Melanocytic activation in HIV-1 disease: HMB-45 staining in common acquired nevi

Clinical and laboratory studies Melanocytic activation in HIV-l disease: HMB-45 staining in common acquired nevi LTC Kathleen 1. Smith, MC, USA,e CDR Henry G. Skelton, MC, USN,a CPT William Heimer, MC, USA,f LCDR Donald Baxter, MC, USN,d COL Peter Angritt, MC, USA,c LCDR Dennis Frisman, MC, USN,b Kenneth F. Wagner, DO,g and Military Medical Consortium for the Advancement of Retroviral Research Washington, D.C. Background: An increase inpigmented lesions hasbeenreported in HIV-L-infected patients. In a study of HIV-l-positive patients, we have seen patients who noticed new or changing pigmented lesions. Objective: The goal of this study was to determine to what degree these pigmented lesions showed evidence of significant melanocytic proliferation as opposed to increased pigment production without significant rnelanocytic proliferation. Methods: Biopsy specimens were studied with routine light microscopy and immunohistochemical stains including S-100 protein, HMB-45, and proliferating cell nuclearantigen. Results: The lesions included two malignant melanomas and 42 benign melanocytic lesions. Significant staining of dermal melanocytes with HMB-45 was present in two of three melanomas and in 19 of 42 nevi. With stains for proliferating cell nuclear antigen there wasa positive reaction in the dermal component of both melanomas and a negative reaction in the dermal cells of the nevi. Conclusion: In someHIV-l-infected patients thereis stimulation ofmelanosome production without significant melanocyte proliferation. (J AM ACAD DERMATOL 1993;29:539-44.)

Eruptive dysplastic nevi(DN) have been reported in HIV-l-positive patients, as well as an increased number and darkening of common acquired nevi. 1,2 In addition, localized and diffuse areas of hyperpigmentation sometimes associated with zidovudine therapy have also been reported in patients with HIV-1 disease.l? As a part of a large HIV protocol within the military population, we have observed more than 600 patients for up to 28 months; during that period biopsies of several pigmented lesions have been performed. We have stained most of them From the Departments ofDermatology" and Cellular Pathology" and the AIDS Registry," National Naval Medical Center, Bethesda, Department of Dermatology'; Walter Reed Army Institute of Research": Walter Reed Army Medical Center, Department of Dermatology': and the Henry M. Jackson Foundatlon.s Supported in part by National Institutes ofHealth, NIAMS interagency agreement YOIAR90008 and YOIAR00014. The opinions or assertions contained herein are the private views ofthe authors and are not to be considered as official or as reflecting the views ofthe department of the Army, Navy, or of the Department of Defense. Accepted for publication March 27,1993. No reprints available. 16/1/47477

for S-lOO, HMB-45, and proliferating cell nuclear antigen (PCNA).6-8 S-lOO protein is a sensitive marker for cells of melanocytic origin but is not specific and does not provide information on the production of melanin or cellular proliferation. HMB-45 is specific for cellsof melanocytic origin and stains the cells of DN, cellular blue nevi, deep penetrating nevi, congenital nevi, and nevifound in hormonally responsive areas.f 9, 10 In addition, melanocytes in a variety of reactive conditions and nevi in close proximity to neoplastic proliferations have also stained for HMB-45 in the dermal component.'! However, the dermal component of common acquired nevi and melanocytes in clinically noninflamed skin do not stain for HMB45.6, 7, 10 PCNA stains the nuclei of proliferating cells and thus gives an indication of cellular proliforation." MATERIAL AND METHODS

A total of 21 patients in Walter Reed (WR) stages 1 through 6 had pigmented lesions that were subjected to biopsy fordiagnosis or wereexcised for cosmetic reasons at the request of thepatient (Fig. 1;Table1). Routine he-

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1 Fig. 1. Patient 3. Numerous macular and papular pigmented lesions on the lower back, which range from O. I to 1.5 em in diameter.

3. Fig. 2. Patient 3. Irregular large pigmented nevi on dorsum of right foot. Fig. 3. Section of acquired compound nevus stained for HMB-45 shows positive reaction not only in nevus cells at the dermoepidermal junction but also in dermal nevus cells. (HMB-45 stain; X75.) matoxylin-and-eosin-stained sections were examined by one of us (K. J. S.) for diagnosis. Immunohistochemical stains for S-100 protein (I :800 Dako; ABC method), HMB-45 (1 :20 Enzo; ABC method), and PCNA (a DNA polymerase-associated protein, cyelin) (1 :40Dako; ABC method) were performed. These stains were evaluated by one of us (I-I. G. S.) before knowing the history. RESULTS (Table 1)

Four patients had an increased number (more than 100) of large, irregular, widely distributed nevi

(Figs. I and 2). Three reported that new lesions had developed since the diagnosis of HIV-l disease and these same three had family members with similar lesions. There was no history of malignant melanoma (MM) in the family members of these three patients; however, a MM has developed in one of the three patients. This patient has an HIV-I-negative identical twin with similar nevi but no MM. Biopsy specimens of these lesions showed nevi with architectural atypia, but minimal if any cytologic atypia. The architectural atypia consisted of large irregular

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Table I. HMB-45 and PCNA staining in common acquired nevi, dysplastic nevi, and malignant melanomas in HIV-L-positive patients Patient No.

Locationt 35

2

3

W

29 31

H

31

W

M

M

M

32 4 5 6 7 8 9 10 II

41

H W W

42 36 29 34 26

W W W W W

33

28

M M M

M M M M M

12

34

W

M

13 14 15 16 17

29 28 26 68 30

W W W W W

M

18 19 20

29 27 42

W W W

M M M

N M M

F

5 5 5 5 5 5 2 2 2 2 2 2 2 2 5 5 I

2 3 3 3 4 5 2 1 2 2 2 2 2 6 6 6 6 2 3 2 3 2 2 3 2 I

IDN DPN DPN DPN DPN MM

DPN DPN DPN BCC DPN BCC DPN DPN BCC BCC CPN ePN ePN ePN ePN IDN ACN ePN ePN DPN IDN ePN DPN DPN ePN IDN ePN ePN AeN FLP ePN FLP ePN ePN FLP ePN MM

Rt ear Rt side of back Mid back Lt side of back Cheek Back Neck Mid back Back Face Rt side of back Shoulder Lt side of back Chest Shoulder, recurrent Neck Face Scalp Rt side of back Back, inf Lt side of back Back Rt thigh Back Scalp Lt leg Lt leg, inf Leg Back, ant Back, inf Rt arm Rt side of chest Mid chest Lt arm Inguinal Back Nose Chest Foot Foot Foot Nose Back

peNA

0.40 mm

0.70 mm

+ + + + + + + + +

+ +

+

+

+ +

+ +

+ + + + + + + + + + + + + + + + + + + + + + + + + + +

+ + + +

+f

+ + + + + + +

+ +t +t + + +t +

+f

ACN, Atypical compound nevus; alii, anterior; BCC, basal cell carcinoma; CPN, compound nevus; DPN, dysplastic nevus; +f, focal; FLP, focal lentiginous proliferation; lDN, intradermal nevus; irf, inferior; Lt, left; MM, malignant melanoma; RI, right; WR, Waller Reed stage. [Positive staining in the junctional melanocytes that do not stain in normal skin. :J:Thickness in melanomas only. Staining for HMB-45 is reported for the dermal component only except in thecases offocallentiginous proliferation.

junctional nests, "bridging" of nests between rete ridges, lamellar fibrosis, lateral extension of the nevus cell at the dermoepidermal junction, and a lymphoid infiltrate. These features have been described in DN but, in our experience, also occur in acquired nevi in adults.

In one of the three patients with DN and in the patient with multiple large nevi but no family history of MM basal cell carcinomas (BeCs) also developed. During the period of this study, one patient with DN and BCC has had two primary BCes one lesion recurred after excision. Two additional pa-

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J ournal o f the American Academy of Der mat ology October 1993

Smith et al.

4

•

• · oJ •

..

'.

#

. ...

.' .!".. -- .. . .-:. - . ~

.

,,-

" Fig. 4. Section from second acquired compound nevus shows positive reaction for HMB-45 in nevus cells in dermal component. (HMB-45 stain ; X 150.) Fig. 5. Section of lentiginous lesion shows strong positive reaction for HMB-45 in nevus cells in lesion at dermoepidermal junction. (HMB-45 stain; X150.)

tients had MM, but neither had DN. Two patients had a large (> 10 mm) irregular, variably pigmented solitary lesion. The biopsy specimens showed features similar to the aforementioned nevi and were classified as atypic al compound nevi. Biopsies were performed in four additional patients with focal macular hyperpigmentation; early lentiginous changes were detected but with no definite nesting. The remaining patients had lesions consistent with common acquired nevi with no significant atypia. IMMUNOHISTOCHEMICAL STAINING (Table f)

All melanocytic lesions were diffusely positive when stained with S-IOO protein , as were the melanocytes at the dermoepidermal junction in the surrounding epidermis. HMB-45 focally stained the dermal component in lOaf the 13 lesions from patients with multiple DN, in two of two MMs, in 6 of 15 common acquired nevi, and in both lesions diagnosed as acquired compound nevus (Figs. 3 and 4).

HMB-45 stained the melanocytes of the four lentiginous lesions (Fig. 5). Only rare melanocytes in the skin around these lesionsshowed a positive reaction for HMB-45 (Fig. 4). None of the various nevi in this study showed significant PCNA staining of the melanocytes, whereas both of the MMs demonstrated a positive reaction in 15% to 50% of the nuclei. In all nevi, peNA staining was seen in keratinocyte nuclei most prominent within the basal cell layer in the epidermis above the nevi. In many lesions with a junctional component there was additional staining of keratinocytic nuclei above the basal layer. DISCUSSION

The biopsy specimens of the melanocytic lesions from HIV-l -infected patients showed features similar to those in HIV-I-negative patients. However, the lesionsshowed increased staining of the dermal component, especially in th e common acquired nevi

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Induction and elickation of contracthypersensitivity

Fig. 6. This diagr am shows how increased levels of IL-I, IL-6, and TN F-a may result in increased levels of a -MS H with increased pigmentation and suppre ssion of hypersensitivi ty reacti ons. a MSH, a -Melanocyte-stimulating hormone; fL-! , interleukin I; /L-6, interleukin 6; M-E, epidermal melanocyte; M-D, dermal melanocyte; TNFa , tumor nec rosis factor-a.

in which the dermal component is usually negative for HMB -45.6, 7,10 In a previous study of HMB-45, we found that HMB-45 stained the dermal component in none of 30 common acquired nevi, although most melanocytic lesions showed some staining of the the junctional component. 10 HMB-45 did stain the dermal component in 3 of 20 congenital nevi, 2 of 30 spindle cell and epithelioid cell nevi, all of 30 deep penetrating nevi and 25 cellular blue nevi, and 28 of 30 MMs . 10 HMB-45 stains melanosomes in early stages of development.12Thus HMB-45 seems to be more closely related to activation of melanocytes, th at is, the production of premelanosomes rather than their proliferation," 9, 12 HMB-45 has been reported to sta in melanomas and Spitz nevi that produce little or no pigment. In these lesions, the melanosomes are in their ea rly stage of development or a re defective. However, H MB-45 sta ining is also seen in pigmented melanocytic lesions and in areas of hyperpigmentation secondary to stimulation by local inflammation or neoplasrn.P- 7,9,10 Smoller et a1. 7 have shown that some combinations of growth factors that do not support the growth ofmelanocytes may be sufficient to stimulate the production of melanosomesand thus increase HMB-45 staining in melanocytes, They postulat ed that because rnelano-

cytes do not normally proliferate in adult skin, their growth requirements may be more highly regulated than cell populations that do proliferate in the skin. Of the lesions we examined, only the two melanomas showedsignificant positive peNA staining in melanocytes. These results reinforce the concept that positive HMB-45 staining is associated with activation and not necessarily with cellular proliferation. lO• I I In the benign melanocytic nevi the finding of increased HMB-45 staining without significant staining for peNA suggests th at the melanocytes have been stimulated to produce melanosomes but not to proliferate. In HIV-l disease as patients become immune suppressed, they also become immune activated as well as immune dysregulated . This is supported by elevation of cytokinessuch as interleu kins (IL) 1 and 6 and tumor necrosis factor-a (T N F-a ).LJ-17 Elevations of IL-I , IL-6, and TNF-a may induce inhibitors of these cytokines.P : 19 Increased blood levels of IL-I, IL-6 , and TNF-a induce a febrile response through the hypoth almus, and the neuropeptide a-melanocyte-stimulating hormone (MSH), when released from the anterior pituitary, is a potent natural antipyretic agent that antagonizes the fever induced by IL-l, IL-6, an d TNF-a. 20-24 In addition,

544 Smith et al. IL-l upregulates a-MSH receptor expression by melanocytes and melanoma cells as well as melanin production in the presence of MSH, and IL-l acts directly on the adrenal gland to augment steroid synthesis.P a-MSH is a potent stimulant for melanocytic activation and pigmentation; however, this is not the only effect of a-MSH. a-MSH receptors are widespread, and a-MSH has inhibitory effects on other functions of IL-l, IL-6, and TNF-a. 20·24 The antiinflammatory effects of a-MSH include an inhibitory effect on both the sensitization and elicitation limbs of the cutaneous immune response to contact sensitizers.22- 24 Thus the body's natural mechanism for controlling fever with the release of a-MSH from the anterior pituitary may be contributing not only to the increased development of pigmented lesions clinically but also to the decline in some immunologic features used by some for staging of HIV-l disease (e.g., elicitation of delayed hypersensitivity reactions) (Fig. 6).22-24 HMB-45 staining of pigmented lesions in HIV-I-positive patients, indicative of early melanocytic activation, is evidence, albeit indirect, that a-MSH levels may be increased in HIV-l disease. Support for increased levels of a-MSH in some HIV-l-positive patients comes from a recent report of a patient with diffuse hyperpigmentation and normal cortisol levels as well as a normal corticotropin stimulation test who showed increased levels of an MSH-like hormone.! The results of this study suggest that in some HIV-l-infected patients there is stimulation of melanocytes to increase the production of premelanosomes, as indicated by increased staining with HMB-45 without significant proliferation as detected by peNA staining. REFERENCES 1. DuvicM, Lowe L, Rapini RP, et al. Eruptivedysplastic nevi associated with human immunodeficiency virus infection. Arch Dermatol1989; 125:397-401. 2. Poizot-Martin I, Gobb JJ, Dhiver C, et al. Benign melanocytic nevi and AIDS: a case control study. Sixth International Conferenceon AIDS, 1990:357 [Abstract]. 3. Greenberg RG, Berger TG. Nail and mucocutaneous hyperpigmentation withazidothymidine therapy. J AMACAD DERMATOL 1990;22:327-30. 4. Gamby T, DhiverC, Lafeuillade A, et al. Hyperpigmentation of skin in HIV-infected patients. Sixth International Conferenceon AIDS, 1990:357 [Abstract]. 5. Brigitte M, Dutatre H, Huard A, et a1. Skin hyperpigmentation in AIDS patients: Hypophyseal dysfunction? Seventh International Conference on AIDS 1991:230 [Abstract]. 6. Gown AM, Vogel AM, Hoale DH, et al. Monoclonal antibodies specific for melanocytic tumors distinguish sub-

Journal of the American Academy of Dermatology October 1993

populations of rnelanocytes, Am J Pathol 1986;123:195203. 7. Smoller BR, McNutt NS, Hsu A. HMB-45 staining of dysplastic nevi: support for a spectrum of progression toward melanoma. Am J Surg Pathol 1989;13:680-4. 8. Garcia RL, Coltrera MD, Gown AM. Analysis of proliferative grade using anti-PeNA/cyelin monoclonal antibodies in fixed, embedded tissues:comparison with flow cytometric analysis. Am J Pathol 1989;134:733-9. 9. Benz G, Holzel D, Schmoeckel C. Inflammatory cellular infiltrates in melanocytic nevi.Am J Dermatopathol 1991; 13:538-42. 10. Skelton HG, Smith KJ, Barrett TL, et al. HMB-45 staining in benign and malignant melanocytic lesions: a reflection of cellular activation. Am J Dermatopathol 1991; 13:543-50. II. Smoller BR, McNutt NS, Hsu A. HMB-45 recognizes stimulated melanocytes. J Cutan Pathol 1988;16:49-53. 12. Schaumberg-Lever G, Metzler G, Kaiserling E. Ultrastructural localization of HMB-45 binding sites. J Cutan Pathol 1991;18:432-5. 13. Ascher MS, Sheppard HW. AIDS as immune system activation. II: The panergicimnesia hypothesis. J Acquir Immune Defic 1990;3:177-91. 14. Honda M, Kitamura K, Mizutani Y, et al. Quantitative analysis of serum IL-6 and its correlation with increased levels of serum IL-2R in HIY-induced diseases. J Immunol 1990;145:4059-64. 15. Breen EC, Rezai AR, Nakajima K, et a!. Infection with HIY is associated withelevated IL-6levels and production. J Immunol 1990;144:480-4. 16. Hess G, RossolS, RossolR, et a!.Tumor necrosisfactor and interferon as prognostic markers in human immunodeficiency virus (I-IIY) infection. I Med Klin PolikJin 1991; 19(suppl, 2):S93-7. 17. Haber D, Haque A, Wattre P, et aI: Production of tumour necrosis factor-alpha (TNF-alpha) andinterleukin-I (IL-I) in patients with AIDS: enhanced levelofTNF-alpha is related to a higher cytotoxic activity. Clin Exp Immunol 1989;78:329-33. 18. Locksley AM, Crowe S, Sadick MD, et al. Release of interleukin 1 inhibitory activity (contra-Il--l ) by human monocyte-derived macrophages infected with human immunodeficiency virus in vitro and in vivo. J Ciin Invest 1988;82:2097-105. 19. Swope VB, Abdel-Malek Z, Kassem LM, et a!. Interleukins l alpha and 6 and tumor necrosis factor alpha are paracrine inhibitors ofhuman melanocyte proliferation and melanogenesis. J Invest DermatoI1991;96:180-5. 20. Martin L W, Catania A, Hiltz ME, et a!. Neuropeptide alpha-MSH antagonizes IL-6 and TNF-induced fever. Peptides 1991;12:297-9. 21. LiptonJM. Modulationof host defense by the neuropeptide alpha-MSI-I. Yale J BioI Med 1990;63:173-82. 22. Rheins LA, Cotleur AL, Kleier RS, et al. Alpha-melanocyte stimulating hormone modulates contact hypersensitivity responsiveness in C57/BL6 mice. J Invest Dermatol 1989;93:511-7. 23. Robertson B, Dostal K, Daynes RA. Neuropeptide regulation of inflammatory and immunologic responses. J ImmunoI1988;140:4300-7. 24. Martin LW, Lipton JM. Acute phase responseto endotoxins: rise in plasma alpha-MSH and effects of alpha-MSH injection. Am J Physiol 1990;259:768-72. 25. Schwarz T, Luger TA. Pharmacology of cytokines in the skin. In: Mukhtar H, ed, Pharmacology of the skin. Ann Arbor: CRC Press, 1992:283-313.