- Email: [email protected]
Immunophenotypic and genotypic analysis in cutaneous lymphoid hyperplasias
Immunophenotypic and genotypic analysis in cutaneous lymphoid hyperplasias Elisabeth Hammer, MD, Omar Sangueza, MD, Pan Suwanjindar, MD, Clifton R. White, Jr., MD, and Rita M. Braziel, MD Portland, Oregon Background: The clinical utility of immunophenotyping and Southern blot analysis in the evaluation of patients with cutaneous lymphoid hyperplasia (CLH) is controversial. Objective: Our purpose was to determine whether adjunctive immunophenotyping and Southern blot analysis are of diagnostic and prognostic value in patients with CLH. Methods: Immunophenotyping was performed on skin biopsy specimens from 26 patients with a routine histologic diagnosis of CLH. Southern blot analysis for immunoglobulin (Ig) gene rearrangements was done on 13 of 26 cases. Results: Twenty-four of26 patients had polyclonal CLH on immunophenotyping; 2 of26 had monoclonal lymphoma. Two of II patients with polyc1onal CLH studied by Southern blot analysis had clonal Ig gene rearrangements. In both, lymphoma developed within I to 6 years; comparison of CLH and malignant lymphoma demonstrated overlapping and different clonal bands. Two additional patients with polyclonal CLH developed lymphoma. No clonal gene rearrangements were detected in the CLH or lymphoma from one; the other was not studied. Conclusion: Immunophenotyping will identify some patients with lymphoma with nondiagnostic histologic features. Southern blot analysis will predict some patients with polyclonal CLH in whom malignant lymphoma will develop and who may benefit from definitive therapy. (J AM ACAD DERMATOL 1993;28:426-33.)
The accurate diagnosis of cutaneous lymphoid hyperplasia (CLH) or pseudolymphoma has important therapeutic and prognostic implications. However, the distinction between benign CLH and malignant lymphoma (ML) may be difficult based only on clinical features and routine histopathologic evaluation.l'> With the advent of antibody immunophenotyping, additional information has become available to define cell lineage and to establish more reliably the biologically benign or malignant nature of a particular lymphoid infiltrate. In particular, the demonstration of monoclonal immunoglobulin (Ig) light chain expression in B cells, or aberrant antigen expression in B or T cells is helpful in the diagnosis of ML,6-19 The availability of gene rearrangement analysis has also provided a powerful diagnostic tool From the Department of Pathology, Oregon Health Sciences University. Accepted for publication Sept. 8, 1992. Reprint requests: Rita M. Braziel, MD, Department of Pathology, UHS-19, Oregon Health Sciences University, 3181 Sam Jackson Park Rd., Portland, OR 97201. Copyright @ 1993 by the American Academy of Dermatology, Inc. 0190-9622/93 $ 1.00 + .10
426
16/1/42403
that allows detection of clonal populations that comprise only I% to 5% of all cells. Histologically benign and immunologically polyclonal CLH sometimes contains clonal B cells on genotypic analysis.20-23 We correlated the clinical course of 26 patients with a histologic diagnosis of CLH with results of frozen and/or paraffin section immunophenotyping and, if sufficient tissue, with results of Southern blot analysis for clonal Ig heavy chain gene rearrangements to determine how often these adjunctive studies provide information of value in patient care (i.e., whether they are helpful in diagnosis and in predicting those patients with CLH in whom ML will eventually develop). PATIENTS, MATERIAL, AND METHODS Patients Our study was performed on skin biopsy specimens from 26 patients, ranging in age from 25 to 76 years, with cutaneous lesions that were clinically and histologically consistent with CLH. All patients had plaques, nodules, or tumors on different areas of the skin. None of the patients had signs or symptoms of systemic involvement at
Journal of the American Academy of Dermatology Volume 28, Number 3
Hammer et al. 427
Table I. Antibodies, specificities, and sources Antibody
CD
Name
Frozen-section IP (Leu-5) CD2 (Leu-4) CD3 (Leu-3) CD4 (Leu-I) CD5 (Leu-9) CD? (Leu-2) CD8 CD19 (Leu-l 2) (Leu-16) CD20 Paraffin-section IP (LCA) CD45 CD3 (CD3) CD45RO (UCHL-l) CD20 (L-26) (LN-l) CD?5 Frozen and paraffin section IP IgG IgM IgD IgA K
A
Specificity"
Source
T cells, E-rosette receptor T cells, T-cell receptor T helper cells Pan-T cells Pan-T cells T suppressor cells Pan-B cells Pan-B cells
BD* BD BD BD BD BD BD BD
Leukocyte common antigen T cells, T-cell receptor T cells B cells B cells
Dako Dako Dako ICNt
Ig Ig Ig Ig Ig Ig
Dako Dako Dako Dako DakojBD DakojBD
heavy chain on B cells heavy chain on B cells heavy chain on B cells heavy chain on B cells light chain on B cells light chain on B cells
Dako]
CD, Cluster of differentiation; JP, immunoperoxidasestaining. "Becton Dickinson, Sunnyview,Calif. tCarpentaria, Calif. *Costa Mesa, Calif.
the time of initial presentation and systemic laboratory studies were normal in all patients. The follow-up period ranged from I to 6 years.
Biopsy specimens AII biopsy specimens had a superficial, or a superficial and deep, dense lymphoid infiltrate that spared the epidermis. In a few cases, lymphoid follicles with prominent germinal centers were seen. The cellular composition of the infiltrate was variable, with a predominance of small lymphocytes admixed with a variable number of plasma cells, eosinophils, and histiocytes. The specimens were processed as follows: 6 mm punches or elliptical specimens were bisected; half was fixed in B-5 and paraffin embedded for routine histology and the remaining half was snap-frozen for immunologic and genotypic analysis.
Immunologic analysis Immunologic analysis was performed on frozen and in some cases on paraffin-embedded sections with a standard avidin-biotin complex (ABC) immunoperoxidase technique.e'- 25 Primary antibody binding was detected by the Vector ABC Elite kit. The antibodies used, sources,
and specificities are shown in Table 1. Although the antibody panel varied slightly from case to case, every specimen was studied with antibodies directed against pan-B ceIl antigens, pan-T cell antigens, and Ig light chains.
Genotypic analysis Southern blot analysis for Ig gene rearrangement was performed on DNA extracted from frozen sections by a standard method/" Clonal bands were detected by auto-radiography. Clonal gene rearrangements were considered to be present only when at least two clonal bands were seen, either on the same restriction enzyme digest or on different enzyme digests. RESULTS
Clinical course During the follow-up period of 1 to 6 years, B-cell lymphomas developed in four patients, 9 months to 4 years after the initial biopsy. The histologic subtypes included one diffuse, large cell, one mixed small cleaved and large cell, and two follicular small cleaved cell ML. Both patients with small cleaved cell lymphomas had systemic involvement. In the
428 Hammer et al.
Journal of the American Academy of Dermatology March 1993
Fig. 1. Typical histologic features of CLH. A, This low-power view shows superficial and deep, dense lymphoid infiltrate that spares epidermis. B, At higher power, the infiltrate is predominantly composed of small lymphocytes, admixed with a few larger lymphoid cells and histiocytes, CA, X12; B,: X3000.)
Fig. 2. Malignant lymphoma (ML). This large cell ML (case 1) is characterized by large atypical lymphoid cells with vesicular nuclei and prominent nucleoli. Occasional mitotic figures are present. (X3000.)
other two the malignant process has remained confined to the skin. Representative photomicrographs of a typical CLH biopsy specimen and of an ML are shown in Figs. 1 and 2, respectively. Details of the clinical findings of patients in whom ML developed are presented in Table II.
Immunologic results The skin specimens from 26 patients were successfully immunophenotyped. The initial specimens from 24 patients, including the four patients in whom an ML subsequently developed, showed a reactive, nonneoplastic pattern of antibody staining: T
Journal of the American Academy of Dermatology Volume 28, Number 3
Hammer et al. 429
Table II. Clinical course of patients with CLH and progression to ML Case No.: Bx
Clinical course
M
51 53 55
2
F
48 49 50
3
F
49
51
4
F
69 70
1987: Periumbilical nodule, 3-4 cm,
related to tattoo 1989: Same lesion enlarged to 6 cm; resolved with topical steroid tx 1990/1991: Recurrence at same site and subsequent development of lymphomatous nodules at other sites;excised 1987: Parasternal and L eyebrow nodules; excised 1988: Nodule R orbit, significantly fluctuating in size; excised 1989: Inguinal lymphadenopathy; systemic chcmotx; in remission 1989: Violaceous lump, 3.5 X 2.0 ern, then plaque with substantial alopecia on mid scalp. Partial resolution with steroid injection 1991: Same nodule significantly increased in size with progressive alopecia; excised 1990: Urticaria-like lesion on face and neck; topical steroid tx; no response 1991: Lesions on face and neck, cervical lymphadenopathy and BM involvement
Histologic diagnosis
CLH CLH ML,D,LC (skin) CLH CLH ML,F,SC (LN) CLH
ML, F,MCT (skin) CLH ML,F,SC (LN and BM)
BM, Bone marrow; Bx, biopsy; ML, D, LC, malignant lymphoma, diffuse, large cell; ML, F, MeT, malignant lymphoma, follicular, mixed cell type;
ML, F, SC, malignant lymphoma, follicular, small cleaved cell; tx, therapy.
lymphoid cells predominated with a variable helper/ suppressor (CD4/CD8) ratio, B cells tended to occur in clusters, even when no suggestion of germinal center formation was seen on routine sections, and the B cells were polyclonal with the K and A. light chain antibodies. In the remaining two patients, a monoclonal B-cell population was detected in the original CLH specimen by immunologic analysis, although the histologic findings were similar to the specimens from patients with polydonal CLH. One patient had a monoclonal plasma cell population confined to the skin. Retrospective immunologic analysis of two previous, histologically benign CLH lesions from this same patient, taken 2 years earlier, also revealed monoclonal plasma cells, but there was discordant light chain expression in the simultaneous specimens from different sites. Although histologically almost identical, the plasma cells were x-positive in one lesion and X-positive in the other, a result confirmed
by multiple repeat stainings. Germinalcenters within the infiltrate were polyclonal. In the second case, which was believed to be malignant at the time of the initial biopsy, the immunophenotypic results were equivocal, but a larger repeat biopsy specimen of the same lesion showed a surface Ig-negative follicular large cell lymphoma. In retrospect, the initial biopsy specimen had been taken from the edge of the lesion, where there were sufficient reactive T lymphoid cells to obscure the infrequent, small collections ofsurface Ig-negative B cells.
Genotypic results Of the 24 patients with polyclonal CLH on immunologic analysis of the original biopsy specimen, tissue for gene rearrangements studies was available in II cases, including 3 of 4 patients in whom lymphoma developed. Clonal gene rearrangements were detected in two patients with polyc1onal CLH, in
J ournal o f the American Academy of Dermatology March 1993
430 Hammer et at.
had a surface Ig-negative follicular, large celllymphoma. Faint clonal bands were detected with the JH probe in both the EeaRI and BamHI/HindIII digests (not shown).
Table III. Results of immunophenotypic analysis Genotype Case No.: Ox
JH
I
JH B-H
Immunophenotype
HenRI
CLH ML
Polyclonal Monoclonal, IgG, "A
R R
R R
CLH ML
Polyclonal Monoclonal, IgG,
R R
R R
CLH ML
Polyclonal Monoclonal, A
G G
G G
CLH ML
Polyclonal Monoclonal, K
/
I
2 K
3
4
t. Insufficient DNA available
for gene rearrangement studies; B-H. BamHI/HilldlII restriction enzyme double digest; Dx, diagnosis; G. gerrnline; JH, DNA probe for the joining region of the Ig heavy chain gene; R, rearranged.
both of whom B-celilymphomas subsequently developed (cases I and 2 in Tables II and III). Comparative genotypic analysis of the earlier, immunologically polyclonal and histologically benign skin lesions and the malignant B-cell lymphomas was carried out. In case 1, a patient in whom multiple large cell lymphomas confined to skin developed, clonal B-cell populations were detected at varying intensities with the JH probe in both specimens. There were some clonal bands that were present in DNA from both specimens, and some bands that were different (Fig. 3, lanes A and B). In the second case, a patient in whom systemic involvement by a follicular small cleaved cell lymphoma developed, comparative genotypic analysis of the CLH and the ML again showed overlapping and different clonal JH bands, similar to the previous case (Fig. 3, lanes C and D). Of the remaining two patients with polyclonal CLH in whom ML developed (cases 3 and 4), gene rearrangement analysis was performed on one (case 3) and no clonal bands were detected in either the CLH or ML biopsy specimens. In the other patient (case 4), no tissue was available for Southern blot analysis. Table III shows detailed genotypic and immunophenotyping results of the cases with progression to a B-cell malignancy. Of the two patients with monoclonal populations of their initial CLH, we were able to perform gene rearrangements studies in the case of the patient who
DISCUSSION
The many terms historically applied to CLH (lymphadenosis benigna cutis, lymphocytoma cutis, and Spiegler-Fendt sarcoid, to name a few2, 4, 27) reflect the morphologic diversity of this lesion as well . as the limitations of routine microscopy in the prediction of its biologic behavior. The application of new techniques to diagnostic evaluation has made it clear that the distinction between benign and malignant lymphoid infiltrates in the skin is often impossible on a histologic basis alone. Although the information gained from the use of these special techniques is of interest to researchers, it is reasonable to question whether the information provided is of sufficient clinical value to justify its cost to the patient. More specifically, is the clinical course of patients who are found to have monoclonal B-cell populations by immunophenotypic or genotypic analysis different from that of patients with no detectable monoclonality? Our results, correlating the clinical course of patients with a histologicdiagnosis of CLH with results of immunophenotyping and genotyping studies, suggest that this is the case. In all but two of our patients with CLH, immunologic analysis showed the characteristic findings of a reactive lymphoid infiltrate or pseudolymphoma, that is, a predominance of T cells with a variable CD4/CD8 ratio and small clusters of polyclonal B cells with or without germinal center formation." 22 Two patients had B-cell lymphomas , rather than CLH, on immunologic analysis of their initial CLH biopsy specimens, again highlighting the failure of histologic criteria alone to differentiate reliably benign from malignant lymphoid skin infiltrates. Both these patients had disease confined to the skin, both received radiation therapy to the lesions, and both are free of disease at I and 2 years after therapy. Four patients with histologically benign and immunologically polyclonal CLH had overt B-cell lymphomas within 1to 4 years oftheir original CLH biopsy. Only one of these patients had what was believedto be a relatively insignificant lesion,a solitary, mixed, small cleaved, and large cell lymphoma confined to skin. The other three patients had more clinically aggressive disease. One had multiple, widespread, diffuse, large cell lymphomas confined
Journal of the American Academy of Dermatology Volume 28, Number 3
PL
A
PL
B
A
Hammer et at. 431
•
24 -
::0
PL
B
C
D
PL
C
D
24 -
=
!lll- _
•
0.0- .
-
• R1 BAM HI +HINDIII .....- -ECO------
ECO -R1
BAM HI + HIND III
Fig. 3. Southern blot analysis (SBA) of cutaneouslymphoidhyperplasia (CLH) and malignant lymphoma (ML). Autoradiograph compares genotypic results of initial CLH and subsequent ML from twopatients (cases 1 and 2). SBA for Ig gene rearrangement was performed on DNA extracted from frozen tissue, digested with the restriction endonucleases EeoRI and BamHI/HindIII, sizefractionedby electrophoresis, transferred to a nylon membrane (Oncor), then hybridizedwith32P-Iabeled probefor joiningregion ofthe Ig heavychain gene (JH). Lanes labeled"PL" representthe placental control, lane A the CLH and lane B the ML of the ftrst patient (case 1); similarly, lanes C and D represent CLH and ML, respectively, ofthe secondpatient (case 2). Sizesofnonrearrangedgermlinebands are indicated in numbers, measured in kilobases. Both casesdemonstratesimilar results. There are several clonalbands (setsof corresponding arrows inadjacent lanesA-Band C-D), whichare present in both digestsof CLH and ML. An additional,unique band (solitary arrows) is seen in both cases of CLH, Bamiii]HindIII digests only, and these bands are absent in the ML.
to skin, and the other two had systemic involvement with follicular, small cleaved cell lymphoma. A comparative genotypic analysis ofthe previous CLH biopsy specimen and the ML in two of these four patients identified clonal rearrangements of the Ig gene not only in the lymphomas, but also in the immunologically polyclonal CLH specimens. Of particular interest was the pattern of clonal bands seen in these cases, with some identical and some different bands. These results suggest that the development of overt lymphoma in these cases is not just from the slow accumulation of a clonal B-cell population with eventual overgrowth and obliteration of reactive cells and development ofovert immunologic monoclonality, but that some other genetic change or clonal evolution has taken place. This evolution was accompanied in both patients with the development of more aggressive disease. The presence of "transient" clonal bands, detected in the polyclonal CLH but not in the malignant lymphomas of these two patients, lends support to the hypothesis that these patients had an abnormal immune response that led to the development of multiple B-cell clones, one or more of which eventu-
ally escaped the control of the patient's immunoregulatory mechanisms. Alternatively, different clonal bands could also be explained by additional alterations of DNA sequences by somatic mutations of the Ig genes 28 or gene deletion and subsequent development of additional clones. The finding of clonal bands on Southern blot analysis of CLH has been reported before. 2o , 23 Wood et a1.20 performed a study similar to ours, but with much shorter follow-up in many of the cases. They found clonal bands in 5 of 14 cases with histologically diagnosed, immunologically polyclonal CLH. One of their patients also had ML, 11 years after the initial CLH, with identical clonal JH bands in both the CLH and the ML. The detection of clonal bands in histologically benign, immunologically polydonal lymphoid proliferations has also been reported in other extranodal sites, particularly the ocular adnexa and salivary glands. In these sites, the percentage of immunologically polyclonal lymphoid proliferations containing clonal B-cell populations on genotypic analysis is almost 100%. This percentage is clearly much higher than that in ours or other pu blished series of CLH. The reason for this
432
Hammer et al.
difference is not clear. It is possible that there are true biologic differences in the cutaneous lymphoid infiltrates, but the difference could also be explained by simple differences in lymphoid cell density or other technical considerations. Histologically benign, immunologically polyc1onal lymphoid infiltrates from other extranodal sites also have an increased incidence of subsequent progression to ML. This progression is particularly well documented in orbital pseudolymphornas-v '! and lymphoepithelial lesions from patients with Sjogren's syndrome and Mikulicz's disease. 32- 38 In our study, the findings of discordant light chain phenotypes in simultaneous lymphoma biopsy specimens and "transient" clonal bands in polyclonal CLH support the concept that these patients initially have an abnormal immune state that leads to the presence of multiple B-cell clones, with development of overt, sometimes synchronous ML after additional transformations or clonal evolution. The clinical significance of the finding of a clonal Ig gene rearrangement in a histologically benign and immunologically polyclonal CLH and, therefore, the clinical utility of genotypic analysis in CLH will continue to be debated until sufficient numbers of patients with CLH with and without clonal gene rearrangement have been followed up for sufficient time to determine the actual frequency of development of ML. Until these longitudinal studies are completed to establish the actual risk of lymphoma, it would be premature to label these patients with a diagnosis of occult lymphoma. At the present time, they are not considered to have ML until they have developed an immunologically monoclonal B-ce1l population. It is also premature to suggest that all patients with polyclonal CLH should have genotypic analysis, but it would certainly be reasonable to perform the test in patients with multiple or recurrent lesions. The association of clonal Ig gene rearrangements with progression to lymphoma in our study does provide some support for the concept that gene rearrangement analysis may provide a way to identify those patients with CLH who are at highest risk for the development of lymphoma, that is, a means of selecting those patients with CLH who are most likely to benefit from early therapeutic intervention, most commonly local irradiation. Although the natural history of most low-grade B-cel1 lymphomas involving only extranodal sites is to remain localized for prolonged periods, the potential for development
Journal of the American Academy of Dermatology March 1993
of systemic and incurable disease, as seen in two of our patients, clearly exists. REFERENCES 1. Rijlaarsdarn lU, Meijer C.JL, Willemze R. Differentiation between lymphadenosis benigna cutis and primary cutaneous follicular center cell lymphoma. Cancer 1990; 65:2301-6. 2. Evans HL, Winkelmann RK, Banks PM. Differential diagnosis of malignant and benign cutaneous lymphoid infiltrates. Cancer 1979;44:699-717. 3. Lange Wantzin G, Hou-Jensen K, Nielson M, et al. Cutaneous lymphocytomas: clinical and histological aspects. Acta Derm Venereol (Stockh) 1982;62:119-24. 4. Caw W A, Helwig EB. Cutaneous lymphoid hyperplasia. Cancer 1969;24:487-502. 5. MacDonald DM. Histopathological differentiation of benign and malignant cutaneous lymphocytic infiltrates. Br J Dermatol 1982;107:715-8. 6. Knowles DM, Halper JP, Jacobiec FA. The immunologic characterization of 40 extranodal lymphoid infiltrates. Cancer 1982;49:2321-35. 7. Wirt DP, Grogan TM, Jolley CS, et al. Theimmunoarchitecture of cutaneous pseudolyrnphoma, Hum Pathol 1985; 6:492-510. 8. Garcia CF, Weiss LM, Warnke RA, et al. Cutaneous follicular lymphoma. Am J Surg Pat hoi 1986; 10:454-63. 9. Ralflciaer E, Saati TA, Bosq J, et al. Immunocytochemical characterization of cutaneous lymphomas other than mycosis fungoides. J Clin Pathol 1986;39:553-63. 10. WiJlemze R, deGraalT-Reitsma CB, Cnossen J, et al, Characterization of T-cell subpopulations in skin and peripheral blood of patients with cutaneous T-ceJllymphomas and benign inflammatory dermatoses. J Invest Dermatol 1983;80:60-6. 11. Payne CM, Grogan TM, Lynch PJ. An ultrastructural morphometric and immunohistochemical analysis of cutaneous lymphomas and benign lymphocytic infiltrates of skin. Arch Dermatol 1986;122: 1139-54. 12. Medeiros LJ, Picker LJ, Abel EA, et al. Cutaneous lymphoid hyperplasia. J AM ACAD DERMATOL 1989; 21:929-42. 13. WiJlemze R, Vermeer BJ, Meijer CJLM. Immunohistochemical studies in lymphocytic infiltration of the skin (Jessner) and discoid lupus erythematosus. J AM ACAD DERMATOL 1984;11:832-40. 14. Rallkiaer E, Lange Wantzin G, Mason DY, et al. Characterization of benign cutaneous lymphocytic infiltrates by monoclonal antibodies. Br J Dermatol 1984; III :635-45. 15. Weiss LM, Crabtree GS, Rouse RV, et al. Morphologic and immunologic characterization of 50 peripheral T-cell lymphomas. Am J Pathol 1985;118:316-24. 16. Picker LJ, Weiss LM, Medeiros LJ, et al. Irnmunophenotypic criteria for the diagnosis of non-Hodgkin's lymphoma. Am J PathoI1987;128:181-201. 17. Wood GS, Abel EA, Hoppe RT, et al. Leu-8 and Leu-9 antigen phenotypes: immunologic criteria for the distinction of mycosis fungoides from cutaneous inflammation. J AM ACAD DERMATOL 1986;14:1006-13. 18. Wood GS, Burke IS, Horning S, et al, The immunologic and clinicopathologic heterogeneity of cutaneous lymphomas other than mycosis fungoides. Blood 1983;62:464-72. 19. Warnke RA, Rouse RV. Limitations encountered in the application of tissue section immunodiagnosis to the study
Journal of the American Academy of Dermatology Volume 28, Number 3
20.
21. 22. 23. 24. 25.
26. 27. 28.
29.
of lymphomas and related disorders. Hum Pathol 1985; 16:326-31. Wood GS, Ngan BY, Ting R, et al. Clonal rearrangements of immunoglobulin genes and progression to B-cell lymphoma in cutaneous lymphoid hyperplasia. Am J Pathol 1989;135:13-9. Bignon YJ, Souteyrand P, Roger H, et al. Dual genotype in cutaneous T-cell lymphomas and pseudolymphomas, Hum Genet 1990;84:177-8. WechslerJ, BagotM, HenniT, et aJ.Cutaneous pseudolymphomas: immunophenotypical and immunogenotypical studies. Curr Probl Dermatol 1990;19:183-8. Griesser H, Feller AC, Sterry W. T-cell receptor and immunoglobulin gene rearrangements in cutaneous T-cellrich pseudolymphomas. J Invest Dermatol 1990;95:292-5. Bindl J, Warnke RA. Advantage of detecting monoclonal antibody binding to tissue sections with biotin and avidin agents in Coplin jars. Am J Clin Pathol 1986;85:490-3. Cartun RW, Redersen CA. An immunocytochemicaltechnique offeringincreased sensitivityand loweredcost with a streptavidinhorseradish peroxidaseconjugate. J Histotechnol 1989;12:273-5. Southern EM. Detection ofspecific sequencesamong DNA fragments separated by gel electrophoresis. J Mol Bioi 1975;98:503-17. Ackerman AB, ed. Histologic diagnosis of inflammatory skin diseases. Philadelphia: Lea & Febiger, 1978:215-7, 442-7,494-5. Cleary ML, Meeker TC, LevyS, et al. Clusteringof extensivesomatic mutations in the variable region of an immunoglobulin heavy chain gene from a human B-cell lymphoma. Cell 1986;44:97-106. Chen P, Liu JH, Lin SH, et al. Rearrangement of immu-
Hammer et al. 433
30.
31.
32. 33.
34. 35. 36. 37.
38.
noglobulin gene and oncogenes in ocular adnexal pseudolyrnphomas. Curr Eye Res 1991;10:547-55. Neri A, lakobiec FA, Pelicci P-G, et a1. Immunoglobulin and T-cellreceptor iJ-chain gene rearrangement analysis of ocular adnexal lymphoid neoplasms: clinical and biologic implications. Blood 1987;70:1519-29. KnowlesOM, Athan E, Ubriaco A, et al. Extranodal noncutaneous lymphoid hyperplasias represent a continuous spectrum of B-cell neoplasia: demonstration by molecular genetic analysis. Blood 1989;73:1635-45. FishlederA, Tubbs R, Hesse B, et al. Uniform detection of immunoglobulingene rearrangement in benign lyrnphoepithelial lesions. N Engl J Moo 1987;316:1118-21. Falzon M, Isaacson PG. The natural history of benign lymphoepitheliallesion of the salivary gland in which there is a monoclonal population of B cells. Am J Surg Pathol 1991;15:58-65. Zulman 1, laffe R, Talal N. Evidence that the malignant lymphoma of Sjogren's syndrome is a monoclonal B-ceJl neoplasm. N Engl J Med 1978;299:1215-20. Bridges AJ, England MD. Benign lympboepithelial lesion: relationshipto Sjogren's syndrome and evolving malignant lymphoma. Semin Arthritis Rheum 1989;19:201-8. Azzopardi lG, Evans DJ. Malignant lymphoma of parotid associated with Mikulicz disease (benign lymphoepithelial lesion). J Clin Pathol 1971;24:744-52. Hyman GA, Wolff M. Malignant lymphomas of the salivary glands: reviewof the literature and report of 33 new cases, including four cases associated with the lyrnphoepithclial lesion. Am J Clin Pathol 1976;65:421-38. Colby TV, Dorfman RF. Malignant lymphomas involving the salivary glands. Pathol Annu 1979;l4(part 2):307-24.
BOUND VOLUMES AVAILABLE TO SUBSCRIBERS
Bound volumes oftheJOURNAL or:THE AMERICAN ACADEMY or DERMATOLOGY areavailable tosubscribers (only) for the 1993 issues from the Publisher at a costof$69.00 fordomestic, $91.83 forCanadian, and$87.00 for international forvolume 28(January-June) andvolume 29 (July-December). Shipping charges are included. Each bound volume contains a subject and author index and all advertising is removed. Copies are shipped within 60 days after publication of thelast issue in the volume. The binding is durable buckram with the journal name, volume number, and year stamped in gold on thespine. Payment III/1St accompany all orders. ContactMosby, Subscription Services, 11830 Westline Industrial n-, SI. Louis, MO 63146-3318. USA: phone (800) 325-4177, ext. 4351; (314) 453-4351. Subscriptions must be in force to qualify. Bound volumes are not available ill place of' a regular journal subscription.
The accurate diagnosis of cutaneous lymphoid hyperplasia (CLH) or pseudolymphoma has important therapeutic and prognostic implications. However, the distinction between benign CLH and malignant lymphoma (ML) may be difficult based only on clinical features and routine histopathologic evaluation.l'> With the advent of antibody immunophenotyping, additional information has become available to define cell lineage and to establish more reliably the biologically benign or malignant nature of a particular lymphoid infiltrate. In particular, the demonstration of monoclonal immunoglobulin (Ig) light chain expression in B cells, or aberrant antigen expression in B or T cells is helpful in the diagnosis of ML,6-19 The availability of gene rearrangement analysis has also provided a powerful diagnostic tool From the Department of Pathology, Oregon Health Sciences University. Accepted for publication Sept. 8, 1992. Reprint requests: Rita M. Braziel, MD, Department of Pathology, UHS-19, Oregon Health Sciences University, 3181 Sam Jackson Park Rd., Portland, OR 97201. Copyright @ 1993 by the American Academy of Dermatology, Inc. 0190-9622/93 $ 1.00 + .10
426
16/1/42403
that allows detection of clonal populations that comprise only I% to 5% of all cells. Histologically benign and immunologically polyclonal CLH sometimes contains clonal B cells on genotypic analysis.20-23 We correlated the clinical course of 26 patients with a histologic diagnosis of CLH with results of frozen and/or paraffin section immunophenotyping and, if sufficient tissue, with results of Southern blot analysis for clonal Ig heavy chain gene rearrangements to determine how often these adjunctive studies provide information of value in patient care (i.e., whether they are helpful in diagnosis and in predicting those patients with CLH in whom ML will eventually develop). PATIENTS, MATERIAL, AND METHODS Patients Our study was performed on skin biopsy specimens from 26 patients, ranging in age from 25 to 76 years, with cutaneous lesions that were clinically and histologically consistent with CLH. All patients had plaques, nodules, or tumors on different areas of the skin. None of the patients had signs or symptoms of systemic involvement at
Journal of the American Academy of Dermatology Volume 28, Number 3
Hammer et al. 427
Table I. Antibodies, specificities, and sources Antibody
CD
Name
Frozen-section IP (Leu-5) CD2 (Leu-4) CD3 (Leu-3) CD4 (Leu-I) CD5 (Leu-9) CD? (Leu-2) CD8 CD19 (Leu-l 2) (Leu-16) CD20 Paraffin-section IP (LCA) CD45 CD3 (CD3) CD45RO (UCHL-l) CD20 (L-26) (LN-l) CD?5 Frozen and paraffin section IP IgG IgM IgD IgA K
A
Specificity"
Source
T cells, E-rosette receptor T cells, T-cell receptor T helper cells Pan-T cells Pan-T cells T suppressor cells Pan-B cells Pan-B cells
BD* BD BD BD BD BD BD BD
Leukocyte common antigen T cells, T-cell receptor T cells B cells B cells
Dako Dako Dako ICNt
Ig Ig Ig Ig Ig Ig
Dako Dako Dako Dako DakojBD DakojBD
heavy chain on B cells heavy chain on B cells heavy chain on B cells heavy chain on B cells light chain on B cells light chain on B cells
Dako]
CD, Cluster of differentiation; JP, immunoperoxidasestaining. "Becton Dickinson, Sunnyview,Calif. tCarpentaria, Calif. *Costa Mesa, Calif.
the time of initial presentation and systemic laboratory studies were normal in all patients. The follow-up period ranged from I to 6 years.
Biopsy specimens AII biopsy specimens had a superficial, or a superficial and deep, dense lymphoid infiltrate that spared the epidermis. In a few cases, lymphoid follicles with prominent germinal centers were seen. The cellular composition of the infiltrate was variable, with a predominance of small lymphocytes admixed with a variable number of plasma cells, eosinophils, and histiocytes. The specimens were processed as follows: 6 mm punches or elliptical specimens were bisected; half was fixed in B-5 and paraffin embedded for routine histology and the remaining half was snap-frozen for immunologic and genotypic analysis.
Immunologic analysis Immunologic analysis was performed on frozen and in some cases on paraffin-embedded sections with a standard avidin-biotin complex (ABC) immunoperoxidase technique.e'- 25 Primary antibody binding was detected by the Vector ABC Elite kit. The antibodies used, sources,
and specificities are shown in Table 1. Although the antibody panel varied slightly from case to case, every specimen was studied with antibodies directed against pan-B ceIl antigens, pan-T cell antigens, and Ig light chains.
Genotypic analysis Southern blot analysis for Ig gene rearrangement was performed on DNA extracted from frozen sections by a standard method/" Clonal bands were detected by auto-radiography. Clonal gene rearrangements were considered to be present only when at least two clonal bands were seen, either on the same restriction enzyme digest or on different enzyme digests. RESULTS
Clinical course During the follow-up period of 1 to 6 years, B-cell lymphomas developed in four patients, 9 months to 4 years after the initial biopsy. The histologic subtypes included one diffuse, large cell, one mixed small cleaved and large cell, and two follicular small cleaved cell ML. Both patients with small cleaved cell lymphomas had systemic involvement. In the
428 Hammer et al.
Journal of the American Academy of Dermatology March 1993
Fig. 1. Typical histologic features of CLH. A, This low-power view shows superficial and deep, dense lymphoid infiltrate that spares epidermis. B, At higher power, the infiltrate is predominantly composed of small lymphocytes, admixed with a few larger lymphoid cells and histiocytes, CA, X12; B,: X3000.)
Fig. 2. Malignant lymphoma (ML). This large cell ML (case 1) is characterized by large atypical lymphoid cells with vesicular nuclei and prominent nucleoli. Occasional mitotic figures are present. (X3000.)
other two the malignant process has remained confined to the skin. Representative photomicrographs of a typical CLH biopsy specimen and of an ML are shown in Figs. 1 and 2, respectively. Details of the clinical findings of patients in whom ML developed are presented in Table II.
Immunologic results The skin specimens from 26 patients were successfully immunophenotyped. The initial specimens from 24 patients, including the four patients in whom an ML subsequently developed, showed a reactive, nonneoplastic pattern of antibody staining: T
Journal of the American Academy of Dermatology Volume 28, Number 3
Hammer et al. 429
Table II. Clinical course of patients with CLH and progression to ML Case No.: Bx
Clinical course
M
51 53 55
2
F
48 49 50
3
F
49
51
4
F
69 70
1987: Periumbilical nodule, 3-4 cm,
related to tattoo 1989: Same lesion enlarged to 6 cm; resolved with topical steroid tx 1990/1991: Recurrence at same site and subsequent development of lymphomatous nodules at other sites;excised 1987: Parasternal and L eyebrow nodules; excised 1988: Nodule R orbit, significantly fluctuating in size; excised 1989: Inguinal lymphadenopathy; systemic chcmotx; in remission 1989: Violaceous lump, 3.5 X 2.0 ern, then plaque with substantial alopecia on mid scalp. Partial resolution with steroid injection 1991: Same nodule significantly increased in size with progressive alopecia; excised 1990: Urticaria-like lesion on face and neck; topical steroid tx; no response 1991: Lesions on face and neck, cervical lymphadenopathy and BM involvement
Histologic diagnosis
CLH CLH ML,D,LC (skin) CLH CLH ML,F,SC (LN) CLH
ML, F,MCT (skin) CLH ML,F,SC (LN and BM)
BM, Bone marrow; Bx, biopsy; ML, D, LC, malignant lymphoma, diffuse, large cell; ML, F, MeT, malignant lymphoma, follicular, mixed cell type;
ML, F, SC, malignant lymphoma, follicular, small cleaved cell; tx, therapy.
lymphoid cells predominated with a variable helper/ suppressor (CD4/CD8) ratio, B cells tended to occur in clusters, even when no suggestion of germinal center formation was seen on routine sections, and the B cells were polyclonal with the K and A. light chain antibodies. In the remaining two patients, a monoclonal B-cell population was detected in the original CLH specimen by immunologic analysis, although the histologic findings were similar to the specimens from patients with polydonal CLH. One patient had a monoclonal plasma cell population confined to the skin. Retrospective immunologic analysis of two previous, histologically benign CLH lesions from this same patient, taken 2 years earlier, also revealed monoclonal plasma cells, but there was discordant light chain expression in the simultaneous specimens from different sites. Although histologically almost identical, the plasma cells were x-positive in one lesion and X-positive in the other, a result confirmed
by multiple repeat stainings. Germinalcenters within the infiltrate were polyclonal. In the second case, which was believed to be malignant at the time of the initial biopsy, the immunophenotypic results were equivocal, but a larger repeat biopsy specimen of the same lesion showed a surface Ig-negative follicular large cell lymphoma. In retrospect, the initial biopsy specimen had been taken from the edge of the lesion, where there were sufficient reactive T lymphoid cells to obscure the infrequent, small collections ofsurface Ig-negative B cells.
Genotypic results Of the 24 patients with polyclonal CLH on immunologic analysis of the original biopsy specimen, tissue for gene rearrangements studies was available in II cases, including 3 of 4 patients in whom lymphoma developed. Clonal gene rearrangements were detected in two patients with polyc1onal CLH, in
J ournal o f the American Academy of Dermatology March 1993
430 Hammer et at.
had a surface Ig-negative follicular, large celllymphoma. Faint clonal bands were detected with the JH probe in both the EeaRI and BamHI/HindIII digests (not shown).
Table III. Results of immunophenotypic analysis Genotype Case No.: Ox
JH
I
JH B-H
Immunophenotype
HenRI
CLH ML
Polyclonal Monoclonal, IgG, "A
R R
R R
CLH ML
Polyclonal Monoclonal, IgG,
R R
R R
CLH ML
Polyclonal Monoclonal, A
G G
G G
CLH ML
Polyclonal Monoclonal, K
/
I
2 K
3
4
t. Insufficient DNA available
for gene rearrangement studies; B-H. BamHI/HilldlII restriction enzyme double digest; Dx, diagnosis; G. gerrnline; JH, DNA probe for the joining region of the Ig heavy chain gene; R, rearranged.
both of whom B-celilymphomas subsequently developed (cases I and 2 in Tables II and III). Comparative genotypic analysis of the earlier, immunologically polyclonal and histologically benign skin lesions and the malignant B-cell lymphomas was carried out. In case 1, a patient in whom multiple large cell lymphomas confined to skin developed, clonal B-cell populations were detected at varying intensities with the JH probe in both specimens. There were some clonal bands that were present in DNA from both specimens, and some bands that were different (Fig. 3, lanes A and B). In the second case, a patient in whom systemic involvement by a follicular small cleaved cell lymphoma developed, comparative genotypic analysis of the CLH and the ML again showed overlapping and different clonal JH bands, similar to the previous case (Fig. 3, lanes C and D). Of the remaining two patients with polyclonal CLH in whom ML developed (cases 3 and 4), gene rearrangement analysis was performed on one (case 3) and no clonal bands were detected in either the CLH or ML biopsy specimens. In the other patient (case 4), no tissue was available for Southern blot analysis. Table III shows detailed genotypic and immunophenotyping results of the cases with progression to a B-cell malignancy. Of the two patients with monoclonal populations of their initial CLH, we were able to perform gene rearrangements studies in the case of the patient who
DISCUSSION
The many terms historically applied to CLH (lymphadenosis benigna cutis, lymphocytoma cutis, and Spiegler-Fendt sarcoid, to name a few2, 4, 27) reflect the morphologic diversity of this lesion as well . as the limitations of routine microscopy in the prediction of its biologic behavior. The application of new techniques to diagnostic evaluation has made it clear that the distinction between benign and malignant lymphoid infiltrates in the skin is often impossible on a histologic basis alone. Although the information gained from the use of these special techniques is of interest to researchers, it is reasonable to question whether the information provided is of sufficient clinical value to justify its cost to the patient. More specifically, is the clinical course of patients who are found to have monoclonal B-cell populations by immunophenotypic or genotypic analysis different from that of patients with no detectable monoclonality? Our results, correlating the clinical course of patients with a histologicdiagnosis of CLH with results of immunophenotyping and genotyping studies, suggest that this is the case. In all but two of our patients with CLH, immunologic analysis showed the characteristic findings of a reactive lymphoid infiltrate or pseudolymphoma, that is, a predominance of T cells with a variable CD4/CD8 ratio and small clusters of polyclonal B cells with or without germinal center formation." 22 Two patients had B-cell lymphomas , rather than CLH, on immunologic analysis of their initial CLH biopsy specimens, again highlighting the failure of histologic criteria alone to differentiate reliably benign from malignant lymphoid skin infiltrates. Both these patients had disease confined to the skin, both received radiation therapy to the lesions, and both are free of disease at I and 2 years after therapy. Four patients with histologically benign and immunologically polyclonal CLH had overt B-cell lymphomas within 1to 4 years oftheir original CLH biopsy. Only one of these patients had what was believedto be a relatively insignificant lesion,a solitary, mixed, small cleaved, and large cell lymphoma confined to skin. The other three patients had more clinically aggressive disease. One had multiple, widespread, diffuse, large cell lymphomas confined
Journal of the American Academy of Dermatology Volume 28, Number 3
PL
A
PL
B
A
Hammer et at. 431
•
24 -
::0
PL
B
C
D
PL
C
D
24 -
=
!lll- _
•
0.0- .
-
• R1 BAM HI +HINDIII .....- -ECO------
ECO -R1
BAM HI + HIND III
Fig. 3. Southern blot analysis (SBA) of cutaneouslymphoidhyperplasia (CLH) and malignant lymphoma (ML). Autoradiograph compares genotypic results of initial CLH and subsequent ML from twopatients (cases 1 and 2). SBA for Ig gene rearrangement was performed on DNA extracted from frozen tissue, digested with the restriction endonucleases EeoRI and BamHI/HindIII, sizefractionedby electrophoresis, transferred to a nylon membrane (Oncor), then hybridizedwith32P-Iabeled probefor joiningregion ofthe Ig heavychain gene (JH). Lanes labeled"PL" representthe placental control, lane A the CLH and lane B the ML of the ftrst patient (case 1); similarly, lanes C and D represent CLH and ML, respectively, ofthe secondpatient (case 2). Sizesofnonrearrangedgermlinebands are indicated in numbers, measured in kilobases. Both casesdemonstratesimilar results. There are several clonalbands (setsof corresponding arrows inadjacent lanesA-Band C-D), whichare present in both digestsof CLH and ML. An additional,unique band (solitary arrows) is seen in both cases of CLH, Bamiii]HindIII digests only, and these bands are absent in the ML.
to skin, and the other two had systemic involvement with follicular, small cleaved cell lymphoma. A comparative genotypic analysis ofthe previous CLH biopsy specimen and the ML in two of these four patients identified clonal rearrangements of the Ig gene not only in the lymphomas, but also in the immunologically polyclonal CLH specimens. Of particular interest was the pattern of clonal bands seen in these cases, with some identical and some different bands. These results suggest that the development of overt lymphoma in these cases is not just from the slow accumulation of a clonal B-cell population with eventual overgrowth and obliteration of reactive cells and development ofovert immunologic monoclonality, but that some other genetic change or clonal evolution has taken place. This evolution was accompanied in both patients with the development of more aggressive disease. The presence of "transient" clonal bands, detected in the polyclonal CLH but not in the malignant lymphomas of these two patients, lends support to the hypothesis that these patients had an abnormal immune response that led to the development of multiple B-cell clones, one or more of which eventu-
ally escaped the control of the patient's immunoregulatory mechanisms. Alternatively, different clonal bands could also be explained by additional alterations of DNA sequences by somatic mutations of the Ig genes 28 or gene deletion and subsequent development of additional clones. The finding of clonal bands on Southern blot analysis of CLH has been reported before. 2o , 23 Wood et a1.20 performed a study similar to ours, but with much shorter follow-up in many of the cases. They found clonal bands in 5 of 14 cases with histologically diagnosed, immunologically polyclonal CLH. One of their patients also had ML, 11 years after the initial CLH, with identical clonal JH bands in both the CLH and the ML. The detection of clonal bands in histologically benign, immunologically polydonal lymphoid proliferations has also been reported in other extranodal sites, particularly the ocular adnexa and salivary glands. In these sites, the percentage of immunologically polyclonal lymphoid proliferations containing clonal B-cell populations on genotypic analysis is almost 100%. This percentage is clearly much higher than that in ours or other pu blished series of CLH. The reason for this
432
Hammer et al.
difference is not clear. It is possible that there are true biologic differences in the cutaneous lymphoid infiltrates, but the difference could also be explained by simple differences in lymphoid cell density or other technical considerations. Histologically benign, immunologically polyc1onal lymphoid infiltrates from other extranodal sites also have an increased incidence of subsequent progression to ML. This progression is particularly well documented in orbital pseudolymphornas-v '! and lymphoepithelial lesions from patients with Sjogren's syndrome and Mikulicz's disease. 32- 38 In our study, the findings of discordant light chain phenotypes in simultaneous lymphoma biopsy specimens and "transient" clonal bands in polyclonal CLH support the concept that these patients initially have an abnormal immune state that leads to the presence of multiple B-cell clones, with development of overt, sometimes synchronous ML after additional transformations or clonal evolution. The clinical significance of the finding of a clonal Ig gene rearrangement in a histologically benign and immunologically polyclonal CLH and, therefore, the clinical utility of genotypic analysis in CLH will continue to be debated until sufficient numbers of patients with CLH with and without clonal gene rearrangement have been followed up for sufficient time to determine the actual frequency of development of ML. Until these longitudinal studies are completed to establish the actual risk of lymphoma, it would be premature to label these patients with a diagnosis of occult lymphoma. At the present time, they are not considered to have ML until they have developed an immunologically monoclonal B-ce1l population. It is also premature to suggest that all patients with polyclonal CLH should have genotypic analysis, but it would certainly be reasonable to perform the test in patients with multiple or recurrent lesions. The association of clonal Ig gene rearrangements with progression to lymphoma in our study does provide some support for the concept that gene rearrangement analysis may provide a way to identify those patients with CLH who are at highest risk for the development of lymphoma, that is, a means of selecting those patients with CLH who are most likely to benefit from early therapeutic intervention, most commonly local irradiation. Although the natural history of most low-grade B-cel1 lymphomas involving only extranodal sites is to remain localized for prolonged periods, the potential for development
Journal of the American Academy of Dermatology March 1993
of systemic and incurable disease, as seen in two of our patients, clearly exists. REFERENCES 1. Rijlaarsdarn lU, Meijer C.JL, Willemze R. Differentiation between lymphadenosis benigna cutis and primary cutaneous follicular center cell lymphoma. Cancer 1990; 65:2301-6. 2. Evans HL, Winkelmann RK, Banks PM. Differential diagnosis of malignant and benign cutaneous lymphoid infiltrates. Cancer 1979;44:699-717. 3. Lange Wantzin G, Hou-Jensen K, Nielson M, et al. Cutaneous lymphocytomas: clinical and histological aspects. Acta Derm Venereol (Stockh) 1982;62:119-24. 4. Caw W A, Helwig EB. Cutaneous lymphoid hyperplasia. Cancer 1969;24:487-502. 5. MacDonald DM. Histopathological differentiation of benign and malignant cutaneous lymphocytic infiltrates. Br J Dermatol 1982;107:715-8. 6. Knowles DM, Halper JP, Jacobiec FA. The immunologic characterization of 40 extranodal lymphoid infiltrates. Cancer 1982;49:2321-35. 7. Wirt DP, Grogan TM, Jolley CS, et al. Theimmunoarchitecture of cutaneous pseudolyrnphoma, Hum Pathol 1985; 6:492-510. 8. Garcia CF, Weiss LM, Warnke RA, et al. Cutaneous follicular lymphoma. Am J Surg Pat hoi 1986; 10:454-63. 9. Ralflciaer E, Saati TA, Bosq J, et al. Immunocytochemical characterization of cutaneous lymphomas other than mycosis fungoides. J Clin Pathol 1986;39:553-63. 10. WiJlemze R, deGraalT-Reitsma CB, Cnossen J, et al, Characterization of T-cell subpopulations in skin and peripheral blood of patients with cutaneous T-ceJllymphomas and benign inflammatory dermatoses. J Invest Dermatol 1983;80:60-6. 11. Payne CM, Grogan TM, Lynch PJ. An ultrastructural morphometric and immunohistochemical analysis of cutaneous lymphomas and benign lymphocytic infiltrates of skin. Arch Dermatol 1986;122: 1139-54. 12. Medeiros LJ, Picker LJ, Abel EA, et al. Cutaneous lymphoid hyperplasia. J AM ACAD DERMATOL 1989; 21:929-42. 13. WiJlemze R, Vermeer BJ, Meijer CJLM. Immunohistochemical studies in lymphocytic infiltration of the skin (Jessner) and discoid lupus erythematosus. J AM ACAD DERMATOL 1984;11:832-40. 14. Rallkiaer E, Lange Wantzin G, Mason DY, et al. Characterization of benign cutaneous lymphocytic infiltrates by monoclonal antibodies. Br J Dermatol 1984; III :635-45. 15. Weiss LM, Crabtree GS, Rouse RV, et al. Morphologic and immunologic characterization of 50 peripheral T-cell lymphomas. Am J Pathol 1985;118:316-24. 16. Picker LJ, Weiss LM, Medeiros LJ, et al. Irnmunophenotypic criteria for the diagnosis of non-Hodgkin's lymphoma. Am J PathoI1987;128:181-201. 17. Wood GS, Abel EA, Hoppe RT, et al. Leu-8 and Leu-9 antigen phenotypes: immunologic criteria for the distinction of mycosis fungoides from cutaneous inflammation. J AM ACAD DERMATOL 1986;14:1006-13. 18. Wood GS, Burke IS, Horning S, et al, The immunologic and clinicopathologic heterogeneity of cutaneous lymphomas other than mycosis fungoides. Blood 1983;62:464-72. 19. Warnke RA, Rouse RV. Limitations encountered in the application of tissue section immunodiagnosis to the study
Journal of the American Academy of Dermatology Volume 28, Number 3
20.
21. 22. 23. 24. 25.
26. 27. 28.
29.
of lymphomas and related disorders. Hum Pathol 1985; 16:326-31. Wood GS, Ngan BY, Ting R, et al. Clonal rearrangements of immunoglobulin genes and progression to B-cell lymphoma in cutaneous lymphoid hyperplasia. Am J Pathol 1989;135:13-9. Bignon YJ, Souteyrand P, Roger H, et al. Dual genotype in cutaneous T-cell lymphomas and pseudolymphomas, Hum Genet 1990;84:177-8. WechslerJ, BagotM, HenniT, et aJ.Cutaneous pseudolymphomas: immunophenotypical and immunogenotypical studies. Curr Probl Dermatol 1990;19:183-8. Griesser H, Feller AC, Sterry W. T-cell receptor and immunoglobulin gene rearrangements in cutaneous T-cellrich pseudolymphomas. J Invest Dermatol 1990;95:292-5. Bindl J, Warnke RA. Advantage of detecting monoclonal antibody binding to tissue sections with biotin and avidin agents in Coplin jars. Am J Clin Pathol 1986;85:490-3. Cartun RW, Redersen CA. An immunocytochemicaltechnique offeringincreased sensitivityand loweredcost with a streptavidinhorseradish peroxidaseconjugate. J Histotechnol 1989;12:273-5. Southern EM. Detection ofspecific sequencesamong DNA fragments separated by gel electrophoresis. J Mol Bioi 1975;98:503-17. Ackerman AB, ed. Histologic diagnosis of inflammatory skin diseases. Philadelphia: Lea & Febiger, 1978:215-7, 442-7,494-5. Cleary ML, Meeker TC, LevyS, et al. Clusteringof extensivesomatic mutations in the variable region of an immunoglobulin heavy chain gene from a human B-cell lymphoma. Cell 1986;44:97-106. Chen P, Liu JH, Lin SH, et al. Rearrangement of immu-
Hammer et al. 433
30.
31.
32. 33.
34. 35. 36. 37.
38.
noglobulin gene and oncogenes in ocular adnexal pseudolyrnphomas. Curr Eye Res 1991;10:547-55. Neri A, lakobiec FA, Pelicci P-G, et a1. Immunoglobulin and T-cellreceptor iJ-chain gene rearrangement analysis of ocular adnexal lymphoid neoplasms: clinical and biologic implications. Blood 1987;70:1519-29. KnowlesOM, Athan E, Ubriaco A, et al. Extranodal noncutaneous lymphoid hyperplasias represent a continuous spectrum of B-cell neoplasia: demonstration by molecular genetic analysis. Blood 1989;73:1635-45. FishlederA, Tubbs R, Hesse B, et al. Uniform detection of immunoglobulingene rearrangement in benign lyrnphoepithelial lesions. N Engl J Moo 1987;316:1118-21. Falzon M, Isaacson PG. The natural history of benign lymphoepitheliallesion of the salivary gland in which there is a monoclonal population of B cells. Am J Surg Pathol 1991;15:58-65. Zulman 1, laffe R, Talal N. Evidence that the malignant lymphoma of Sjogren's syndrome is a monoclonal B-ceJl neoplasm. N Engl J Med 1978;299:1215-20. Bridges AJ, England MD. Benign lympboepithelial lesion: relationshipto Sjogren's syndrome and evolving malignant lymphoma. Semin Arthritis Rheum 1989;19:201-8. Azzopardi lG, Evans DJ. Malignant lymphoma of parotid associated with Mikulicz disease (benign lymphoepithelial lesion). J Clin Pathol 1971;24:744-52. Hyman GA, Wolff M. Malignant lymphomas of the salivary glands: reviewof the literature and report of 33 new cases, including four cases associated with the lyrnphoepithclial lesion. Am J Clin Pathol 1976;65:421-38. Colby TV, Dorfman RF. Malignant lymphomas involving the salivary glands. Pathol Annu 1979;l4(part 2):307-24.
BOUND VOLUMES AVAILABLE TO SUBSCRIBERS
Bound volumes oftheJOURNAL or:THE AMERICAN ACADEMY or DERMATOLOGY areavailable tosubscribers (only) for the 1993 issues from the Publisher at a costof$69.00 fordomestic, $91.83 forCanadian, and$87.00 for international forvolume 28(January-June) andvolume 29 (July-December). Shipping charges are included. Each bound volume contains a subject and author index and all advertising is removed. Copies are shipped within 60 days after publication of thelast issue in the volume. The binding is durable buckram with the journal name, volume number, and year stamped in gold on thespine. Payment III/1St accompany all orders. ContactMosby, Subscription Services, 11830 Westline Industrial n-, SI. Louis, MO 63146-3318. USA: phone (800) 325-4177, ext. 4351; (314) 453-4351. Subscriptions must be in force to qualify. Bound volumes are not available ill place of' a regular journal subscription.