JOURNAL OF PATHOLOGY, VOL.
161: 3 5 4 0 (1990)
NATURAL KILLER CELLS IN CUTANEOUS MALIGNANT MELANOMA N. M. KERNOHAN, H. F. SEWELL AND F. WALKER
Department of Pathology. University of Aberdeen. Medical School Buildings, Foresterhill. Aberdeen AB9 2 2 0 , U.K. Received 6 September 1989 Accepted 22 November I989
SUMMARY Using immunocytochemical techniques on fresh surgical samples, a series of 16 cases of cutaneous malignant melanoma (CMM) were examined to characterize further the host inflammatory response. Antibodies to the following cluster of differentiation (CD) antigens were used: CD-3, CD-4, CD-8 (T-cell markers), CD-I lb, CD-14 (macrophage marker), CD-16 [an antigen expressed by natural killer (NK) cells and granulocytes],and CD-25. Also examined were a small number of other melanocytic lesions [two cases of lentigo maligna (Hutchinson’smelanotic freckle) and five of intradermal naevi]. The results of the study document a population of cells with the morphological and immunophenotypic characteristics of NK cells in association with 10 of the 16 cases of CMM. These cells were consistently absent from the other melanocytic lesions studied. The presence of NK cells in association with some cases of CMM bears no clear relationship to the Breslow thickness, Clark level, tumour ulceration, or the presence of activated T cells as determined by expression of the CD-25 antigen. Whilst an explanation for the significantnumbers of NK cells in some CMM lesions is unclear, their presence in intimate association with tumour cells does prompt speculation regarding a possible role in determining the biological behaviour of the turnour. Additionally, the study has confirmed and extended previous findingswith respect to the broad characterization of mononuclear cells present in the host infiltrate associated with CMM. KEY
WoRDs-Cutaneous malignant melanoma, natural killer cells. INTRODUCTION
16 is expressed by N K cells and granulocytes and is reco ized as being a reliable marker for N K cells.’>’ In view of their proposed role in host defence against tumour development and metastasisI6 and also as they are the principal cell type giving rise to lymphokine-activated killer (LAK) cell demonstration of N K cells in relation to lesions of CMM may disclose information of prognostic value.
gn
In view of the enigmatic behaviour of CMM,’ the characteristic phenomenon of tumour regression,’ and the current successful application of adoptive immunotherapeutic techniques to the management of disseminated disease,- there has been much interest concerning the potential role of the host immune response in influencing the biological behaviour ofthe tumour. There are now several reports in the literature that document the immunophenotypic characteristics of the host response to CMM, and all present broadly similar findings.%I4In our MATERIALS A N D METHODS attempts to dissect further the immunophenotypic composition of the host lymphocytic reaction to Tissue CMM, antibodies directed against the CD-I l b and CD- 16 antigens were used. The antigen CD- 1 1b is From fresh surgical excision specimens, tissue expressed on natural killer (NK) cells, putative T- bearing a representative portion of a melanocytic suppressor cells, monocytes, and granulocytes; CD- skin lesion along with adjacent normal skin was Addressee for correspondence:Dr N. M. Kernohan, Lkpart- donated for immunohistochemical study. The tismerit of Pathology, University Medical Buildings, Foresterhill, sue, which was 1-2 mm in maximum thickness, was Aberdeen AB9 2ZD. U.K. snap-frozen in Arcton (ICJ)/liquid nitrogen and 0022-34 I7/90/050035-06$05.00 0 1990 by John Wiley & Sons, Ltd.
s.M. KERNOHAN E r AL.
36 Table I-Source,
specificity. and working dilution of the antibodies utilized in this study
Antibody
Specificity
Source
Working dilution
-~
CD-3 CD-4
Oxoid Dakopatts
CD-8 CD-IIb(Leu 15)
Dakopatts Becton-Dickinson
CD-I6(Leu Ilb)
Becton-Dickinson
CD-14 CD-25 Rabbit anti-mouse (RAM) immunoglobulins Alkaline phosphatasei anti-alkaline phosphatase (APAAP complex)
Oxoid Dakopatts Dakopatts Dakopatts
stored at - 20°C double-wrapped in 'Parafilm' until required for sectioning and staining. Immunohistochemical method Cryostat cut sections, 4 - 6 ~thick on chrome alum gelatin-subbed slides, were used throughout. The source, specificity, and working dilution of the antibodies used in this study are listed in Table I. All dilutions and washes were made using 0.005 M Tris-buffered saline (TBS), pH 7.6. Serial sections were cut from the lesions and stained using the antibodies listed in Table I in a standard three-stage APAAP technique,"-*' following fixation in acetone for 20 min, after which they were allowed to air-dry for 5 min. The alkaline phosphatase was developed as previously described'' using Fast Red TR salt (Sigma) as the chromogen, which provided excellent contrast with the light haematoxylin nuclear counterstain and endogenous melanin. Optimal demonstration of CD- 16 required the use of an enhanced APAAP technique, whereby the second and third stages (RAM and APAAP, respectively) were repeated with 15 min incubations prior to development of the enzyme reaction. Assessment of results A negative control slide where TBS was substituted for the monoclonal antibody was available for each case. This was of particular value when assessing sections stained using an enhanced APAAP technique. which, although intensifying specific positive
Pan-T T helperlinducer + some macrophageimonocytes T suppressorjcytotoxic T suppressor cells, NK cells, monocytes, granulocytes Fc IgG receptor on NK cells and neutrophils Monocytes Interleukin-' receptor -
1:200 1:lO
1:lO 1:lO
1:lO 15 1:lO 1 :20 1 :40
staining, may give rise to background staining without rigorous attention to the washing of sections between each stage of the immunohistochemical procedure. Positive control slides were available from other cases whose lymphocytic infiltrate had been characterized previously. Where cells expressing CD- 16 were identified, reference was made to other sections from the case and it was confirmed that the cell population did not express markers indicative of T-cell (CD-3, CD-4, CD-8) or macrophage (CD-14) lineage. As CD-11 b is expressed by putative T-suppressor cells, monocytes, granulocytes, and NK cells. demonstration of apparent co-expression of CD-11 b and CD-16 was taken as evidence that cells so stained were NK cells. Many more cells expressed CD-11 b than CD-16, but as expected the distribution of the excess CD1 1 b positive cells coincided with that of T-cells, macrophages, and granulocytes. As CD-I 1 b and CD- 16 may also be co-expressed by polymorphonuclear granulocytes as well as by mononuclear NK cells, the morphological appearances of the positively stained cells were carefully noted. Furthermore, to ascertain that CD-16/CD-I 1b positively stained cells were NK cells and not granulocytes, sections were exposed for 6 min to 3',3'-diaminobenzidine tetrahydrochloride solution, a substrate for the endogenous peroxidase enzyme present in granulocytes but absent in NK cells. The intense brown peroxidase-substrate reaction product in granulocytes could be easily distinguished from
NATURAL KILLER CELLS IN CUTANEOUS MELANOMA
Table 11-Distribution of CD-16/CD-11b positive cells in the inflammatory infiltrate associated with melanocytic skin lesions correlated with the presence or absence of ulceration of the lesion and CD-25 positive cells in the infiltrate
Lesion
Nos.
CD-16 positive cells
~~
CMM HMF IDN
Ulceration
+
-
Incipient
CD-25 positive cells
1 0 0
16 2 5
~
16 2 5
10 0 0
5 4 0 2 0 5
37
one showed incipient ulceration, and four were non-ulcerated. Of the remaining six cases, five were non-ulcerated and one showed incipient ulceration. Ulceration was defined histologically (Tables I1 and 111) using routinely processed haematoxylin and eosin stained tissue sections. From these sections the histogenetic type of melanoma, its Clark level, and Breslow thickness were also assessed, but none of these parameters correlated with the presence of NK cells (results not shown).
DISCUSSION
In this study a significant population of cells displaying the morphological and immunophenotypic characteristics (CD-1 lb’, CD-16+, CD-3-, CD14-, peroxidase-) that correlate with functional NK cells has been demonstrated in association with 10 of 16 primary CMM lesions. In addition, the absence of such cells from both cases of lentigo RESULTS maligna, a premalignant lesion, and all cases of A population of mononuclear cells expressing benign, intradermal naevi was recorded. the CD-16 antigen was identified in association with NK cells form a distinct subset of lymphoid cells 10 of the 16 cases of CMM (Fig. 1). These cells with the morphological appearance of large granuaccounted for up to 10 per cent of the chronic lar lymphocytes, and for which the CD-16 antigen inflammatory cell infiltrate and were found princi- is recognized as being the most reliable immunopally within the substance of the tumour, either phenotypic marker, in preference to anti-HNK-1 among tumour cells or in the stroma between used in previous studies.” NK cells are capable of groups of tumour cells. A minority of the cells were non-MHC restricted killing of target tumour cells, present in the inflammatory infiltrate deep in the in the absence of specific antibody or antigenic tumour. CD-16 positive cells were also present near stimulation. The identification of such cells in the ulcerated surface of some tumours, but closer association with primary CMM lesions may thereexamination indicated that most of the stained cells fore be seen as a potentially favourable feature of in this instance were granulocytes. No case of LM or the host response to the tumour. Furthermore, the IDN displayed a similar CD- 16 positive cell popu- presence of NK cells, often in intimate association lation in the inflammatory infiltrate (Table 11). with tumour cells (Fig. 1) is of considerable interest Serial sections from each case were examined and in view of the development of adoptive immunoconfirmed that T-cell markers were not expressed by therapy in the treatment of disseminated malignant the CD-16 positive cells (Fig. 2). Co-expression of melanoma. Such therapy requires the administhe CD- 16 and CD- 1 1b antigens was, however, tration ofcombinations of the cytokine interleukin-2 noted but CD-1 l b positive cells were more abun- (IL-2) and autologous lymphokine-activated killer dant than CD-16 positive cells. Immunopheno- cells (LAK cells). NK cells are now known to be typic, histochemical, and morphological control the principal precursors of LAK cell^,'^.'^ and criteria (see above) documented the excess CD-1 l b tumours already containing a significant population cells as a mixture of T-cells, macrophages, and, in of these LAK cell precursors (10 of 16 cases in the some cases, granulocytes. present study) might be expected to respond well Lymphoid cells expressing the CD-25 (inter- to immunotherapeutic approaches as pioneered by leukin-2 receptor) antigen were present in all cases Rosenberg. Despite attempts to refine such treatof CMM, LM, and IDN studied. Of the ten cases of ment rtgimes,* adoptive immunotherapy is associCMM containing CD-16/CD-l1b positive cells in ated with si nificant morbidity and, occasionally, the inflammatory infiltrate, five were ulcerated, Therefore additional information melanin pigment present in some sections. Thus, the criteria used clearly demonstrated the presence, immunophenotypically and morphologically, of CD- 16/CD-11b positive ‘NK’ cells.
38
N. M. KERNOHAN E T A L .
Fig. I X M M associated with mononuclear cells expressing CD-16 (Leu Ilb). Many of these cells lie within a nodule of turnour cells (anti-CD-16. enhanced APAAP)
Fig. 2-Same area from the same case as in Fig. 1 to show T cells; the cell population stained by anti-CD-16 is distinct (anti-CD-3. APAAP)
39
NATURAL KILLER CELLS IN CUTANEOUS MELANOMA
Table 111-Presence or absence of ulceration in cases of CMM correlated with expression of CD-16/CD-I1b by cells in the host inflammatory response CD-I 6/CD-1 I b (+)
CD-i 6/CD-11b ( - )
5
4 1
5
Ulceration No ulceration Incipient ulceration
that may be gleaned by immunohistochemical analysis of pretreatment biopsies might contribute to the rational use of such therapy. NK cells have previously been recognized in as~ sociation with melanoma. Kornstein et ~ 1 . , *using another CD-16 antibody, found NK cells in 1 of 8 primary LMM lesions, 10 of 12 metastatic deposits, and only 3 of 3 1 ‘dysplastic naevi’. The conclusion drawn was that NK cells probably did not play a major role in surveillance preventing the ‘dysplastic’ lesions progressing to invasive melanoma. Using an anti-CD-16 antibody with an enhanced APAAP technique, in this study we have demonstrated NK cells in a higher proportion of primary lesions but, like Kornstein er al., failed to detect them in association with the premalignant lesion, lentigo maligna. A further small series of six cases was reported as part of a study by Cohen et ~ l . , ~where ’ NK cells were rarely found in association with metastatic melanoma lesions either before or after adoptive immunotherapy with rIL-2 and LAK cells. Yamamura et u I . , ~ ‘ however, found that in a case of metastatic melanoma treated by a similar adoptive immunotherapeutic regime NK cells were present in large numbers after treatment in the tumour deposits, a finding contrary to that reported in Cohen et al.’s series. Thus, the biological role of NK cells in relation to primary and metastatic melanoma lesions and their presence in response to immunotherapy is unclear. NK cell recruitment to the site of a tumour could be the result of the release of chemical mediators by non-specific inflammatory and immunological reactions.” Such mediators include activated T-cell derived interleukin-2 and y-interferon. Activated T lymphocytes have been found in association with melanocytic lesions, both benign and malignant, and in this study they were present in all cases. Accordingly, neither the presence of CD-25 positive T-lymphocytes per se nor prognostic factors (such as the histogenetic type of tumour, its Clark level, Breslow thickness, or ulceration) correlated with the presence of NK cells.
1
In conclusion, this study has demonstrated NK cells in association with some cases of CMM. Such cells were absent from the small number of other melanocytic lesions studied including two cases of LM, a premalignant lesion. These findings prompt speculation concerning the possible role of NK cells in determining not only the biological behaviour of melanoma, but also its response to adoptive immunotherapy regimes. ACKNOWLEDGEMENTS
This work was supported through funding by the Scottish Hospitals Endowment Research Trust and by Grampian Health Board, who generously supplied the antibodies used. The authors wish to express their thanks to Mr I. R. Kernohan, who provided fresh excision biopsy specimens for use in this study; and to Mrs I. M. Watson for typing this manuscript. The expert technical assistance of Shirley Sinclair and George King is gratefully acknowledged. REFERENCES I . Elias EG. Didolkar MS. Goel IP, e r a / . A clinico-pathologx study of prognostic factors in cutaneous malignant melanoma. SurR Gynecol Obsrer 1977; 144: 327-334. 2. McGovern VJ. Spontaneous regression of malignant melanoma. In. Melanoma Hisrolagical Diagnosis and Prognosis. New York: Raven Press. 1982: 138-147. 3. Lotzc MT. Chang AE. Seipp CA. er a / . High dose recombinant interleukin-2 in the treatment of patients with disseminated cancer. J Am MedAssoc 1986;u6:3117-3124. 4. Oliver RTD. Theclinical potential of interleukin-2. Br J Cancer 1988: 58:4oM. 5. Rosenberg SA. Adoptive immunotherapy of cancer: accomplishments and prospects. Cancer Treat Rep 1985: 68: 233-255 6 . Rosenberg SA. Lotze MT, Muul LM, er al. Observations on the systemic administration of autologous lymphokine-activated killer cells and recombinant interleukin-2 to patients with metastatic cancer. h‘ EngfJ Med 198%313 1485-1492. 7. Rosenberg SA. Lotze MT. Muul LM. ei a/. A progress report on the treatment of I57 patients with advanced cancer using lymphokineactivated killer cellsand interleukin-? or high dose interleukin-2 alone. N €ng/ J Med 1987: 316 889-897.
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