The Prognostic Significance of Delayed Hypersensitivity toDinitrochlorobenzene and Mechlorethamine Hydrochloride inCutaneous T Cell Lymphoma

Eric C. Vonderheid, Seema K. Ekbote, Kathleen Kerrigan, Jacob D. Kalmanson,† Eugene J. Van Scott,‡Alain H. Rook,* and J. Todd AbramsDepartment of Dermatology, Allegheny University of the Health Sciences, Philadelphia, Pennsylvania, U.S.A.; *Department of Dermatology, University ofPennsylvania, Philadelphia, Pennsylvania, U.S.A.; †Private Practice of Dermatology, Pittsburgh, Pennsylvania, U.S.A.; ‡Private Practice of Dermatology,Abington, Pennsylvania, U.S.A.

Recent studies suggest that cells elaborating type 1cytokines are important mediators of anti-tumor cell-mediated immunity in cutaneous T cell lymphoma. Type1 cell-mediated immune responsiveness was assessed in276 patients with cutaneous T cell lymphoma (mycosisfungoides and Sezary syndrome) using 2,4-dinitrochloro-benzene (DNCB) skin testing as part of the initialevaluation. The overall rate of sensitization after one andtwo DNCB challenges was 32% and 67%, respectively,which is much decreased compared with the expectedrate of more than 95% for normal individuals. Moreover,the frequency of DNCB sensitization and allergic contact

During the past decade, it has been recognized thatCD41 T helper cells can be functionally dividedaccording to their cytokine profile into T-helper-1cells that produce interleukin (IL)-2, interferon-γ, andtumor necrosis factor β; and T-helper-2 cells that

produce IL-4, IL-5, IL-6, IL-10, and IL-13 (Mosmann and Sad, 1996;Romagnoli et al, 1997). Because cells other than CD41 T cells alsocontribute to the cytokine response in vivo (Carter and Dutton, 1996),T-helper-1 and T-helper-2 cytokine patterns are also designated as type1 or type 2 cytokine responses, respectively. Type 1 cytokines enhancecell-mediated immunity, stimulate T cell proliferation, and activatemacrophages; type 2 cytokines augment humoral responses whilesuppressing cell-mediated immunity. IL-4 and IL-10 act to downregul-ate interferon-γ production and interferon-γ represses the type 2response (Gajewski and Fitch, 1988; Peleman et al, 1989; Gaya et al,1991; Parronchi et al, 1992; D’Andrea et al, 1993).

Cutaneous T cell lymphoma (CTCL) is a clonal proliferation ofskin-homing T cells (Diamandidou et al, 1996). Recent studies indicatethat the malignant T cells of CTCL typically have the cytokinesecretion profile of T-helper-2 cells (Vowels et al, 1994; Asadullah et al,1996; Dummer et al, 1996). It has been proposed that the progressionof CTCL is associated with a switch from a type 1 predominant to a

Manuscript received November 1, 1997; revised January 23, 1998; acceptedfor publication January 30, 1998.

Reprint requests to: Dr. Eric Vonderheid, Department of Dermatology,Mailstop 478, Allegheny University, Broad and Vine Streets, Philadelphia,PA 19102.

Abbreviations: MF, mycosis fungoides; HN2, mechlorethamine hydro-chloride.

0022-202X/98/$10.50 · Copyright © 1998 by The Society for Investigative Dermatology, Inc.

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dermatitis to topically applied mechlorethaminedecreased with advancing stage of disease. In addition tothe expected strong correlation with stage, we observedthat patients who were DNCB test positive were signific-antly less likely to experience disease progression and hada better overall prognosis compared with DNCB-negativepatients. These results support the concept that cell-mediated responses are important in cutaneous T celllymphoma, and that augmentation of these responseswould be therapeutically beneficial. Key words: T-helper-1/T-helper-2. J Invest Dermatol 110:946–950, 1998

type 2 predominant cytokine profile in lesional skin, as the number ofmalignant cells increases in the infiltrate relative to reactive T cells(Vowels et al, 1994). If true, localized production of inhibitory type 2cytokines would suppress cell-mediated type 1 anti-tumor responsesthought to be mediated by CD81 cytotoxic T cells, resulting infavorable conditions for tumor growth (Hoppe et al, 1995). IL-10seems to be a more important inhibitor of type 1 responses in CTCLthan IL-4 (Asadullah et al, 1996; Dummer et al, 1996). Moreover, anexcess of type 2 cytokines appears to account for many of theimmunologic abnormalities in advanced CTCL, such as hypereosino-philia, increased serum IgA or IgE, impaired natural killer cell andlymphokine-activated killer cell activity, decreased mitogen-inducedproliferation, and impaired cell-mediated immune responses to antigens(Rook and Heald, 1995; Hansen, 1996). The finding that delayedhypersensitivity responses improve after treatment of the skin withphotochemotherapy supports this hypothesis (Kubba et al, 1980).

Skin testing with 2,4-dinitrochlorobenzene (DNCB) has been util-ized as a measure of cell-mediated immune response for more than50 y. Previous studies showed that immune responsiveness to DNCBbecomes impaired in various cancers and that a positive correlationexists between DNCB reactivity and prognosis (Eilber and Morton,1970; Wells et al, 1973; Bleumink et al, 1974; Bosworth et al, 1975;Hazra et al, 1975; Lee et al, 1975; Pinsky et al, 1976). The initial studyof DNCB reactivity in CTCL concluded that cell-mediated immunitywas normal (Blaylock et al, 1966), but patients in this study wereexposed to much higher sensitizing doses than typically used (10 mginstead of 2 mg or multiple exposures to 2 mg doses three timesweekly). After the DNCB method was standardized by Catalona(1972a, b), three other studies indicated that DNCB reactivity wasimpaired compared with normal individuals, particularly in advanceddisease (Nordqvist and Kinney, 1976; Vonderheid et al, 1977; van der

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Table I. Frequency of delayed hypersensitivity reactions to dinitrochlorobenzene (DNCB) and topical mechlorethamine (HN2)chemotherapy in patients with cutaneous T cell lymphoma according to clinical diagnosis and stage of diseasea

DNCB tested X1 DNCB tested X2 HN2 allergy

No. positive/no. tested (%) No. positive/no. tested (%) No. positive/no. treated (%)

Clinical diagnosisMF-patch phase 45/123 (37) 83/102 (81) 39/91 (43)MF-plaque phase 19/71 (27) 42/68 (62) 25/66 (38)MF-tumor phase 14/40 (35) 18/33 (55) 11/38 (29)MF-erythroderma 10/31 (32) 16/27 (59) 11/28 (39)Sezary syndrome 0/11 (0) 1/10 (10) 1/11 (9)

StageIa 44/97 (45) 73/85 (86) 34/70 (49)Ib 15/66 (23) 39/58 (67) 21/60 (35)IIa 4/27 (15) 12/23 (52) 9/23 (39)IIb 12/30 (40) 16/26 (62) 11/29 (38)III 9/32 (28) 15/28 (54) 11/29 (38)IVabb 4/24 (17) 5/20 (25) 1/23 (4)

Total 88/276 (32) 160/240 (67) 87/234 (37)

aThe frequency of DNCB sensitization is highly significant except for DNCB tested once versus clinical diagnosis (p 5 0.114). The frquency of HN2 sensitization is significant forstage (p 5 0.012), but not clinical diagnosis (p 5 0.189)

bIncludes five patients with stage IVb disease (three tumor MF, two Sezary syndrome).

Table II. Frequency of disease progression and deaths in patients with cutaneous T cell lymphoma according to clinicaldiagnosis and stage of disease

Deaths

Number Progression (%)a CTCL-related (%) Any cause (%)

Clinical diagnosisMF-patch phase 123 9 (7) 9 (7) 47 (38)MF-plaque phase 71 21 (30) 18 (25) 45 (63)MF-tumor phase 40 24 (60) 29 (73) 37 (93)MF-erythroderma 31 7 (23) 9 (29) 27 (87)Sezary syndrome 11 4 (36) 9 (82) 11 (100)

StageIa 97 8 (8) 9 (9) 36 (37)Ib 66 11 (17) 11 (17) 37 (56)IIa 27 8 (30) 4 (15) 15 (56)IIb 30 20 (67) 22 (73) 28 (93)III 32 9 (28) 11 (34) 28 (88)IVabb 24 9 (38) 17 (71) 23 (96)

All patients 276 65 (24) 74 (27) 167 (61)

aDisease progression occurred in 65 patients: stage Ia–IIa, tumors (13 patients), erythroderma (one patient), lymph node involvement (10 patients), and blood involvement (threepatients); stage IIb, lymph node involvement (18 patients) or blood involvement (two patients); stage III erythroderma, skin tumor formation (one patient), lymph node (five patients),or blood involvement (three patients); and stage IV erythroderma, skin tumor formation (two patients) or blood involvement (one patient) or visceral involvement (six patients).

bIncludes five patients with stage IVb disease (three tumor MF, two Sezary syndrome).

Harst-Oostveen and van Vloten, 1978). In addition, we observed inan early review that DNCB-positive patients achieved a completeresponse to therapy sooner than DNCB-negative patients (4 mo versus6 mo, respectively), albeit that both groups had identical response ratesand that DNCB-positive patients had a lower death rate from CTCL-related causes than their DNCB-negative counterparts (Vonderheidet al, 1977). This study extends those previous observations byexamining the relationship of type 1 cell-mediated immune respons-iveness as measured by DNCB and allergic sensitization to therapeutictopical applications of mechlorethamine hydrochloride (HN2) to stageof disease, disease progression, and survival patterns in CTCL.

MATERIALS AND METHODS

Between March 1968 and July 1981, 276 patients (174 men, 102 women) withmycosis fungoides (MF) or Sezary syndrome, excluding other primary cutaneousperipheral T cell lymphomas and lymphomatoid papulosis, were tested withDNCB as a measure of type 1 cell-mediated immune function as part of theirinitial evaluation. The median age of patients in this CTCL cohort was 58 y(range, 13–84 y). Patients with classical MF were classified into patch,

plaque, or tumor phases, and erythrodermic CTCL patients were classified aserythrodermic MF or Sezary syndrome based on whether there were less thanor more than 20 Sezary cells per 100 lymphocytes on blood smears as previouslydescribed (Vonderheid et al, 1985). Staging was based on the tumor-node-blood-metastasis scheme according to recommendations by the MFCGCooperative Group (Bunn and Lamberg, 1979).

DNCB testing was accomplished using a modified method described byCatalona (1972a, b). Each patient was exposed to a 2 mg sensitizing dose ofDNCB (0.1 ml of a 2% solution in acetone) applied to a 2.5 cm diameter siteof normal-appearing skin on the upper arm, which was then covered withsemiporous paper tape for 24 h. Several weeks later, 25 or 50 mcg in 0.1 mlof DNCB was applied to another site as a challenge dose. The test was scoredas positive if the challenge site developed evidence of erythema, edema, orblistering. The sensitization procedure was repeated in 152 of the 188 (81%)patients whose initial skin test was negative. Thus, two attempts at DNCBsensitization were made on a total of 240 patients with CTCL.

The following clinical data were extracted from the medical records ofpatients tested to DNCB: patient’s age, gender, race, clinical diagnosis, skin T-rating and stage of disease, evidence of allergic contact dermatitis in patientstreated with topical HN2 (allergy confirmed by patch testing in almost allinstances), date of disease progression if applicable, and cause and date of death

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if applicable. Disease progression was defined as the development of skintumors in patients with previous patch, plaque, or erythrodermic disease, thedevelopment of nodal or visceral involvement (stages IVa or IVb) in patientswith previous intracutaneous disease (stages I–III), the development of bloodinvolvement (repeated Sezary cell counts . 20 per 100 lymphocytes) in patientswithout previous blood involvement, and the development of visceral disease(stage IVb) in patients with previous involvement limited to lymph nodes (stageIVa). Updated information about the current status of each patient was obtainedby direct communication with the patient, a spouse or close relative, or aphysician. Survival was calculated from the date of initial evaluation to the dateof last contact; patients lost to follow-up were considered to be censored at thetime of last contact.

The Fisher’s exact test for 232 tables and chi-square test for multiway tableswere used as a measure of association among clinical groups. Student’s t testwas utilized to test for differences in age between two groups. Kaplan–Maierestimators and the Cox proportional hazards regression model were used to testfor differences in survival rates between groups. Statistical software used fordata analysis were SYSTAT for Windows, Version 5 (SYSTAT, Evanston, IL),StatXact-Turbo (CYTEL Software, Cambridge, MA), and EGRET (Statisticsand Epidemiology Research, Seattle, WA).

RESULTS

DNCB and topical HN2 reactivity decreases in advanceddisease Table I shows the sensitization rates to DNCB whenapplied once or twice and to topically applied HN2 when administeredas part of the initial treatment regimen. The frequency of sensitizationafter one round of DNCB testing was only 32% (88 of 276), althoughthis increased to 67% (160 of 240) after a second DNCB sensitizingdose. Likewise the frequency of allergic contact dermatitis to topicalHN2, a less potent allergen than DNCB, occurred in 37% (87 of 234)of patients who used it. Patients who became DNCB positive werealmost two times more likely to experience an allergic reaction totopical HN2 compared with DNCB-negative patients (n 5 207, 45%vs 24%, p 5 0.003). Patients testing positively to DNCB after the firstchallenge test were significantly younger (mean age, 54 y) thannonsensitized patients (mean age, 58 y, t test, p 5 0.009), but thisdifference was less apparent in patients challenged twice (mean age,56 y vs 59 y, p 5 0.053). By contrast, the mean age of patientsdeveloping an allergic reaction to topical HN2 was higher than thatof patients who did not become allergic (mean age, 60 y vs 56 y, p 50.04). DNCB sensitization rates did not correlate significantly withpatients’ gender (p . 0.8), race (p 5 0.09), or duration of diseaseprior to testing (p . 0.9).

When correlated to clinical diagnosis and stage, the rate of sensitiza-tion varied significantly for patients grouped according to stage (andT-rating, data not shown) for initial DNCB testing (p 5 0.003) andtopical HN2 (p 5 0.012), but not for patients grouped according toclinical diagnosis (p . 0.1 for each); however, differences in DNCBsensitization rates were more significant after two DNCB challengesfor patients grouped according to both clinical diagnosis and stage/T-rating (p , 0.001 for each).

Sensitization rates for twice-applied DNCB were found to be nearnormal (86%) for patients with CTCL at stage Ia, but were diminishedin later stages of disease down to 25% for stage IV disease (Table I).Similarly, the rate of allergic contact dermatitis to HN2 was highest atstage Ia (49%), and then decreased to only 4% at stage IV.

DNCB reactivity decreases with more extensive skininvolvement The rate of sensitization to twice-applied DNCB wasalso found to vary according to extent of skin involvement in patientswith patch and plaque phase MF without lymph node involvement(stages I–IIa). Of patients with patch phase MF, 58 of 66 patients (88%)with skin lesions involving less than 10% of the skin surface wereDNCB positive, compared with 25 of 35 patients (71%) with greaterthan 10% involvement (Fisher’s test, p 5 0.056). Similarly, 20 of 27patients (74%) with plaque phase and less than 10% skin surfaceinvolvement tested positively to DNCB, compared with 21 of 38patients (55%) with more than 10% involvement (p 5 0.192). Whenpatch and plaque MF results were combined, the difference insensitization rates was significantly higher for patients with limitedinvolvement compared with patients with more extensive disease(respectively, 84% vs 63%, p 5 0.004).

Figure 1. Survival according to DNCB reactivity in CTCL.

Figure 2. Survival according to stage of CTCL. The survival of patientswith cutaneous decreases with advancing stage of disease (p , 0.001).

DNCB reactivity correlates to survival Univariate regressionanalysis identified several parameters that correlated significantly withsurvival (all causes of death) of 240 patients tested twice to DNCB:age, clinical diagnosis, T-rating and stage, DNCB reactivity (p , 0.001for all), and gender (p 5 0.049). Kaplan–Meier survival estimatescorresponding to DNCB reactivity and stage of disease for this cohortof CTCL patients are shown in Figs 1 and 2, respectively. Nocorrelation with survival was found for response to a single applicationof DNCB (p . 0.1) or for allergy to topical HN2 (p . 0.3). Moreover,no additional prognostic information was found when DNCB-positiveand -negative groups were further stratified according to HN2 reactivity(data not shown). Multivariate regression analysis indicated that ofthese parameters, stage correlated most strongly with survival in allCox models, and that DNCB reactivity did retain borderline signific-ance (p 5 0.052) together with stage when both were entered stepwiseinto the model. The reason for this association is shown in Fig 3,which compares survival curves for DNCB-positive and DNCB-negative patients according to stage. Although no significant differencein survival was found for any particular stage, a trend favoring bettersurvival was observed for DNCB-positive patients with stage I, III,and IV disease; however, DNCB reactivity did not retain significancein a three parameter model that included age and stage of disease.Similar correlations were observed in survival analysis using disease-related death as the endpoint, although the relationship of age tosurvival was less apparent in the Cox models as might be expected.

DNCB reactivity correlates to risk of disease progression TableII shows the relationship between clinical diagnosis and stage of CTCLat the time of initial evaluation to the subsequent progression of diseaseand death rate. In addition to its correlation with overall survival, DNCBreactivity was found to correlate to subsequent disease progression inpatients with relatively early CTCL. For patients with patch and plaquephase MF (stage I–IIa), disease progression occurred in 11 of 42 patients

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Figure 3. Survival for each stage of CTCL according to DNCBreactivity.

Figure 4. Risk of disease progression according to DNCB reactivity inearly stage mycosis fungoides.

(26%) who tested negatively to DNCB versus 14 of 124 patients (11%)who were DNCB positive (Fisher’s test, p 5 0.026). Disease progressionwas manifested as development of cutaneous tumors (13 patients),erythroderma (one patient), lymph node involvement (nine patients),or blood involvement (two patients) without apparent differencebetween the two groups (p 5 0.157). The difference in the rate ofdisease progression first became evident about 8–10 y after testing(Fig 4). It is interesting that the risk of progression was almost identicalfor patients who developed contact allergy to topical HN2 comparedwith patients who did not (Fisher’s test, p 5 1.0). Because diseaseprogression strongly correlates with overall and disease-related deaths(p , 0.001), this may explain the positive association of DNCBreactivity to survival.

DISCUSSION

Our results reaffirm earlier studies that showed DNCB reactivity inCTCL is impaired compared with normal individuals, and the sensitiza-tion rate especially decreases in more advanced disease (Nordqvist andKinney, 1976; Vonderheid et al, 1977; van der Harst-Oostveen andvan Vloten, 1978). A single application of 2 mg DNCB followed bya challenge dose of 50 mcg after 2 wk sensitizes more than 95% ofnormal individuals or patients with dermatitis (Catalona et al, 1972a,b; Bleumink et al, 1974). In this series, only 32% (91 of 287 patientstested) developed cutaneous hypersensitivity after a single DNCBapplication, with the highest rate (47%) in patients with stage Ia disease;however, a second DNCB application and challenge yielded an overallsensitization rate of 67% (160 of 240 patients) with 86% of patientswith stage Ia mycosis fungoides becoming positive. Additional exposures

to DNCB provided an even higher sensitization rate (93% overall,data not shown). These observations suggest that the cell-mediatedimmune defect in CTCL is more qualitative than absolute, and explainthe results of an earlier study that concluded that DNCB reactivityin CTCL after repetitive applications was near normal (Blaylocket al, 1966).

The absence of similar correlations between HN2 allergic reactionsand disease progression or overall prognosis is of note. One explanationis that reactivity to weak allergens such as topical HN2 may beinadequate to identify two subsets of patients with differing prognosisin CTCL. Support for this hypothesis is that a single exposure toDNCB, which gave a sensitization rate similar to HN2, also did notcorrelate with disease progression or overall survival. Alternatively, thedichotomy of action of HN2 may make prognostic correlations lessdistinct compared with DNCB. Topical HN2 is not only toxic tomalignant lymphocytes invading the epidermis, but also becomes anallergen when applied to the skin. Consequently, development of anallergic reaction to topical HN2 signifies not only that an intact cell-mediated immune response is present (favorable event), but also thatthe therapeutic benefit of this drug may be compromised because theallergic sensitization hampers its long-term use for topical chemotherapy(unfavorable event); however, the equal therapeutic response rate inpatients who are or who are not allergic to topical HN2 would notsupport this interpretation of our results (Vonderheid et al, 1977).

In the context of the proposed immunopathogenic model of CTCLdiscussed in the introduction (Rook and Heald, 1995; Hansen, 1996),abnormal DNCB skin testing might be identifying a subset of patientswho have developed sufficient type 2 cytokine excess in lesional skinthat systemic anti-tumor responses become impaired. In addition todiminished sensitization rates in advanced stages of CTCL, DNCBsensitization also decreases in patients with early stages of CTCL whohave more extensive skin involvement (T2 rating) compared withpatients with limited involvement (T1 rating). Such patients aresignificantly more likely to develop progression of disease and have aworse overall survival compared with patients who had intact DNCBresponses even when the effect of stage was considered; however, interms of long-term survival, age and stage remain the major prognosticindicators in CTCL, and DNCB reactivity provides only supportiveinformation.

These results provide indirect evidence that cell-mediated immunemechanisms are important in controlling progression of CTCL, spe-cifically responses mediated by type 1 cells. In this regard, it is knownthat type 1 cytokines promote the generation of CD81 cytotoxic Tcells, and CD81 cells isolated from patients with advanced CTCLmediate lysis of autologous tumor cells in vitro (Berger et al, 1997).Cell-mediated responses also may function to reduce or eliminatepotential foreign antigens attracting the predominant clone to theepidermis. Hoppe et al (1995) observed that high numbers of CD81cells in lesional skin correlates with improved survival. Methods toenhance anti-tumor type 1 responses may therefore be effective intreatment. For example, previous reports indicate beneficial effects ofdelayed hypersensitivity reaction superimposed on lesions of CTCL(Ratner et al, 1968; Klein et al, 1976; Vonderheid et al, 1977; Cohenand Bekierkunst, 1979). Current strategies involve direct administrationof type 1 cytokines (interferon-γ, IL-2, IL-12) which in initial trialshave shown activity in CTCL (Kaplan et al, 1990; Rybojad et al, 1992;Marolleau et al, 1995; Horikoshi et al, 1996; Rook et al, 1996).

This work was supported by The Leonard and Ruth Levine Skin Research Foundation.

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