinguishing it from mycosis fungoides and Hodgkin's disease, but also, because as many as 20% of patients with lymphomatoid papulosis actually develop malignant lymphoma, most commonly, mycosis fungoides 48. Weiss et al . 50 recently examined the lesions of lymphomatoid papulosis occurring

in six patients for the presence of clonal rearrangements of the beta and gamma T cell receptor genes . They found clonal Tß and/or T, gene rearrangements in the lymphomatoid papulosis lesions occurring in five of six patients . Furthermore, the lesion in one patient appeared to contain at least two distinct clonal T cell populations . Three separate lesions occurring in another patient contained different T cell clones . These results strongly suggest that lymphomatoid papulosis represents a clonal T cell proliferation . These findings may explain why a high proportion of such patients develop clinically manifest malignant lymphoma . However, the events which result in the progression of lymphomatoid papulosis to frank malignant lymphoma remain unclear .



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D . M . Knowles et al.

Hodgkin's disease Malignant lymphoid neoplasms are broadly divided by clinical, morphologic and immunophenotypic criteria into two major categories : (i) non-Hodgkin's lymphoma (NHL) and lymphoid leukaemias (LL) and (ii) Hodgkin's disease (HD)' . Nearly all NHLs and LLs represent clonal expansions of either B or T cells, as discussed above . In contrast, the lineage derivation and clonal origin of HD have remained controversial since the origin and nature of the Reed-Sternberg (RS) cell, the diagnostic cell of HD, remains unknown . Morphologic and immunophenotypic studies have left suggestions that RS cells may derive from B or T cells, monocytes, dendritic cells, or an as yet undefined cell population" - ", unresolved . The failure to resolve this issue has contributed to the controversy concerning the pathogenesis of HD and whether or not it represents a clonal proliferation . We investigated the organization of the immunoglobulin heavy chain and the Tß gene loci in 15 cases of lymphocyte predominant, nodular sclerosis and mixed cellularity HD" . None of the 15 cases exhibited obvious clonal immunoglobulin heavy chain or T, gene rearrangement bands upon hybridization of EcoRl, Hind III and Barn HI digested DNA to immunoglobulin heavy chain and T ß gene DNA probes . However, the utilization of certain sensitive experimental conditions permitted the detection of barely perceptible clonal immunoglobulin and/or Ta gene rearrangement bands in three of the 15 samples . These weak hybridization signals suggested a small clonal population representing < 1 % of the total DNA extracted from each pathologic sample . In order to determine if these weak hybridization signals were related to RS cells, we investigated three cases of lymphocyte-depleted, RS cell-rich HD containing 25% to greater than 50% RS cells and RS cell variants . However, despite the large proportion of RS cells in these three pathologic samples, we found no evidence of clonal immunoglobulin heavy chain or T ß gene rearrangements, suggesting that the weak hybridization signals probably do not come from RS cells but, rather, from other clonai lymphoid populations within the samples" . These studies demonstrate first, that the variable lymphoid cell populations seen in HD are predominantly polyclonal B and T cells and second, that RS cells and their morphologic variants do not represent clonal B or T cell populations . Further, we found that RS cell-rich cases of lymphocyte-depleted HD exhibit a decrease in the intensity of the EcoRl 12 . 0 kb band, a finding commonly seen in polyclonal T cell populations" . This finding suggests the provocative, albeit preliminary, suggestion that RS cells may represent polyclonal T cells or a T cell related population .

SUMMARY AND CONCLUSIONS The application of DNA probes to demonstrate clonal rearrangements of the immunoglobulin heavy and light chain and T cell receptor beta chain gene loci by Southern blot hybridization analysis has led to significant advances in our ability to diagnose, classify and investigate the lymphoproliferative disorders . This approach has allowed us to conclusively determine the B or T cell lineage derivation and the clonal nature of the vast majority of lymphoid neoplasms, resulting in a new level of understanding of the biology of lymphoid neoplasia . Further application of these DNA probes to other poorly defined and controversial lymphoproliferative disorders



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should clarify their nature as well . Eventually, antigen receptor gener rearrangement analysis may become routinely employed in diagnosing lymphoid malignancies, monitoring the effects of chemotherapy, detecting early subclinical relapses and identifying disease progression, especially with respect to detecting the emergence of new clonal populations . ACKNOWLEDGEMENTS We thank Drs T . Mak and S . Korsmeyer for the T cell receptor beta chain and immunoglobulin DNA probes, Dr A . Neri for his contribution to portions of these studies, to Angela Ubriaco, Laurie McFall and Tom Taylor for excellent technical assistance, and to Margaret Walden for help in preparing the manuscript. These studies were partially supported by National Institutes of Health grants EY06337 (DMK), CA37165 (RDF), CA37295 (RDF), the New York State AIDS Institute (DMK) and the Bernard and Frances Laterman Project Chai Philanthropic Trust (DMK) . Dr Riccardo Dalla-Favera is a Scholar of the Leukemia Society of America .

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