Gene Rearrangement Analysis in L ymphoid Neoplasia BRIAN D. ZELICKSON, MD MARGOT S. PETERS, MD MARK R. PITTELKOW, MD
C
utaneous lymphoma and pseudolymphoma comprise a broad clinical spectrum of disorders that require evaluation by the dermatologist. Variousmethods,includingroutinehistologic analysis, electron microscopy, immunophenotyping, and flow cytometry, have been used to determine T- versus B-cell lineage and to attempt to establish the neoplastic or benign nature of cutaneous lymphoid infiltrates. Although these techniques are useful in the diagnosis of lymphoid neoplasms, each has shortcomings. Histopathologic examination of early lesions of cutaneous T-cell lymphoma (CTCL) may be nonspecific and difficult to distinguish from dermatitis. Disorders such as lymphocytoma, large plaque parapsoriasis, or lymphomatoid papulosis may present atypical cytologic features despite exhibiting a benign or nonaggressive clinical course.1-4 Certain microscopic morphologic characteristics, including cell shape, size, and convolution of nuclei, may suggest T- or B-cell lineage; however, these structural indicators are at times unreliable.5*6 The use of immunophenotyping for diagnosis of neoplastic versus benign lymphoid infiltrates is limited by various factors, including 1) immature T- and B-cell malignancies expressing aberrant or incomplete sets of surface markers; 2) admixtures of malignant neoplastic and benign reactive lymphoid cells within the infiltrate; and 3) frequently, the T-cell helper phenotype and other various activation markers being shared by both benign and malignant infiltrates.
Principles and Interpretation of Gene Rearrangement Analysis in Lymphoid Disease The molecular technology of gene rearrangement analysis has the potential to identify both lineage and clonality From the Department of Dermatology, Mayo Clinic and Mayo Foundation, Rochester, Minnesota. Address correspondence to: Mark R Pittelkow, M.D., Mayo Clinic, 200 First Street SW, Rochester, MN 55905.
0 2991 by Elsevier Science Publishing Co., Inc.
l
0738-081x/91/$3.50
of neoplastic processes, particularly malignant lymphoproliferative diseases. DNA of all nucleated cells contains numerous genetic sequences that direct RNA transcription and translation of proteins. Among lymphoid cells, immunoglobulins and T-cell (antigen) receptor (TCR) proteins are characteristic, lineage-specific, antigen& markers found on the surface of B and T cells, respectively. The genes that encode these proteins consist of multiple variable, diversity, joining, and constant regions that are found in a germline or nonrearranged configuration in all nonlymphoid cells. In lymphoid cells, each receptor protein is translated from a specific genetic sequence that is formed by rearranging these genes during differentiation and maturation of the cell (Figure 1). The resulting configurations of DNA are highly variable from cell to cell and contribute to the diversity and specificity of immune cell populations in recognizing antigenic substances. They also serve as genetic markers of individual lymphocytes and represent the virtually unique genotypic attributes of a cell or population of cells. Once this rearrangement occurs, each daughter cell or progeny will contain the identical genetic pattern. The DNA hybridization or Southern blot procedure allows identification and relative quantitation of the frequency of a particular gene rearrangement within the DNA isolated from a population of lymphoid cells.’ Development, proliferation, and expansion of a specific unique population generate a cellular clone. Clonality correlates closely with malignancy, although benign neoplastic processes occasionally develop clonal populations of cells within a specific lineage. DNA is harvested from fresh or frozen tissue or fluid samples containing intact nuclei of cells (Figure 2). For Tor B-cell analysis, adequate numbers of lymphoid cells should be present in the specimen. DNA is digested with restriction enzymes (endonucleases), which split the DNA at specific nucleotide sequences (Figure 1). The resulting DNA fragments are separated by agarose gel electrophoresis and transferred to a nylon membrane. This nylon membrane is then incubated with radioactive- or
119
Clinics in Dermatology 1991;9:119-228
120 ZELICKSON ET AL
Germline
Allele
Rearranged
“PI
gene
“6
RBHHHHRH
R
R
H
R
lIIlllll
I
I
I
I
DP,
JPI
n w
n
“PI
%*
%I
%, JP2
JP2
%*
%*
U
4 mRNA 4 fi -chain protean
Figure 1. T-cell receptor /I-chain rearrangement. Genome of the T-cell receptor P-chain with schematic representation of gene rearrangement. This includes the combination of different variable (V), diversity (D),joining (f,I,and constant (C) regions with subsequent formation of ml7NA and P-chain protein. The locations of the BamHI, EcoRI, and Hind111restriction endonuclease sites are indicated by the letters B, R, and H, respectively. From Zelickson et a1.j3 By permission of Mayo Foundation. nonradioactive-labeled fragments of nucleic acids complementary to the appropriate portion of the immunoglobulin or TCR genes. Specific hybridizing fragments of immunoglobulin or TCR DNA are identified and visualized as bands of specific molecular size. Three basic banding patterns are detected by Southern blot analysis (Figure 3): germline, polyclonal, and monoclonal. A germline pattern, as previously mentioned, represents genes that have not undergone rearrangement and includes the genes of all nonlymphoid cells. A polyclonal pattern will appear as multiple indistinct bands, often as a “smear” or as no additional non-germline bands. This pattern is produced by multiple, benign lymphoid cells, each containing a different rearrange-
ment site. If the lymphoid population contains cells that have undergone identical DNA rearrangement and expansion, a clonal, neoplastic pattern is observed. The rearrangement appears as additional non-germline bands. One or two non-germline bands represent rearrangement of one or both alleles. This technique routinely detects a clonal cell population that represents a minimum of 0.5%-5% of the total cellular population yielding DNA.* Some pitfalls that may result in false-negative results include sampling error, which may result in an insufficient number of clonal cells. In addition, corn&ration occurs when a TCR gene rearrangement produces a DNA restriction fragment approximately the same size as the germline fragment; this clonal fragment migrates to the same location in the gel as the germline band and therefore will not be detected. This problem may be resolved by digesting the DNA with several different restriction enzymes. The TCR is composed of a cluster of separate polypeptide subunits. Each subunit is encoded by separate genes, which are designated (Y,/I,y, or 6. Although an infrequent occurrence, some clonal proliferations of T cells express y/6 T-cell receptors instead of (Y or j? receptors. Thus, in selected instances, negative (Y and p studies may be further examined by y and 6 gene rearrangement analysis. With progression of the neoplasia, chromosomal loss can occur, which may eliminate the rearranged TCR genes. Another potential technical pitfall may occur with incomplete digestion of DNA by endonucleases. This procedural error causes formation of a “pseudoclone,” which could produce a false-positive result.9 Gene rearrangement analysis is performed in most university and tertiary referral centers. The procedure usually takes 1 - 2 weeks to perform. Currently, fresh or frozen tissue is required; however, formalin-fixed tissue and special amplification procedures (see polymerase chain
Specific DNA
Figure 2. Gene rearrangement analysis performed by the Southern blot technique. High-molecular-weight DNA is extracted from a cell suspension obtained from involved tissue or peripheral blood. The DNA is then digested to completion with endonucleases and fractioned by gel electrophoresis. The DNA is then transferred to a nylon filter and incubated with specific 32P-radiolabeled probes. The hybridization pattern is then revealed by autoradiography (not shown).
w
Recombmant plasmid
” P radlolabeled or nonradloactive probe
I I
\
Hybridization
Peripheral blood
“Bands”
/
k/
/-I
Filter
Southern transfer
/ Twue
DNA isolation
/?I molecular weight DNA
clease digestion
electrophoresis
Transfer
Get
Clinics in Dermatology 1991;9:129-128
ZELICKSON ET AL GENE REARRANGEMENT ANALYSIS IN LYMPHOID NEOPLASIA
121
and suggest specific guidelines for use of this technology in a clinical dermatology practice.
Cutaneous T-Cell Lymphoma
Figure 3. Banding patterns detected by Southern blot analysis after EcoRI digestion of TCR gene and hybridization with ],Q probe: germline @), monoclonal (O), and polyclonal (4 banding patterns. From Zelickson et a1.j3 By permission of Mayo Foundation.
reaction [PCR], see article by Jester) have been used with success to overcome these limitations.*0 For tissue specimens, depending on the density of the infiltrate, a 4-mm punch biopsy specimen is adequate; however, a larger sample may allow for more complete and accurate results. We usually obtain a 5- to 6-mm punch biopsy specimen for each assay. A frozen section stained with hematoxylin-eosin should be obtained from the specimen, and all normal-appearing (that is, lymphoid-free) tissue should be trimmed from the block. This processing step decreases the likelihood that the DNA of the clonal, neoplastic cellular population will be proportionately underrepresented and become “masked’ by germline DNA of other epidermal or dermal cells. There have been numerous reports in the literature of the application of this technique to cutaneous T- and B-cell neoplasms. We have compiled the results of gene rearrangement analyses previously reported for CTCL, peripheral T-cell lymphoma (PTCL), malignant or potentially neoplastic T-cell infiltrates (including large plaque parapsoriasis, lymphomatoid papulosis, follicular mucinosis, and granulomatous slack skin syndrome), cutaneous B-cell lymphoma, and pseudolymphoma. We review illustrative cases that have been reported, compare these observations with data accumulated at our institution,
CTCL, including mycosis fungoides and Sezary syndrome, encompasses a group of chronic and progressive, malignant lymphoproliferative disorders primarily involving the skin in early stages of the disease. Many studies have demonstrated the clonality of these related disorders (Table 1). The overall percentage of patients with CTCL who have gene rearrangements detected in tissue or peripheral blood varies with the type of sample analyzed. In our studies, the percentage of positive results is greater in skin samples of mycosis fungoides, whereas peripheral blood samples of patients with Sezary syndrome more frequently contain a clonal population. Although there are conflicting reports concerning the findings of rearrangements in patients with early (stage I-II) mycosis fungoides,“-**,” we believe that this technique is helpful for detecting early CTCL. False-negative results may be minimized by evaluating multiple gene segments with different DNA probes and by examining more than one specimen. We have observed patients with SCzary syndrome who demonstrated a germline configuration in peripheral blood but clonal rearrangement in a skin sample and also patients with mycosis fungoides who had the converse findings. This evidence stresses the importance of testing multiple samples from each patient, The finding of a clonal population is helpful for distinguishing early CTCL from dermatitis. However, the absence of gene rearrangement does not exclude a diagnosis of CTCL. Furthermore, germline configurations may occur even in patients with advanced disease.6,15,17 This finding may be due to chromosomal loss during neoplastic progression, which eliminates the rearranged gene or results in a “hidden” clone, as discussed earlier. Dual genotypes, the coexistence of bothimmunoglobulin and TCR gene rearrangements, have been detected in patients with CTCL.16 This event may occur as a result of the malignant cell being derived from a pluripotential cell type, multiple chromosomal translocations,‘* or the nonspecificity of the recombinase enzymes, which directs the process of DNA rearrangement.19 However, the presence of an immunoglobulin light-chain rearrangement is highly indicative of a B-cell neoplasm. Rearrangement of immunoglobulin heavy-chain genes may be observed in B- or T-cell neoplasms. 19rzoThere have also been reports of TCR gene rearrangements in patients with B-cell lymphoma.z1,22 TCR gene rearrangement analysis also may be helpful for detecting lymphomatous involvement of enlarged
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Clinics in Dermatology 2991;9:119-128
ZELICKSON ET AL
Table 2. Reported Results of T-Cell Gene Rearrangement Analysis in Cutaneous T-Cell Lumvhomas*6J-16 Site Disorder
Skin
Peripheral blood
Lymph node
Not specified
Mycosis fungoides No. positive
41
11
14
7
No. tested
60
41
23
8
% positive
68
27
60
88
Skzary syndrome No. Positive
43
3
16
No. tested
55
3
16
78
100
100
% uositive
69
lymph nodes in patients with CTCL. A clonal population may be detected in a histologically benign lymph nodeel Gene rearrangement analysis is therefore useful in the diagnosis of CTCL and should be performed in conjunction with routine histologic examination and immunophenotyping. As this technique is further refined, it should be applied in both the diagnosis and the staging of patients. It also may be of benefit in following the course of patients with CTCL. 4~17Our preliminary results of serial TCR gene rearrangement analyses in patients with Sezary syndrome show that changes in Sezary cell counts, erythema, or pruritus were associated with changes in clonal density in many cases. These results are promising; however, more data are needed to evaluate this application. Furthermore, with newer technologies such as PCR, more specific and sensitive methods to identify and monitor the malignant genotypes will likely be forthcoming. There are multiple cutaneous T-cell lymphoproliferative disorders that histologically mimic CTCL. Although most of these diseases are clinically benign, some may progress to or are associated with frank T-cell neoplasia. These include lymphomatoid papulosis, large plaque parapsoriasis, granulomatous slack skin syndrome, preSezary syndrome, follicular mucinosis, and pagetoid reticulosis. Gene rearrangement analyses should be performed on skin biopsy specimens and, in the appropriate settings, on peripheral blood.
Pre-!.%zary Syndrome and Large Plaque Parapsoriasis Pre-Sezary syndrome, as defined by Winkelmann et a1,23 and large plaque parapsoriasis each represent a diagnostic dilemma. Ralfkiaer et all3 reported eight cases suspected to represent early CTCL, all of which had a germline configuration. We have detected a clonal
rearrangement in four of eight patients with pre-Sezary syndrome but in none of four patients with large plaque parapsoriasis. As with the data of Ralfkiaer et al, sufficient duration of follow-up to establish disease progression was lacking for these patients. However, the finding of a clonal population in patients with these diseases, despite indeterminate histopathologic results and immunotype, may herald the evolution to T-cell neoplasia. The morphologic distinction between Sezary cells and Sezary-like cells is difficult. Bendelac et al6 reported a germline banding pattern of isolated Sezary-like cells in a patient with CTCL. We did not observe clonal rearrangement in peripheral blood lymphocytes after nonspecific mitogenic stimulation with the lectin phytohemagglutinin, which induces formation of blastogenic, Sezarylike cells in culture.
Granulomatous Slack Skin Syndrome, Follicular Mucinosis, and Pagetoid Reticulosis Granulomatous slack skin syndrome is a distinctive disorder with bulky, flexural, cutaneous lesions consisting of a dense granulomatous infiltrate. It has been associated with Hodgkin’s disease24,25 and mycosis fungoides.’ Only three cases with TCR gene rearrangement analysis have been reported in the literature, all of which had a clonal rearrangement detected.26 Follicular mucinosis and pagetoid reticulosis are disorders that have been associated with mycosis fungoides. 27,28 LeBoit et a129 detected a clonal rearrangement in the peripheral blood of a patient with erythrodermic follicular mucinosis who had 40% circulating Sezary cells. We have studied skin samples from three young patients with follicular mucinosis, without evidence for CTCL, all of whom had a clonal rearrangement. A single case of pagetoid reticulosis with a clonal rearrangement has been reported.** These studies verify that
Clinics in Dermatology 2991;9:119-228
GENE REARRANGEMENT
clonal populations of T cells develop in the skin and peripheral blood of patients with these uncommon disorders; however, the clinical significance of this finding is not yet known.
Lymphomatoid Papulosis Lymphomatoid papulosis is a recurrent papulonodular ulcerative disease that may progress to CTCL in 10 -20% of cases.3o Previous studies have correlated malignant progression with the type A histology classified by Willemze et aP1 and aneuploidy detected by flow cytometry. 32 Gene rearrangement analysis has shown clonal rearrangement in 65% of 35 patients tested.33-36 Four of these patients had the concomitant diagnosis of T-cell lymphoma, and two patients eventually developed Ki-1 (CD30) lymphoma. 34 Four of five patients with multiple lesions had the identical clone detected in each sample. The presence of a clone did not correlate with the histologic type or a characteristic clinical presentation. Additional, long-term surveillance of these patients will be required to determine whether a clonal rearrangement marks a distinct clinical course. However, lymphomatoid papulosis is one of several diseases that illustrates the potential problem of considering clonality as synonymous with malignancy and emphasizes the importance of correlating all gene rearrangement studies with the clinical findings, histologic findings, and immunophenotypic profile before establishing a final diagnosis.
Pityriasis Lichenoides et Varioliformis Acuta (PLEVA) PLEVA, a clinically benign disorder with histologic features that may mimic those of lymphomatoid papulosis or CTCL, has been shown to consist of clonal T cells by TCR gene rearrangement analysis.37 Two patients were noted to have the same clone in separate lesions. We evaluated one patient with PLEVA and one with small plaque parapsoriasis, both of whom had germline configurations. However, the results demonstrate that, despite a benign clinical course, some cases of PLEVA display clonality. The finding of TCR rearrangement in selected cases distinguishes this disorder from other benign inflammatory disorders, including lichen planus, nummular eczema, psoriasis, pityriasis rosea, and other reactive inflammatory cutaneous diseases in which clonal rearrangements have not been detected.33,M,37 Lymphomatoid papulosis and PLEVA share certain features, although immunophenotypic evaluation may be able to separate these diseases in most cases.38 The fact that patients with benign clinical outcomes have a clonal population of T cells is intriguing. These patients may
ZELICKSON ET AL ANALYSIS IN LYMPHOID NEOPLASIA
123
have “malignant” clones that are restrained and suppressed by the host defenses and cause them to appear clinically and biologically benign. Alternatively, the clone may reflect a limited expansion of T cells with a rearranged TCR gene that has been activated by antigen or other mitogenic stimuli.
Peripheral T-Cell Lymphomas PTCL, including angioimmunoblastic lymphadenopathy with dysproteinemia (AILD), AILD-like lymphoma, T-cell non-Hodgkin’s lymphoma, HTLV- 1 -associated T-cell leukemia/lymphoma, and lymphoproliferative disorders of large granular lymphocyte/natural killer cells, contained TCR gene rearrangement in the absence of immunoglobulin rearrangement in the majority of caSeS 1720.33-49 This technique appears to be helpful in establishing T-cell lineage and clonality of these disorders. Again, one should not rely solely on molecular genetics to determine cell lineage because TCR rearrangements have been detected in B-cell malignancies, including B-cell non-Hodgkin’s lymphomas,22 and lesions of ill-defined or incompletely characterized lineage, such as regressing atypical histiocytosis. 5oNevertheless, demonstration of dual genotypes in certain lymphoid malignancies may not only establish the stage of differentiation in clonal populations5’ but also assist in segregating and delineating subsets of disease. Feller et a152 suggested classifying patients with AILD into two different clinical and prognostic groups based on TCR gene rearrangement with or without accompanying immunoglobulin gene rearrangements. Correlation between gene rearrangement patterns and clinical presentation, disease course, and response to therapy is in the early stages of formulation and evaluation.
B-Cell Lymphoma and Pseudolymphoma Another challenging problem for the dermatopathologist is to differentiate primary cutaneous B-cell lymphoma from pseudolymphoma. Immunoglobulin gene rearrangement analysis is very helpful in determining lineage and clonality of B-cell lymphomas.5J’*53,54 This technique also may be helpful for identifying patients at risk for developing B-cell lymphoma. Wood et alS5 described 14 patients with cutaneous lymphoid hyperplasia diagnosed by histologic analysis and immunophenotyping. Five of these patients had an immunoglobulin gene rearrangement, one of whom developed a B-cell lymphoma within 2 years. However, monoclonality of these disorders, as determined by immunophenotyping, already has shown disparity and incomplete correlation between clonality and clinical aggressiveness. 56,57These results appear to be
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ZELICKSON ET AL
Table 2. Reported Results of Gene Rearrangement Analysis in Primary B-Cell Lymphoma and Pseudolumahoma B-cell lymphoma
Reference Liang et al5 Souteyrand et al” Delia et a153 Mayo datat Wood et als5 Kadin58 Weiss et aP Whittaker et alI5
No. of patients 1 3 14 13
TCR
l/13
lg
Pseudolymphoma*
Progression to malignant disease
l/l 213 14/14 5/13
No. of patients
TCR
lg
Progression to malignant disease
ND ND ND O/13$
3 14 11 2 1
l/3 o/11
o/3 5/14 l/11
o/2 l/l
O/3$ l/14 ND ND ND
Abbreviations: Ig, immunoglobulin gene rearrangement; ND, no date; TCR T-cell gene receptor rearrangement. * Pseudolymphoma includes cutaneous lymphoid hyperplnsia and lymphocytoma cutis. f Unpublished data. #Average duration offollow-upwas 2 years.
similar to the findings of gene rearrangement analysis. There are few reports of gene rearrangement analysis of pseudolymphomas, including cutaneous lymphoid hyperplasia and lymphocytoma cutis, and primary cutaneous B-cell lymphoma (Table 2).5~15~17~37*53~55 A clonal rearrangement was seen in approximately one-quarter of pseudolymphomas and in approximately three-quarters of patients with primary cutaneous lymphomas. None of the patients with malignant lymphoma who were followed for an average of 2 years had progression of their disease. This technique may help to distinguish reactive from malignant neoplastic lymphoid lesions. However, because of the nonaggressive nature of many primary B-cell cutaneous neoplasms, longer surveillance is needed to judge the clinical significance of clonal rearrangements within the immunoglobulin loci in these disorders.
antigenic markers has been associated with Hodgkin’s disease, and the Ki-1 (CD30) phenotype is characteristi~.~~-~ The discovery of Ki-1 -positive cells in diseases such as lymphomatoid papulosis, mycosis fungoides, Hodgkin’s disease, and non-Hodgkin’s lymphomas has widened the spectrum of ‘W-l lymphomas.“5K”-68 Herbst et aP9 described 39 patients with Hodgkin’s disease: six had TCR gene rearrangement, three had immunoglobulin gene rearrangement, and one had both types of rearrangements. Of the 22 patients with Ki-1 -positive lymphoma, 12 had TCR gene rearrangement, one had immunoglobulin gene rearrangement, and two had both. Although the results of gene rearrangement analysis and immunophenotyping were congruous in most cases, several deviations were noted. This may have occurred as a result of the immaturity of clonal cell populations.
Miscellaneous Disorders Sjtigren’s Syndrome
Patient-Specific Gene Probes
Sjogren’s syndrome, along with other chronic autoimmune diseases, has been associated with the development of non-Hodgkin’s lymphomas.59,60 Gene rearrangement analysis has been reported in nine patients with Sjogren’s syndrome. Eight patients had immunoglobulin gene rearrangements of involved salivary tissue, three of whom had concurrent non-Hodgkin’s lymphoma.61 One patient had a TCR gene rearrangement detected in a histologically benign lesion that progressed into a pleomorphic T-cell lymphoma.
Recognizing the virtually unlimited variations of TCR and immunoglobulin gene rearrangements, the ability to identify patient-specific gene sequences is currently being evaluated. Lessin et a170 isolated RNA from the malignant cells of a patient with CTCL. After selected gene amplification with PCR, a specific DNA alteration was able to be identified, and a unique probe for the specific TCR gene rearrangement from this patient was cloned. Similar studies have been performed in patients with follicular lymphomas. 71*72The identification of patient-specific gene rearrangements holds significant potential for following the clinical course and managing treatment. However, problems inherent to malignancy, including genomic instability and the development of ad-
Hodgkin’s Disease and Ki-1 -Positive
Lymphomas
The origin of the Reed-Sternberg cell has been extensively studied. Expression of T-cell, B-cell, and monocytic
Clinics in Dermatology 2991:9:119-128
ZELICKSON GENE
REARRANGEMENT
Table 3. Uses for Gene Rearrangement Analysis in Clinical Dermatolow Current Uses 1. Diagnosis of CTCL 2. Staging of CTCL 3. Determination of lineage in neoplastic lymphoid infiltrates of ambiguous phenotypes 4. Defining the nosology of poorly classified or indeterminate neoplasms (for example, “histiocytic” disorders-histiocytic lymphomas, regressing atypical histiocytosis, lymphoproliferative disorders associated with the skin diseases-follicular mucinosis) Proposed future uses: Southern blot analysis, in conjunction with PCR technology 1. Monitor treatment of CTCL/PTCL 2. Detect relapse of CTCL/PTCL 3. Detect and delineate subgroups of patients with PTCL 4. Differentiate B-cell lymphoma from pseudolymphoma Abbreviations: CTCL, cutaneous T-cell lymphomn; PTCL peripheral T-cell lymphoma.
ANALYSIS
needed to determine rearrangements.
IN LYMPHOID
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NEOPLASIA
the clinical significance
of clonal
References 1. Samman PD. The natural history of parapsoriasis en plaques (chronic superficial dermatitis) and prereticulotic poikiloderma. Br J Dermatol 1972;87:405 - 11.
2. Black MM, Jones EW. “Lymphomatoid”
pityriasis lichenoides; a variant with histological features simulating a lymphoma: a clinical and histopathological study of 15 cases with details of long term follow up. Br J Dermatol 1972;86:329-47.
3. Kim R, Winkelmann RK. Follicular mucinosis (alopecia mucinosa). Arch Dermatol 1962;85:490-98.
4. Ackerman AB. Histologic Diagnosis of Inflammatory Skin Diseases: A Method by Pattern Analysis. Philadelphia, Lea & Febiger, 1978, pp 483-85.
5. Liang G, Pardo RJ, Voigt W, et al. Studies of immunoglobuditional new rearrangements during progression of the disease, create potential difficulties with this new technologY.9
Summary Current uses for gene rearrangement analysis in clinical dermatology are listed in Table 3. This technique is useful for determining the existence of clonal populations within a background of polyclonal lymphoid cells;73*74 therefore, it is helpful in the diagnosis and staging of patients with CTCL and PTCL.9,75 Although dual genotypes do occur, this technique is usually capable of determining lineage in a clonal lymphoid infiltrate9,76,77 and elucidating and characterizing the etiopathogenesis of certain neoplasms. ‘*,xJ On the basis of this review of the and our own experience, we conclude that gene rearrangement analysis shows great promise for monitoring response to therapy and detecting progression or relapse in patients with CTCL and PTCL. With the recent technology of PCR, it is possible to amplify specific sequences of DNA, detect molecular alterations in individual malignant T cells, and even identify exogenous retroviral gene sequences in tissues of patients with CTCL.*O Although gene rearrangement analysis has supported or established the clonal nature of lymphomatoid papulosis, pre-S6zary syndrome, granulomatous slack skin syndrome, and follicular mucinosis, the clinical significance of these findings is not yet clear. In the case of primary cutaneous B-cell lymphoma and its benign counterpart, further investigation will be B-cell pseudolymphoma,
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M, Sundeen J, et al. Molecular genetics and the diagnosis of lymphoma. Arch Path01 Lab Med 1988;112:117-27.
10. Bugawan TL, Saiki RK, Levenson CH, et al. The use of non-radioactive oligonucleotide probes to analyze enzymatically amplified DNA for prenatal diagnosis and forensic HLA typing. Biotechnology 1988;6:943-47. 11.
Dosaka N, Tanaka T, Fujita M, et al. Southern blot analysis of clonal rearrangements of T-cell receptor gene in plaque lesion of mycosis fungoides. J Invest Dennatol 1989; 93:626-29.
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Bignon YJ, Roger H, Souteyrand P, et al. Study of T-cell antigen receptor gene rearrangement: A useful tool for early diagnosis of mycosis fungoides. Acta Derm Venereol (Stockh) 1989;69:217-22.
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Ralfkiaer E, O’Connor NTJ, Crick J, et al. Genotypic analysis of cutaneous T-cell lymphomas. J Invest Dermatol 1987;88:762-65.
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Tanaka T, Takahashi K, Ideyama S, et al. Demonstration of clonal proliferation of T lymphocytes in early neoplastic disease. J Am Acad Dermatol 1989;21:218-23.
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T-cell receptor genes in cutaneous T-cell lymphomas. Br J Dermatol 1989;120:295. 16. Berger CL, Eisenberg A, Soper L, et al. Dual genotype in cutaneous T cell lymphoma: immunoglobulin gene rearrangement in clonal T cell malignancy. J Invest Dermato1 1988:90:73 - 77. 17. Souteyrand I’, Bignon YJ, Roger H, et al. Evaluation of gene rearrangement studies for the diagnosis of cutaneous lymphomas and pseudolymphomas: Analysis of 47 cases. Br J Dermatol 1989;121(Suppl34):20-21. 18. Edelson RL, Berger CL, Raafat J, Warburton D. Karyotype studies of cutaneous T cell lymphoma: evidence for clonal origin. J Invest Dermatol 1979;73:548-50. 19. Finger LR, Harvey RC, Moore RCA, et al. A common mechanism of chromosomal translocation in T- and B-cell neoplasia. Science 1986;234:982-85. 20. Pelicci PG, Knowles DM II, Favera RD. Lymphoid tumors displaying rearrangements of both immunoglobulin and T cell receptor genes. J Exp Med 1985;162:1015-24. 21. Park JK, McKeithan TW, Le Beau MM, et al. An (8;14)(q24;qll) translocation involving the T-cell receptor a-chain gene and the MYC oncogene 3’ region in a B-cell lymphoma. Genes Chromosomes & Cancer 1989;1:15 -22. 22. Leber BF, Amlot I’, Hoffbrand AV, Norton JD. T-cell receptor gene rearrangement in B-cell non-Hodgkin’s lymphoma: Correlation with methylation and expression. Leukemia Res 1989;13:473-81. 23. Winkelmann RK, Buechner SA, Diaz-Perez JL. Pre-Sezary syndrome. J Am Acad Dermatol 1984;10:992-99. 24. Convit J, Kerdel F, Goihman M, et al. Progressive, atrophying, chronic granulomatous dermohypodermitis. Arch Dermatol 1973;107:27174. 25. Le T, Pierard I’. Granulomatous slack skin syndrome and Hodgkin’s disease. Ital Gen Rev Dermatol 1986;23:4849. 26. LeBoit PE, Beckstead JH, Bond B, et al. Granulomatous slack skin: Clonal rearrangement of the T-cell receptor p gene is evidence for the lymphoproliferative nature of a cutaneous elastolytic disorder. J Invest Dermatol 1987;89:183-86. 27. Rivers JK, Norris PG, Greaves MW, Smith NP. Follicular mucinosis in association with Sezary syndrome. Clin Exp Dermatol 1987;12:207-10. 28. Wood GS, Weiss LM, Hu C-H, et al. T-cell antigen deficiencies and clonal rearrangements of T-cell receptor genes in pagetoid reticulosis (Woringer-Kolopp disease). N Engl J Med 1988;318:164-67. 29. LeBoit PE, Abel EA, Cleary ML, et al, Clonal rearrangement of the T cell receptor p gene in the circulating lymphocytes of erythrodermic follicular mucinosis. Blood 1988;71:1329-33. 30. Sanchez NP, Pittelkow MR, Muller SA, et al. The clinicopathologic spectrum of lymphomatoid papulosis: study of 31 cases. J Am Acad Dermatol 1983;8:81-94. 31. Willemze R, Meyer CJLM, van Vloten WA, Scheffer E. The
clinical and histologic spectrum of lymphomatoid sis. Br J Dermatol 1982;107:131-44.
papulo-
32. Wantzin GL, Thomsen K, Larsen JK, et al. DNA analysis by flow cytometry in lymphomatoid papulosis. Clin Exp Dermatol 1983;8:505 - 12. 33. Zelickson BD, Peters MS, Muller SA, et al. Analysis of T-cell receptor gene rearrangements in cutaneous T-cell lymphoma premalignant and benign dermatoses. In Parapsoriasis: Proceedings of the First International Parapsoriasis Symposium Rochester, MN, Mayo Foundation, pp 37-42, 1990. 34. Whittaker SJ, Smith NP, Russell-Jones R, Luzzatto L. Lymphomatoid papulosis-A genotypic study. Br J Dermatol 1989;121(Suppl34):21-22. 35. Kadin ME, Vonderheid EC, Sako D, et al. Clonal composition of T cells in lymphomatoid papulosis. Am J Path01 1987;126:13-17. 36. Weiss LM, Wood GS, Trela M, et al. Clonal T-cell populations in lymphomatoid papulosis: evidence of a lymphoproliferative origin for a clinically benign disease. N Engl J Med 1986;315:475-79. 37. Weiss LM, Wood GS, Ellisen LW, et al. Clonal T-cell populations in pityriasis lichenoides et varioliformis acuta (Mucha-Habermann disease). Am J Path01 1987;126: 417-21. 38. Barga FJ, Vonderheid EC, Olbricht SM, Kadin ME. Immuno-histochemical distinction of lymphomatoid papulosis and pityriasis lichenoides et varioliformis acuta. Am J Path01 1990;136:979-87. 39. Sheibani K, Wu A, Ben-Ezra J, et al. Rearrangement of Kchain and T-cell receptor P-chain genes in malignant lymphomas of “T-cell” phenotype. Am J Path01 1987;129:201-207. 40. Waldman TA, Davis MM, Bongiovanni KF, Korsmeyer SJ. Rearrangement of genes for the antigen receptor on T cells as markers of lineage and clonality in human lymphoid neoplasms. N Engl J Med 1985;313:776-83. 41. Knowles DM II, Pelicci PG, Dalla-Favera R. T-cell receptor beta chain gene rearrangements: genetic markers of T-cell lineage and clonality. Hum Path01 1986;17:546 - 51. 42. Kimura N, Takihara Y, Akiyoshi T, et al. Rearrangement of T-cell receptor 6 chain gene as a marker of lineage and clonality in T-cell lymphoproliferative disorders. Cancer Res 1989;49:4488-92. 43. Aisenberg AC, Krontiris TG, Mak TW, Wilkes BM. Rearrangement of the gene for the beta chain of the T-cell receptor in T-cell chronic lymphocytic leukemia and related disorders. N Engl J Med 1985;313:529-33. 44. Weiss LM, Strickler JG, Dorfman RF, et al. Clonal T-cell populations in angioimmunoblastic lymphadenopathy and angioimmunoblastic lymphadenopathy-like lymphoma. Am J Path01 1986;122:392-97. 45. O’Connor NTJ, Crick JA, Wainscoat JS, et al. Evidence for monoclonal T lymphocyte proliferation in angioimmunoblastic lymphadenopathy. J Clin Path01 1986;39:1229-32.
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46. Griesser H, Feller A, Lennert K, et al. Rearrangement of the /Ichain of the T cell antigen receptor and immunoglobulin genes in lymphoproliferative disorders. J Clin Invest 1986;78:1179-84.
61. Fishleder A, Tubbs R, Hesse B, Levine H. Uniform detection of irnmunoglobulin-gene rearrangement in benign lymphoepithelial lesions. N Engl J Med 1987;316:111821.
47. Weiss LM, Wood GS, Nickoloff BJ, Sklar J. Gene rearrangement studies in lymphoproliferative disorders of skin. Adv Dermatol 1988;3:141-57.
62. Stein H, Mason DY, Gerdes J, et al. The expression of the Hodgkin’s disease associated antigen Ki-1 in reactive and neoplastic lymphoid tissue: evidence that Reed-Stemberg cells and histiocytic malignancies are derived from activated lymphoid cells. Blood 1985;66:848-58.
48. Rambaldi A, Pelicci P-G, Allavena I’, et al. T cell receptor /.I chain gene rearrangements in lymphoproliferative disorders of large granular lymphocytes/natural killer cells. J Exp Med 1985;162:2156-62. 49. Zelickson BD, Tefferi A, Gertz MA, et al. Transient acantholytic dermatosis associated with lymphomatous angioimmunoblastic lymphadenopathy. Acta Derm Venereol (Stockh) 1989;69:445-48.
63. Stein H, Hansmann ML, Lennert K, et al. Reed-Stemberg and Hodgkin cells in lymphocyte-predominant Hodgkin’s disease of nodular subtype contain J chain. Am J Clin Path01 1986;86:292-97.
50. Headington JT, Roth MS, Ginsburg D, et al. T-cell receptor gene rearrangement in regressing atypical histiocytosis. Arch Dermatol 1987;123:1183-87.
64. Falini B, Stein H, Pileri S, et al. Expression of lymphoid-associated antigens on Hodgkin’s and Reed-Stemberg cells of Hodgkin’s disease. An immunocytochemical study on lymph node cytospins using monoclonal antibodies. Histopathology 1987;11:1229-42.
51. Leber BF, Amlot I’, Hoffbrand AV, Norton JD. T-cell receptor gene rearrangement in B-cell non-Hodgkin’s lymphoma: Correlation with methylation and expression. Leukemia Res 1989;13:473-81.
65. Kadin ME, Muramoto L, Said J. Expression of T-cell antigens on Reed-Stemberg cells in a subset of patients with nodular sclerosing and mixed cellularity Hodgkin’s disease. Am J Path01 1988;130:345-53.
52. Feller AC, Griesser H, Schilling CV, et al. Clonal gene rearrangement patterns correlate with immunophenotype and clinical parameters in patients with angioimmunoblastic lymphadenopathy. Am J Path01 1988;133:549-56.
66. Agnarsson BA, Kadin ME. Ki-1 positive large cell lymphoma: A morphologic and immunologic study of 19 cases. Am J Surg Path01 1988;12:264-74.
53. Delia D, Borrelo MG, Berti E, et al. Clonal immunoglobulin gene rearrangements and normal T-cell receptor, bcl-2, and c-myc genes in primary cutaneous B-cell lymphomas. Cancer Res 1989;49:4901-05. 54. Arnold A, Cossman J, Bakhshi A, et al. Immunoglobulingene rearrangements as unique clonal markers in human lymphoid neoplasms. N Engl J Med 1983;309:1593-99. 55. Wood GS, Ngan B-Y, Tung R, et al. Clonal rearrangements of immunoglobulin genes and progression to B cell lymphoma in cutaneous lymphoid hyperplasia. Am J Path01 1989;135:13-19. 56. Turner RR, Egbert P, Wamke RA. Lymphocytic infiltrates of the conjunctiva and orbit: Immunohistochemical staining of 16 cases. Am J Clin Path01 1984;81:447-52. 57. Knowles DM II, Jakobiec FA. Cell marker analysis of extranodal lymphoid infiltrates: to what extent does the determination of mono- or polyclonality resolve the diagnostic dilemma of malignant lymphoma v pseudolymphoma in an extranodal site? Semin Diagn Path01 1985;2:163 -68. 58. Kadin ME. The spectrum of Ki-1+ cutaneous lymphomas. Curr Probl Dermatol 1990;19:132-43.
67. Bums BF, Dardick I. K&l-positive non-Hodgkin’s lymphomas: An immunophenotypic, ultrastructural, and morphometric study. Am J Clm Path01 1990;93:327-32. 68. Kaudewitz I’, Stein H, Dallenbach F, et al. Primary and secondary cutaneous K&l+ (CD30+) anaplastic large cell lymphomas: Morphologic, immunohistologic, and clinical characteristics. Am J Path01 1989;135:359-67. 69. Herbst H, Tippelmann G, Anagnostopoulos I, et al. Immunoglobulin and T-cell receptor gene rearrangements in Hodgkin’s disease and Ki-1 -positive anaplastic large cell lymphoma: Dissociation between phenotype and genotype. Leukemia Res 1989;13:103-16. 70. Lessin SR, Rook AH, Rovera G. Molecular diagnosis of cutaneous T-cell lymphoma: polymerase chain reaction amplification of T-cell antigen receptor /?-chain gene rearrangements. J Invest Dermatol 1991;96:299-302. 71. Ngan BY, Nourse J, Cleary ML. Detection of chromosomal translocation t( 14;18) within the minor cluster region of bd-2 by polymerase chain reaction and direct genomic sequencing of the enzymatically amplified DNA in follicular lymphomas. Blood 1989;73:1759-62. 72. Lee M-S, Chang K-S, Cabanillas F, et al. Detection of minimal residual cells carrying the t(14;18) by DNA sequence amplification. Science 1987;237:175 - 78.
59. van der Valk PGM, Hollema H, van Voorst Vander PC, et al. SjBgren’s syndrome with specific cutaneous manifestations and multifocal clonal T-cell populations progressing to a cutaneous pleomorphic T-cell lymphoma. Am J Clin Path01 1989;92:357-61.
73. Knowles DM, Wamke R. Society for hematopathology annual meeting: T cell neoplasia. Am J Surg Path01 1988;12:158-63.
60. Moutsopoulos HM, Chused TM, Mann DL, et al. Sjogren’s syndrome (sicca syndrome): Current issues. Ann Intern Med 1980;92:212-26.
74. Bertness V, Kirsch I, Hollis G, et al. T-cell receptor gene rearrangements as clinical markers of T-cell lymphomas. N Engl J Med 1985;313:534-38.
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75. Weiss LM, Hu E, Wood GS, et al. Clonal rearrangements of T-cell receptor genes in mycosis fungoides and dermatopathic lymphadenopathy. N Engl J Med 1985;313:53944. 76. Williams ME, Innes DJ, Borowitz MJ, et al. Immunoglobulin and T cell receptor gene rearrangements in human lymphoma and leukemia. Blood 1987;69:79-86. 77. Sklar J, Weiss LM. Applications of antigen receptor gene rearrangements to the diagnosis and characterization of lymphoid neoplasms. Ann Rev Med 1988;39:315-34.
78. Bleicher PA. T-cell receptor gene rearrangements: and promise. Arch Dermatol 1988;124:359-63.
progress
79. Bignon YJ, Dastugue B, Plagne R. Molecular biology diagnosis in oncology. Biomed Pharmacother 1988;42:65359. 80. Whittaker SJ, Smith NP, Jones RR, Luzzatto L. HTLV-1 and cutaneous T-cell lymphoma (abstr). J Invest Dermatol 1990:94:590.
C
utaneous lymphoma and pseudolymphoma comprise a broad clinical spectrum of disorders that require evaluation by the dermatologist. Variousmethods,includingroutinehistologic analysis, electron microscopy, immunophenotyping, and flow cytometry, have been used to determine T- versus B-cell lineage and to attempt to establish the neoplastic or benign nature of cutaneous lymphoid infiltrates. Although these techniques are useful in the diagnosis of lymphoid neoplasms, each has shortcomings. Histopathologic examination of early lesions of cutaneous T-cell lymphoma (CTCL) may be nonspecific and difficult to distinguish from dermatitis. Disorders such as lymphocytoma, large plaque parapsoriasis, or lymphomatoid papulosis may present atypical cytologic features despite exhibiting a benign or nonaggressive clinical course.1-4 Certain microscopic morphologic characteristics, including cell shape, size, and convolution of nuclei, may suggest T- or B-cell lineage; however, these structural indicators are at times unreliable.5*6 The use of immunophenotyping for diagnosis of neoplastic versus benign lymphoid infiltrates is limited by various factors, including 1) immature T- and B-cell malignancies expressing aberrant or incomplete sets of surface markers; 2) admixtures of malignant neoplastic and benign reactive lymphoid cells within the infiltrate; and 3) frequently, the T-cell helper phenotype and other various activation markers being shared by both benign and malignant infiltrates.
Principles and Interpretation of Gene Rearrangement Analysis in Lymphoid Disease The molecular technology of gene rearrangement analysis has the potential to identify both lineage and clonality From the Department of Dermatology, Mayo Clinic and Mayo Foundation, Rochester, Minnesota. Address correspondence to: Mark R Pittelkow, M.D., Mayo Clinic, 200 First Street SW, Rochester, MN 55905.
0 2991 by Elsevier Science Publishing Co., Inc.
l
0738-081x/91/$3.50
of neoplastic processes, particularly malignant lymphoproliferative diseases. DNA of all nucleated cells contains numerous genetic sequences that direct RNA transcription and translation of proteins. Among lymphoid cells, immunoglobulins and T-cell (antigen) receptor (TCR) proteins are characteristic, lineage-specific, antigen& markers found on the surface of B and T cells, respectively. The genes that encode these proteins consist of multiple variable, diversity, joining, and constant regions that are found in a germline or nonrearranged configuration in all nonlymphoid cells. In lymphoid cells, each receptor protein is translated from a specific genetic sequence that is formed by rearranging these genes during differentiation and maturation of the cell (Figure 1). The resulting configurations of DNA are highly variable from cell to cell and contribute to the diversity and specificity of immune cell populations in recognizing antigenic substances. They also serve as genetic markers of individual lymphocytes and represent the virtually unique genotypic attributes of a cell or population of cells. Once this rearrangement occurs, each daughter cell or progeny will contain the identical genetic pattern. The DNA hybridization or Southern blot procedure allows identification and relative quantitation of the frequency of a particular gene rearrangement within the DNA isolated from a population of lymphoid cells.’ Development, proliferation, and expansion of a specific unique population generate a cellular clone. Clonality correlates closely with malignancy, although benign neoplastic processes occasionally develop clonal populations of cells within a specific lineage. DNA is harvested from fresh or frozen tissue or fluid samples containing intact nuclei of cells (Figure 2). For Tor B-cell analysis, adequate numbers of lymphoid cells should be present in the specimen. DNA is digested with restriction enzymes (endonucleases), which split the DNA at specific nucleotide sequences (Figure 1). The resulting DNA fragments are separated by agarose gel electrophoresis and transferred to a nylon membrane. This nylon membrane is then incubated with radioactive- or
119
Clinics in Dermatology 1991;9:119-228
120 ZELICKSON ET AL
Germline
Allele
Rearranged
“PI
gene
“6
RBHHHHRH
R
R
H
R
lIIlllll
I
I
I
I
DP,
JPI
n w
n
“PI
%*
%I
%, JP2
JP2
%*
%*
U
4 mRNA 4 fi -chain protean
Figure 1. T-cell receptor /I-chain rearrangement. Genome of the T-cell receptor P-chain with schematic representation of gene rearrangement. This includes the combination of different variable (V), diversity (D),joining (f,I,and constant (C) regions with subsequent formation of ml7NA and P-chain protein. The locations of the BamHI, EcoRI, and Hind111restriction endonuclease sites are indicated by the letters B, R, and H, respectively. From Zelickson et a1.j3 By permission of Mayo Foundation. nonradioactive-labeled fragments of nucleic acids complementary to the appropriate portion of the immunoglobulin or TCR genes. Specific hybridizing fragments of immunoglobulin or TCR DNA are identified and visualized as bands of specific molecular size. Three basic banding patterns are detected by Southern blot analysis (Figure 3): germline, polyclonal, and monoclonal. A germline pattern, as previously mentioned, represents genes that have not undergone rearrangement and includes the genes of all nonlymphoid cells. A polyclonal pattern will appear as multiple indistinct bands, often as a “smear” or as no additional non-germline bands. This pattern is produced by multiple, benign lymphoid cells, each containing a different rearrange-
ment site. If the lymphoid population contains cells that have undergone identical DNA rearrangement and expansion, a clonal, neoplastic pattern is observed. The rearrangement appears as additional non-germline bands. One or two non-germline bands represent rearrangement of one or both alleles. This technique routinely detects a clonal cell population that represents a minimum of 0.5%-5% of the total cellular population yielding DNA.* Some pitfalls that may result in false-negative results include sampling error, which may result in an insufficient number of clonal cells. In addition, corn&ration occurs when a TCR gene rearrangement produces a DNA restriction fragment approximately the same size as the germline fragment; this clonal fragment migrates to the same location in the gel as the germline band and therefore will not be detected. This problem may be resolved by digesting the DNA with several different restriction enzymes. The TCR is composed of a cluster of separate polypeptide subunits. Each subunit is encoded by separate genes, which are designated (Y,/I,y, or 6. Although an infrequent occurrence, some clonal proliferations of T cells express y/6 T-cell receptors instead of (Y or j? receptors. Thus, in selected instances, negative (Y and p studies may be further examined by y and 6 gene rearrangement analysis. With progression of the neoplasia, chromosomal loss can occur, which may eliminate the rearranged TCR genes. Another potential technical pitfall may occur with incomplete digestion of DNA by endonucleases. This procedural error causes formation of a “pseudoclone,” which could produce a false-positive result.9 Gene rearrangement analysis is performed in most university and tertiary referral centers. The procedure usually takes 1 - 2 weeks to perform. Currently, fresh or frozen tissue is required; however, formalin-fixed tissue and special amplification procedures (see polymerase chain
Specific DNA
Figure 2. Gene rearrangement analysis performed by the Southern blot technique. High-molecular-weight DNA is extracted from a cell suspension obtained from involved tissue or peripheral blood. The DNA is then digested to completion with endonucleases and fractioned by gel electrophoresis. The DNA is then transferred to a nylon filter and incubated with specific 32P-radiolabeled probes. The hybridization pattern is then revealed by autoradiography (not shown).
w
Recombmant plasmid
” P radlolabeled or nonradloactive probe
I I
\
Hybridization
Peripheral blood
“Bands”
/
k/
/-I
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Southern transfer
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/?I molecular weight DNA
clease digestion
electrophoresis
Transfer
Get
Clinics in Dermatology 1991;9:129-128
ZELICKSON ET AL GENE REARRANGEMENT ANALYSIS IN LYMPHOID NEOPLASIA
121
and suggest specific guidelines for use of this technology in a clinical dermatology practice.
Cutaneous T-Cell Lymphoma
Figure 3. Banding patterns detected by Southern blot analysis after EcoRI digestion of TCR gene and hybridization with ],Q probe: germline @), monoclonal (O), and polyclonal (4 banding patterns. From Zelickson et a1.j3 By permission of Mayo Foundation.
reaction [PCR], see article by Jester) have been used with success to overcome these limitations.*0 For tissue specimens, depending on the density of the infiltrate, a 4-mm punch biopsy specimen is adequate; however, a larger sample may allow for more complete and accurate results. We usually obtain a 5- to 6-mm punch biopsy specimen for each assay. A frozen section stained with hematoxylin-eosin should be obtained from the specimen, and all normal-appearing (that is, lymphoid-free) tissue should be trimmed from the block. This processing step decreases the likelihood that the DNA of the clonal, neoplastic cellular population will be proportionately underrepresented and become “masked’ by germline DNA of other epidermal or dermal cells. There have been numerous reports in the literature of the application of this technique to cutaneous T- and B-cell neoplasms. We have compiled the results of gene rearrangement analyses previously reported for CTCL, peripheral T-cell lymphoma (PTCL), malignant or potentially neoplastic T-cell infiltrates (including large plaque parapsoriasis, lymphomatoid papulosis, follicular mucinosis, and granulomatous slack skin syndrome), cutaneous B-cell lymphoma, and pseudolymphoma. We review illustrative cases that have been reported, compare these observations with data accumulated at our institution,
CTCL, including mycosis fungoides and Sezary syndrome, encompasses a group of chronic and progressive, malignant lymphoproliferative disorders primarily involving the skin in early stages of the disease. Many studies have demonstrated the clonality of these related disorders (Table 1). The overall percentage of patients with CTCL who have gene rearrangements detected in tissue or peripheral blood varies with the type of sample analyzed. In our studies, the percentage of positive results is greater in skin samples of mycosis fungoides, whereas peripheral blood samples of patients with Sezary syndrome more frequently contain a clonal population. Although there are conflicting reports concerning the findings of rearrangements in patients with early (stage I-II) mycosis fungoides,“-**,” we believe that this technique is helpful for detecting early CTCL. False-negative results may be minimized by evaluating multiple gene segments with different DNA probes and by examining more than one specimen. We have observed patients with SCzary syndrome who demonstrated a germline configuration in peripheral blood but clonal rearrangement in a skin sample and also patients with mycosis fungoides who had the converse findings. This evidence stresses the importance of testing multiple samples from each patient, The finding of a clonal population is helpful for distinguishing early CTCL from dermatitis. However, the absence of gene rearrangement does not exclude a diagnosis of CTCL. Furthermore, germline configurations may occur even in patients with advanced disease.6,15,17 This finding may be due to chromosomal loss during neoplastic progression, which eliminates the rearranged gene or results in a “hidden” clone, as discussed earlier. Dual genotypes, the coexistence of bothimmunoglobulin and TCR gene rearrangements, have been detected in patients with CTCL.16 This event may occur as a result of the malignant cell being derived from a pluripotential cell type, multiple chromosomal translocations,‘* or the nonspecificity of the recombinase enzymes, which directs the process of DNA rearrangement.19 However, the presence of an immunoglobulin light-chain rearrangement is highly indicative of a B-cell neoplasm. Rearrangement of immunoglobulin heavy-chain genes may be observed in B- or T-cell neoplasms. 19rzoThere have also been reports of TCR gene rearrangements in patients with B-cell lymphoma.z1,22 TCR gene rearrangement analysis also may be helpful for detecting lymphomatous involvement of enlarged
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Clinics in Dermatology 2991;9:119-128
ZELICKSON ET AL
Table 2. Reported Results of T-Cell Gene Rearrangement Analysis in Cutaneous T-Cell Lumvhomas*6J-16 Site Disorder
Skin
Peripheral blood
Lymph node
Not specified
Mycosis fungoides No. positive
41
11
14
7
No. tested
60
41
23
8
% positive
68
27
60
88
Skzary syndrome No. Positive
43
3
16
No. tested
55
3
16
78
100
100
% uositive
69
lymph nodes in patients with CTCL. A clonal population may be detected in a histologically benign lymph nodeel Gene rearrangement analysis is therefore useful in the diagnosis of CTCL and should be performed in conjunction with routine histologic examination and immunophenotyping. As this technique is further refined, it should be applied in both the diagnosis and the staging of patients. It also may be of benefit in following the course of patients with CTCL. 4~17Our preliminary results of serial TCR gene rearrangement analyses in patients with Sezary syndrome show that changes in Sezary cell counts, erythema, or pruritus were associated with changes in clonal density in many cases. These results are promising; however, more data are needed to evaluate this application. Furthermore, with newer technologies such as PCR, more specific and sensitive methods to identify and monitor the malignant genotypes will likely be forthcoming. There are multiple cutaneous T-cell lymphoproliferative disorders that histologically mimic CTCL. Although most of these diseases are clinically benign, some may progress to or are associated with frank T-cell neoplasia. These include lymphomatoid papulosis, large plaque parapsoriasis, granulomatous slack skin syndrome, preSezary syndrome, follicular mucinosis, and pagetoid reticulosis. Gene rearrangement analyses should be performed on skin biopsy specimens and, in the appropriate settings, on peripheral blood.
Pre-!.%zary Syndrome and Large Plaque Parapsoriasis Pre-Sezary syndrome, as defined by Winkelmann et a1,23 and large plaque parapsoriasis each represent a diagnostic dilemma. Ralfkiaer et all3 reported eight cases suspected to represent early CTCL, all of which had a germline configuration. We have detected a clonal
rearrangement in four of eight patients with pre-Sezary syndrome but in none of four patients with large plaque parapsoriasis. As with the data of Ralfkiaer et al, sufficient duration of follow-up to establish disease progression was lacking for these patients. However, the finding of a clonal population in patients with these diseases, despite indeterminate histopathologic results and immunotype, may herald the evolution to T-cell neoplasia. The morphologic distinction between Sezary cells and Sezary-like cells is difficult. Bendelac et al6 reported a germline banding pattern of isolated Sezary-like cells in a patient with CTCL. We did not observe clonal rearrangement in peripheral blood lymphocytes after nonspecific mitogenic stimulation with the lectin phytohemagglutinin, which induces formation of blastogenic, Sezarylike cells in culture.
Granulomatous Slack Skin Syndrome, Follicular Mucinosis, and Pagetoid Reticulosis Granulomatous slack skin syndrome is a distinctive disorder with bulky, flexural, cutaneous lesions consisting of a dense granulomatous infiltrate. It has been associated with Hodgkin’s disease24,25 and mycosis fungoides.’ Only three cases with TCR gene rearrangement analysis have been reported in the literature, all of which had a clonal rearrangement detected.26 Follicular mucinosis and pagetoid reticulosis are disorders that have been associated with mycosis fungoides. 27,28 LeBoit et a129 detected a clonal rearrangement in the peripheral blood of a patient with erythrodermic follicular mucinosis who had 40% circulating Sezary cells. We have studied skin samples from three young patients with follicular mucinosis, without evidence for CTCL, all of whom had a clonal rearrangement. A single case of pagetoid reticulosis with a clonal rearrangement has been reported.** These studies verify that
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GENE REARRANGEMENT
clonal populations of T cells develop in the skin and peripheral blood of patients with these uncommon disorders; however, the clinical significance of this finding is not yet known.
Lymphomatoid Papulosis Lymphomatoid papulosis is a recurrent papulonodular ulcerative disease that may progress to CTCL in 10 -20% of cases.3o Previous studies have correlated malignant progression with the type A histology classified by Willemze et aP1 and aneuploidy detected by flow cytometry. 32 Gene rearrangement analysis has shown clonal rearrangement in 65% of 35 patients tested.33-36 Four of these patients had the concomitant diagnosis of T-cell lymphoma, and two patients eventually developed Ki-1 (CD30) lymphoma. 34 Four of five patients with multiple lesions had the identical clone detected in each sample. The presence of a clone did not correlate with the histologic type or a characteristic clinical presentation. Additional, long-term surveillance of these patients will be required to determine whether a clonal rearrangement marks a distinct clinical course. However, lymphomatoid papulosis is one of several diseases that illustrates the potential problem of considering clonality as synonymous with malignancy and emphasizes the importance of correlating all gene rearrangement studies with the clinical findings, histologic findings, and immunophenotypic profile before establishing a final diagnosis.
Pityriasis Lichenoides et Varioliformis Acuta (PLEVA) PLEVA, a clinically benign disorder with histologic features that may mimic those of lymphomatoid papulosis or CTCL, has been shown to consist of clonal T cells by TCR gene rearrangement analysis.37 Two patients were noted to have the same clone in separate lesions. We evaluated one patient with PLEVA and one with small plaque parapsoriasis, both of whom had germline configurations. However, the results demonstrate that, despite a benign clinical course, some cases of PLEVA display clonality. The finding of TCR rearrangement in selected cases distinguishes this disorder from other benign inflammatory disorders, including lichen planus, nummular eczema, psoriasis, pityriasis rosea, and other reactive inflammatory cutaneous diseases in which clonal rearrangements have not been detected.33,M,37 Lymphomatoid papulosis and PLEVA share certain features, although immunophenotypic evaluation may be able to separate these diseases in most cases.38 The fact that patients with benign clinical outcomes have a clonal population of T cells is intriguing. These patients may
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123
have “malignant” clones that are restrained and suppressed by the host defenses and cause them to appear clinically and biologically benign. Alternatively, the clone may reflect a limited expansion of T cells with a rearranged TCR gene that has been activated by antigen or other mitogenic stimuli.
Peripheral T-Cell Lymphomas PTCL, including angioimmunoblastic lymphadenopathy with dysproteinemia (AILD), AILD-like lymphoma, T-cell non-Hodgkin’s lymphoma, HTLV- 1 -associated T-cell leukemia/lymphoma, and lymphoproliferative disorders of large granular lymphocyte/natural killer cells, contained TCR gene rearrangement in the absence of immunoglobulin rearrangement in the majority of caSeS 1720.33-49 This technique appears to be helpful in establishing T-cell lineage and clonality of these disorders. Again, one should not rely solely on molecular genetics to determine cell lineage because TCR rearrangements have been detected in B-cell malignancies, including B-cell non-Hodgkin’s lymphomas,22 and lesions of ill-defined or incompletely characterized lineage, such as regressing atypical histiocytosis. 5oNevertheless, demonstration of dual genotypes in certain lymphoid malignancies may not only establish the stage of differentiation in clonal populations5’ but also assist in segregating and delineating subsets of disease. Feller et a152 suggested classifying patients with AILD into two different clinical and prognostic groups based on TCR gene rearrangement with or without accompanying immunoglobulin gene rearrangements. Correlation between gene rearrangement patterns and clinical presentation, disease course, and response to therapy is in the early stages of formulation and evaluation.
B-Cell Lymphoma and Pseudolymphoma Another challenging problem for the dermatopathologist is to differentiate primary cutaneous B-cell lymphoma from pseudolymphoma. Immunoglobulin gene rearrangement analysis is very helpful in determining lineage and clonality of B-cell lymphomas.5J’*53,54 This technique also may be helpful for identifying patients at risk for developing B-cell lymphoma. Wood et alS5 described 14 patients with cutaneous lymphoid hyperplasia diagnosed by histologic analysis and immunophenotyping. Five of these patients had an immunoglobulin gene rearrangement, one of whom developed a B-cell lymphoma within 2 years. However, monoclonality of these disorders, as determined by immunophenotyping, already has shown disparity and incomplete correlation between clonality and clinical aggressiveness. 56,57These results appear to be
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ZELICKSON ET AL
Table 2. Reported Results of Gene Rearrangement Analysis in Primary B-Cell Lymphoma and Pseudolumahoma B-cell lymphoma
Reference Liang et al5 Souteyrand et al” Delia et a153 Mayo datat Wood et als5 Kadin58 Weiss et aP Whittaker et alI5
No. of patients 1 3 14 13
TCR
l/13
lg
Pseudolymphoma*
Progression to malignant disease
l/l 213 14/14 5/13
No. of patients
TCR
lg
Progression to malignant disease
ND ND ND O/13$
3 14 11 2 1
l/3 o/11
o/3 5/14 l/11
o/2 l/l
O/3$ l/14 ND ND ND
Abbreviations: Ig, immunoglobulin gene rearrangement; ND, no date; TCR T-cell gene receptor rearrangement. * Pseudolymphoma includes cutaneous lymphoid hyperplnsia and lymphocytoma cutis. f Unpublished data. #Average duration offollow-upwas 2 years.
similar to the findings of gene rearrangement analysis. There are few reports of gene rearrangement analysis of pseudolymphomas, including cutaneous lymphoid hyperplasia and lymphocytoma cutis, and primary cutaneous B-cell lymphoma (Table 2).5~15~17~37*53~55 A clonal rearrangement was seen in approximately one-quarter of pseudolymphomas and in approximately three-quarters of patients with primary cutaneous lymphomas. None of the patients with malignant lymphoma who were followed for an average of 2 years had progression of their disease. This technique may help to distinguish reactive from malignant neoplastic lymphoid lesions. However, because of the nonaggressive nature of many primary B-cell cutaneous neoplasms, longer surveillance is needed to judge the clinical significance of clonal rearrangements within the immunoglobulin loci in these disorders.
antigenic markers has been associated with Hodgkin’s disease, and the Ki-1 (CD30) phenotype is characteristi~.~~-~ The discovery of Ki-1 -positive cells in diseases such as lymphomatoid papulosis, mycosis fungoides, Hodgkin’s disease, and non-Hodgkin’s lymphomas has widened the spectrum of ‘W-l lymphomas.“5K”-68 Herbst et aP9 described 39 patients with Hodgkin’s disease: six had TCR gene rearrangement, three had immunoglobulin gene rearrangement, and one had both types of rearrangements. Of the 22 patients with Ki-1 -positive lymphoma, 12 had TCR gene rearrangement, one had immunoglobulin gene rearrangement, and two had both. Although the results of gene rearrangement analysis and immunophenotyping were congruous in most cases, several deviations were noted. This may have occurred as a result of the immaturity of clonal cell populations.
Miscellaneous Disorders Sjtigren’s Syndrome
Patient-Specific Gene Probes
Sjogren’s syndrome, along with other chronic autoimmune diseases, has been associated with the development of non-Hodgkin’s lymphomas.59,60 Gene rearrangement analysis has been reported in nine patients with Sjogren’s syndrome. Eight patients had immunoglobulin gene rearrangements of involved salivary tissue, three of whom had concurrent non-Hodgkin’s lymphoma.61 One patient had a TCR gene rearrangement detected in a histologically benign lesion that progressed into a pleomorphic T-cell lymphoma.
Recognizing the virtually unlimited variations of TCR and immunoglobulin gene rearrangements, the ability to identify patient-specific gene sequences is currently being evaluated. Lessin et a170 isolated RNA from the malignant cells of a patient with CTCL. After selected gene amplification with PCR, a specific DNA alteration was able to be identified, and a unique probe for the specific TCR gene rearrangement from this patient was cloned. Similar studies have been performed in patients with follicular lymphomas. 71*72The identification of patient-specific gene rearrangements holds significant potential for following the clinical course and managing treatment. However, problems inherent to malignancy, including genomic instability and the development of ad-
Hodgkin’s Disease and Ki-1 -Positive
Lymphomas
The origin of the Reed-Sternberg cell has been extensively studied. Expression of T-cell, B-cell, and monocytic
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ZELICKSON GENE
REARRANGEMENT
Table 3. Uses for Gene Rearrangement Analysis in Clinical Dermatolow Current Uses 1. Diagnosis of CTCL 2. Staging of CTCL 3. Determination of lineage in neoplastic lymphoid infiltrates of ambiguous phenotypes 4. Defining the nosology of poorly classified or indeterminate neoplasms (for example, “histiocytic” disorders-histiocytic lymphomas, regressing atypical histiocytosis, lymphoproliferative disorders associated with the skin diseases-follicular mucinosis) Proposed future uses: Southern blot analysis, in conjunction with PCR technology 1. Monitor treatment of CTCL/PTCL 2. Detect relapse of CTCL/PTCL 3. Detect and delineate subgroups of patients with PTCL 4. Differentiate B-cell lymphoma from pseudolymphoma Abbreviations: CTCL, cutaneous T-cell lymphomn; PTCL peripheral T-cell lymphoma.
ANALYSIS
needed to determine rearrangements.
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NEOPLASIA
the clinical significance
of clonal
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Summary Current uses for gene rearrangement analysis in clinical dermatology are listed in Table 3. This technique is useful for determining the existence of clonal populations within a background of polyclonal lymphoid cells;73*74 therefore, it is helpful in the diagnosis and staging of patients with CTCL and PTCL.9,75 Although dual genotypes do occur, this technique is usually capable of determining lineage in a clonal lymphoid infiltrate9,76,77 and elucidating and characterizing the etiopathogenesis of certain neoplasms. ‘*,xJ On the basis of this review of the and our own experience, we conclude that gene rearrangement analysis shows great promise for monitoring response to therapy and detecting progression or relapse in patients with CTCL and PTCL. With the recent technology of PCR, it is possible to amplify specific sequences of DNA, detect molecular alterations in individual malignant T cells, and even identify exogenous retroviral gene sequences in tissues of patients with CTCL.*O Although gene rearrangement analysis has supported or established the clonal nature of lymphomatoid papulosis, pre-S6zary syndrome, granulomatous slack skin syndrome, and follicular mucinosis, the clinical significance of these findings is not yet clear. In the case of primary cutaneous B-cell lymphoma and its benign counterpart, further investigation will be B-cell pseudolymphoma,
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