Loss of androgen receptor associated protein 70 (ARA70) expression in a subset of HER2-positive breast cancers.

Co-transfection studies indicate that HER2 (erbB-2) overexpression results in the phosphorylation and enhanced transcriptional activity of the androgen receptor (AR). This amplification of AR action is further enhanced by the expression of ARA70, a putative co-activator with a predilection for the AR. Because androgens inhibit the growth of breast cancer cells whereas HER2 overexpression stimulates the growth of these cells, it seems possible that loss of expression of AR or ARA70 in some HER2 overexpressing tumors might confer a growth advantage to these cells. We examined ARA70 and AR expression in 20 HER2-positive (overexpressing) and 21 HER2-negative cases of breast invasive ductal carcinoma (IDC) to determine the relationship between loss of ARA70 and/or AR with HER2 overexpression. Strong ARA70 immunostaining was observed in all normal and breast epithelial cells in fibrocystic change and in in situ carcinoma present in the patient samples. Of the 41 cases of IDC, focal or complete loss of ARA70 protein expression was observed in 46% of the cases, with 60% of HER2-positive versus 33% of HER2-negative cases showing loss. Loss of AR expression was observed in 60% of HER2-positive versus 43% of HER2-negative cases. Remarkably, only 20% of HER2-positive tumors expressed both AR and ARA70, while 43% of HER2-negative tumors expressed both of these elements of the AR signaling pathway. This trend is consistent with a possible clinical relevance of the potential crosstalk between the HER2 and AR signaling pathways. Western blot analysis for ARA70 expression performed on frozen breast biopsies of normal or malignant breast tissue from four patients revealed a 70 kDa immunoreactive band in all four normal tissue samples, with an additional 35 kDa band in two of the breast cancer samples and in human breast cancer MCF-7 cells. This may reflect aberrant splicing in some breast cancers, leading to the emergence of the 35 kDa isoform.

Defining a test for HER-2/neu evaluation in breast cancer in the diagnostic setting.

In breast cancer amplification of the HER-2/neu oncogene and over-expression of the protein product is associated with poor prognosis, predicts response to some chemotherapeutic regimens and is the target for Herceptin treatment. To date there are several methods to assess the amplification/over-expression of HER-2/neu with each having advantages and disadvantages. We have studied amplification and over-expression of HER-2/neu in 250 consecutive cases of breast cancer (220 invasive and 30 in situ carcinomas) presenting to the Department of Pathology at Women's College Campus of Sunnybrook and Women's College Health Sciences Center. Thirty percent of the invasive carcinomas were node positive. HER-2/neu protein over-expression was assessed by immunohistochemistry (IH) using antibody CB11 and amplification of the gene by differential PCR. The percentage of tumor cells showing CB11 staining was determined and the most significant cut off point for positivity was > or =10% moderate or strong complete membranous staining. The gene was considered amplified if the density score of the product was > or =2. There was 94% concordance between the two methods (P value.0001). Both methods were positive in 16% of cases and negative in 78% of cases. Discrepant cases were examined by FISH which confirmed the IH results in 9/11 invasive carcinomas. These results show that there is excellent concordance between IH and PCR. However, immunohistochemistry is easier to perform and cheaper than PCR and could be used in routine assessment of HER-2/neu in breast cancer patients.

Tenascin differentiates dermatofibroma from dermatofibrosarcoma protuberans: comparison with CD34 and factor XIIIa.

Differentiation of dermatofibroma (DF) from dermatofibrosarcoma protuberans (DFSP) can be difficult. CD34 and Factor XIIIa have been used to differentiate DF from DFSP. However, there is overlap and lack of specificity of their expression. Tenascin is an extracellular matrix glycoprotein that is involved in embryogenesis, carcinogenesis, and wound healing. The aim of the study was to assess the role of tenascin in DF and DFSP and compare the results with those obtained with CD34 and Factor XIIIa. Immunohistochemical staining was performed on 20 cases each of DFSP and DF, using antibodies to tenascin, CD34 and Factor XIIIa, and the streptavidin biotin technique. Positivity for all 3 antibodies was assessed within the tumors. Tenascin expression was also assessed at the dermal-epidermal junction. Strong tenascin positivity was noted at the dermal-epidermal junction overlying the lesion in 20 of 20 cases of DF (100%) and was negative over the lesion in 20 of 20 cases DFSP (100%). Tenascin was noted within the lesion of 80% of both DF and DFSP (16/20 cases). CD34 was strongly expressed in 16 of 20 (80%) DFSP and 5 of 20 (25%) DF, whereas Factor XIIIa was strongly expressed in 19 of 20 (95%) DF and 3 of 15 (15%) DFSP. Although CD34 was expressed in 80% DFSP and Factor XIIIa in 95% of DF, there was overlap in their expression in the 2 types of tumors. The increased expression of tenascin at the dermal-epidermal junction overlying the lesion in DF but not in DFSP, differentiated these 2 tumors. In contrast, tenascin expression within the lesion did not differentiate DF from DFSP.

Factors associated with local breast cancer recurrence after lumpectomy alone: postmenopausal patients.

BACKGROUND: We have been following a cohort of patients who underwent a lumpectomy without receiving adjuvant radiotherapy or adjuvant systemic therapy. We now report the experience of a postmenopausal subgroup. METHODS: The postmenopausal subgroup included 244 patients accrued between 1977 and 1986 and followed up. The end point was ipsilateral local breast cancer recurrence. The factors studied were the patient's age in years; tumor size (in mm); nodal status (N-, Nx, N+); estrogen and progesterone receptor status (< 10, - 10 fmol/mg protein); presence or absence of lymphovascular/perineural invasion; presence or absence, and type, of DCIS (none, non-comedo, comedo); percentage of DCIS; histological grade (1,2,3); and nuclear grade (1,2,3). Univariate analyses consisted of Kaplan-Meier plots and the Wilcoxon (Peto-Prentice) test statistic; the multivariate analyses were step-wise Cox and log-normal regressions. RESULTS: The median follow-up of those patients still alive was 9.1 years, and the overall relapse rate was 24% (59/244). The univariate results indicated that the characteristics of smaller tumor size, negative nodes, positive ER status, and no lymphovascular or perineural invasion were associated with significantly (P <.05) lower relapse. From the multivariate analyses, the factors lymphovascular or perineural invasion, age, and amount of DCIS were all significantly associated with local relapse with both Cox and log-normal regressions. Additionally, there was weak evidence of an association between ER (P = .08 in the Cox regression and in the log-normal) and nodal status (P = .09 in the log-normal regression) with local relapse. We also are able to define a low-risk subgroup (N-, age -65, no comedo, ER positive, no emboli) with a crude 10-year local recurrence rate of 9%. CONCLUSION: With longer follow-up, and for postmenopausal patients, there continues to be support for the theory that local relapse is affected by the factors lymphovascular or perineural invasion, age, amount of DCIS, ER, and nodal status. A low risk subgroup has been identified.

Factors affecting distant disease-free survival for primary invasive breast cancer: use of a log-normal survival model.

BACKGROUND: Invasive breast cancer is a frequently diagnosed disease that now comes with an ever expanding array of therapeutic management options. We assessed the effects of 20 prognostic factors in a multivariate context. METHODS: We accrued clinical data for 156 consecutive patients with stage 1-3 primary invasive breast cancer who were diagnosed in 1989-1990 at the Henrietta Banting Breast Center, and followed to 1995. There is complete follow-up for 91% of patients (median follow-up of 4.9 years). The event of interest was distant recurrence (for distant disease-free survival, DFS). We used Cox and log-normal step-wise regression to assess the multivariate effects of the following factors on DFS: age, tumor size, nodal status, histology, tumor and nuclear grade, lymphovascular and perineural invasion (LVPI), ductal carcinoma-in-situ (DCIS) type, DCIS extent, DCIS at edge of tumor, ER and PgR, ERICA, adjuvant systemic therapy, ki67, S-phase, DNA index, neu oncogene, and pRb. RESULTS: There was strong evidence against the Cox assumption of proportional hazards for nodal status, and nodal status was not in the Cox step-wise model. With step-wise log-normal regression, a large tumor size (P < .001), positive nodes (P = .002), high nuclear grade (P = .01), presence of LVPI (P = .03), and infiltrating duct carcinoma not otherwise specified (P = .05) were associated with a reduction in DFS. CONCLUSIONS: For nodal status, there was strong evidence against the Cox assumption of proportional hazards, and it was not included in the Cox model although it was in the log-normal model. Only traditional factors were included in the step-wise models. Thus, this statistical management of prognostic markers in breast cancer appears to be very important.

RT-PCR amplification of CK19 mRNA in the blood of breast cancer patients: correlation with established prognostic parameters.

We optimized the assay for detection of cytokeratin 19 (CK19) mRNA by the reverse transcriptase-polymerase chain reaction (RT-PCR) in blood as an index of circulating tumor cells in breast cancer patients. The limit of detection of < 1 MCF7 tumor cells per 10(6) peripheral blood leukocytes (PBL) was achieved in mixing experiments. We did not detect CK19 mRNA in control bloods (0/30) or in the blood of patients with benign breast disease (0/15). In blood samples from 109 patients with invasive breast cancer, CK19 mRNA was detected in 7/23 patients with node-negative disease, in 21/58 with node-positive disease, and in 20/28 with distant metastases. There was a significant association (P < 0.01) of CK19 positivity with distant metastatic versus both node-negative and node-positive disease, but not with any other histopathological parameter examined. In a small number of patients with distant metastases, increased intensity of the CK19 RT-PCR signal was associated with a reduced survival.

The effects of nicotine on Parkinson's disease.

Post-mortem studies have demonstrated a substantial loss of nicotinic receptors in Parkinson's disease (PD), which may be at least partially responsible for some of the cognitive, motoric, and behavioral deficits seen in this disorder. Epidemiologic studies have suggested that cigarette smoking is a strong negative risk factor for the development of PD. We have previously shown that blockade of central nicotinic receptors produces cognitive impairment in areas of new learning, short-term memory, and psychomotor slowing with increasing dose sensitivity with age and disease. Studies of acute stimulation of nicotinic receptors in Alzheimer's disease with nicotine and the novel agonist ABT-418 in our laboratory and others have shown improvements in several measures of cognitive function. Prior studies of the effects of nicotine in PD have suggested some improvements in clinical symptomatology. We have begun quantitative studies of both acute and chronic nicotine in PD to assess both cognitive and motor effects. Fifteen (15) nondemented subjects (age 66 +/- 5.3; M/F = 11/4) with early to moderate PD (mean Hoehn-Yahr stage = 1.77; MMSE = 28.6) received a dose-ranging study of intravenous nicotine up to 1.25 microg/kg/min, followed by chronic administration of nicotine by transdermal patch with doses ranging up to 14 mg per day for 2 weeks. Testing occurred both during drug administration and up to 2 months after drug cessation to look for prolonged effects. Preliminary analysis shows improvements after acute nicotine in several areas of cognitive performance, particularly measures such as reaction time, central processing speed, and decreased tracking error. Improvements in attention and semantic retrieval were not seen. After chronic nicotine, improvements were seen in several motor measures suggesting improved extrapyramidal functioning. This appeared to be sustained for up to 1 month after drug. The treatment was well tolerated. Nicotinic stimulation may have promise for improving both cognitive and motor aspects of Parkinson's disease.

Evaluation of HER-2/neu (erbB-2) status in breast cancer: from bench to bedside.

Molecular alterations in breast cancer are being incorporated into the development of new treatment strategies. The HER-2/neu oncogene has been extensively investigated as a prognostic factor and recently as a predictor of response to chemotherapy or endocrine therapy. The development of a humanized anti-HER-2 monoclonal antibody (Herceptin) and the encouraging results obtained in the treatment of patients with HER-2 overexpressing metastatic breast cancer with this antibody have resulted in renewed interest in HER-2/neu. This article reviews the current knowledge of HER-2/neu both as a prognostic and a predictive factor. Problems associated with the standardization of the methodology for assessing HER-2/neu status and clinically significant cut-off points are addressed.

Inhibition of norepinephrine-induced cardiac hypertrophy in s100beta transgenic mice.

We have recently reported that the Ca2+-binding protein S100beta was induced in rat heart after infarction and forced expression of S100beta in neonatal rat cardiac myocyte cultures inhibited alpha1-adrenergic induction of beta myosin heavy chain (MHC) and skeletal alpha-actin (skACT). We now extend this work by showing that S100beta is induced in hearts of human subjects after myocardial infarction. Furthermore, to determine whether overexpression of S100beta was sufficient to inhibit in vivo hypertrophy, transgenic mice containing multiple copies of the human gene under the control of its own promoter, and CD1 control mice were treated with norepinephrine (NE) (1.5 mg/kg) or vehicle, intraperitoneally twice daily for 15 d. In CD1, NE produced an increase in left ventricular/body weight ratio, ventricular wall thickness, induction of skACT, atrial natriuretic factor, betaMHC, and downregulation of alphaMHC. In transgenic mice, NE induced S100beta transgene mRNA and protein, but provoked neither hypertrophy nor regulated cardiac-specific gene expression. NE induced hypertrophy in cultured CD1 but not S100beta transgenic myocytes, confirming that the effects of S100beta on cardiac mass reflected myocyte-specific responses. These transgenic studies complement in vitro data and support the hypothesis that S100beta acts as an intrinsic negative regulator of the myocardial hypertrophic response.

S100beta inhibits alpha1-adrenergic induction of the hypertrophic phenotype in cardiac myocytes.

In an experimental rat model of myocardial infarction, surviving cardiac myocytes undergo hypertrophy in response to trophic effectors. This response involves gene reprogramming manifested by the re-expression of fetal genes, such as the previously reported isoform switch from adult alpha- to embryonic beta-myosin heavy chain. We now report the transient re-expression of a second fetal gene, skeletal alpha-actin in rat myocardium at 7 days post-infarction, and its subsequent down-regulation coincident with the delayed induction of S100beta, a protein normally expressed in brain. In cultured neonatal rat cardiac myocytes, co-transfection with an S100beta-expression vector inhibits a pathway associated with hypertrophy, namely, alpha1-adrenergic induction of beta-myosin heavy chain and skeletal alpha-actin promoters mediated by beta-protein kinase C. The induction of beta-myosin heavy chain by hypoxia was similarly blocked by forced expression of S100beta. Our results suggest that S100beta may be an intrinsic negative regulator of the hypertrophic response of surviving cardiac myocytes post-infarction. Such negative regulators may be important in limiting the adverse consequences of unchecked hypertrophy leading to ventricular remodeling and dysfunction.

An improved statistical approach: can it clarify the role of new prognostic factors for breast cancer?

Recently, there has been a proliferation of new biomarkers, some of which may lead to an improved prognostic index or may influence treatment selection. However, there are methodological and statistical issues that require attention in assessing the role and use of these prognostic factors. Between 1977 and 1986, 1097 primary breast cancer patients were accrued for multidisciplinary research at the Henrietta Banting Breast Centre, Women's College Hospital; follow-up to 1990 is complete for 96% of the patients. Data for these patients are used here to illustrate strategies: (1) for the comparison of results from diverse assessments of biomarkers; (2) for the improved comparability of inter-laboratory results; (3) for the examination of the results from monoclonal or polyclonal antibody assays for possible clinically relevant bimodality; (4) for good statistical resolution of overlapping distributions; (5) that involve the use of quantitative values for prognostic factors whenever possible; and (6) for improved multivariate analyses. Good data handling and analyses may enable more accurate and rapid assessment of new prognostic factors, thereby expediting and improving their clinical application.

Alternative multivariate modelling for time to local recurrence for breast cancer patients receiving a lumpectomy alone.

Certain prognostic factors (patient and/or tumour characteristics) may be associated with low (or high) risk for local recurrence. Patients with these characteristics could be candidates for less (or more) adjuvant therapy or a less (or more) aggressive surgical approach. However, the assessment of many factors can be problematic with the standard multivariate technique-a Cox proportional hazards model and step-wise regression. We compared the results obtained when using a Cox model with those from four alternative models (exponential, Weibull, log logistic and log Normal) in step-wise and all subset regressions. Between 1977 and 1986, 293 primary invasive breast cancer patients were treated at the Henrietta Banting Breast Centre with a lumpectomy with or without an axillary dissection, and with no postoperative adjuvant therapy. The variables considered were age, lymph node status, tumour size, estrogen receptor (ER), progesterone receptor (PgR), histologic grade, nuclear grade, carcinoma in situ (CIS), amount of CIS, and presence of tumour emboli. With follow-up to 1991, nodal status was not found to be included in the step-wise Cox model, although it was in the step-wise exponential, Weibull and log Normal models, and in the best all subset models for all model types. The variables tumour emboli, ER, age, CIS and nodal status were consistently included in the best all subset regressions, regardless of model type. In the 1993 follow-up, the variables in the step-wise Cox model were tumour emboli, ER, age, CIS and nodal status. The multivariate consideration of all possible subsets of regression variables led to an earlier indication of the importance of nodal status, while the data strongly supported accelerated failure time models over the Cox model.

Controlling for variability in research on normal aging and neuropsychological populations discourse analysis and applications: studies in adult clinical populations.

There has been an increase in research during the past few years on discourse, with particular focus on normal older adults and clinical populations, e.g., aphasic and head-injured patients and patients with Alzheimer's disease. A recent edited book, Discourse Analysis and Applications: Studies in Adult Clinical Populations, reviews some of this work. Research on normal older adults and clinical populations raise a number of methodological problems, in particular, variability. Such issues are not easily resolved as we currently have no standard experimental designs for this type of research. This review seeks to point out some of these methodological issues that researchers face and provides some possible solutions.

Proliferative activity and p53 expression in adenoid cystic carcinoma of the breast.

Adenoid cystic carcinoma is a rare type of invasive breast carcinoma that has a good prognosis. We studied a series of four cases of adenoid cystic carcinoma in which we correlated the clinical and pathological features. The pathological features examined included light microscopy; electron microscopy; immunohistochemistry using antibodies to keratin, vimentin, S100 protein, actin, estrogen and progesterone receptors, and proliferation marker MiB-1, and p53 suppressor protein; image cytometric analysis for measurement of DNA ploidy; and molecular analysis using polymerase chain reaction single strand conformation polymorphism to assess point mutation of the p53 gene. All of the cases had a low nuclear grade, were negative for estrogen and progesterone receptors, and were DNA diploid. Three of the cases showed no evidence of metastases and had small primary tumors with low proliferative activity and absence of p53 protein expression. In contrast, one of the cases showed axillary lymph node metastases and in this case the primary tumor was large with a higher proliferative activity and expression of p53 protein, suggesting that these factors might play a role in the biological behavior of adenoid cystic carcinoma.

An investigation of cut-points for primary breast cancer oestrogen and progesterone receptor assays.

Oestrogen and progesterone receptor (ER and PgR) assay values are frequently used in medical decision-making for breast cancer patients. We have proposed statistical standardization of receptor assay values to improve inter-laboratory comparability, and now report the use of standardized log units (SLU) to investigate the effects of ER and PgR cut-points on time to first recurrence outside the breast (DFS). Between 1980 and 1986, there were 678 primary breast cancer patients treated at the Henrietta Banting Breast Centre (HBBC). The effects of ER and PgR cut-points were examined with multivariate analyses considering the variables: age, tumour size, nodal status, weight and adjuvant treatment. We considered receptor assay cut-points ranging from - 1.0 to + 1.0 SLU (ER between 7 and 166 fmol/mg protein; PgR between 7 and 181 fmol/mg protein). PgR was included in the multivariate prognostic models more often than ER, although patients had a better prognosis with both larger ER and PgR values. There was no best cut-point for ER or PgR, and there was strong evidence that ER and PgR should be considered as continuous rather than dichotomous (negative, positive) variables. Patient prognosis should also be more comparable with SLU.

MIB-1 proliferative activity is a significant prognostic factor in primary thick cutaneous melanomas.

Although the Breslow measurement of tumor thickness of melanoma is the most significant predictor of survival, the biologic behavior of thick lesions remains unpredictable. MIB-1, a monoclonal antibody to a Ki-67 epitope, recognizes all proliferating cells. Unlike Ki-67 antibody, which requires frozen tissue, MIB-1 can be used on formalin-fixed tissue. Proliferation, measured by MIB-1 expression and mitotic index, was assessed as a prognostic factor in a group of patients with clinical stage I thick cutaneous melanoma (tumor thickness 4 mm or greater), for which predicted survival is low. From a melanoma data base, 97 patients with this type of melanoma were identified. Of these, 64 had lesional tissue available for study. The median follow-up time was 3.8 years (range 0.42-13.6 years). The percentage of MIB-1 reactivity was scored as low at less than 10% (n = 33), intermediate at 10% to 20% (n = 17), and high at greater than 20% (n = 14). Melanomas with high MIB-1 reactivity were associated with significantly poorer cause-specific survival compared with tumors with intermediate (p < 0.0001) or low MIB-1 reactivity (p = 0.0025). Multivariate analysis demonstrated that MIB-1 reactivity was a significant independent prognostic factor related to cause-specific survival (p = 0.0002) and was more sensitive than tumor thickness or mitotic index in this select group of high-risk patients. Identification of individuals with stage I thick cutaneous melanoma who are at risk of recurrent disease may improve patient management as new therapeutic modalities become available.

bcl-2 protein expression in melanocytic neoplasms of the skin.

Since the discovery of bcl-2 proto-oncogene in follicular lymphomas, the protein product has been detected in a variety of normal tissues including skin, where it is expressed in basal keratinocytes. Recent studies indicate that bcl-2 protein is detected in nonlymphoid malignancies such as neuroblastoma and carcinomas of the lung and prostate. This study investigates the presence of bcl-2 protein in benign and malignant melanocytic neoplasms of the skin. Immunohistochemical analysis of bcl-2 protein expression was performed on 39 nevi and 60 malignant melanomas, including 21 metastases. There was diffuse strong immunopositivity for bcl-2 protein in 100% of nevi and 65% (43/60) of primary and metastatic melanomas. bcl-2 protein was diffusely expressed in 67% (30/39) of primary melanomas and 54% (11/21) of metastases. Although bcl-2 immunoreactivity was observed in all levels of primary cutaneous malignant melanomas, in 43% (9/21) of deep melanomas (Clark level > or = III), and 100% (7/7) of thick tumors (thickness > or = 4.00 mm), there was focal loss of immunoreactivity. Metastatic melanomas showed focal loss of bcl-2 expression in 10% (2/21) of cases and total loss of bcl-2 protein in 39% (8/21). We conclude from our results that bcl-2 protein is expressed by benign and malignant melanocytic tumors of the skin, but there is loss of bcl-2 protein expression with increasing tumor progression.

"Aberrant elastic" in elastofibroma: an immunohistochemical and ultrastructural study.

Elastofibroma is a rare lesion characterized by the presence of abundant abnormal elastic fibers with a unique morphology, fibroblastic proliferation, and collagen deposition. Whether the altered morphology of the elastic fibers is a degenerative phenomenon or is due to abnormal elastogenesis is controversial. We studied fetal skin and three cases of elastofibroma by light microscopy and immunohistochemistry using an antibody to lysozyme, and one case of elastofibroma by electron microscopy (EM). Our previous studies have shown that normal elastic fibers in adult skin do not stain for lysozyme whereas abnormal elastic fibers in solar elastosis and pseudoxanthoma elasticum react positively for lysozyme. In the fetal skin and all three cases of elastofibroma the elastic fibers were negative for lysozyme. EM showed the abnormal flowerlike configuration of the elastic fibers, which consisted of a central core of normal or degenerating elastin surrounded by radiating spokes of granular and filamentous material of variable electron densities, suggesting that the structure and organization of the microfibrils is abnormal. The absence of lysozyme in the aberrant elastic did not differentiate whether there was excessive production of fetal or adult elastic. However, the excessive amount of microfibrils seen at the ultrastructural level suggests that there may be excessive fetal elastic production. The elastic fibers were intimately related to the fibroblasts and were often present within their caveolae, suggesting that the abnormal elastic fibers are produced by the fibroblast. Our study suggests that abnormal elastogenesis with subsequent degeneration plays a role in the production of the abnormal elastic fibers in elastofibroma.

Characterization of the mononuclear infiltrate involved in regression of halo nevi.

Halo nevi are characterized by progressive degeneration of nevus cells surrounded by a mononuclear cell infiltrate. We studied the morphological features of the nevus cells and the composition of the mononuclear cell infiltrate in 15 cases of halo nevi using immunohistochemical techniques and a battery of antibodies to different subsets of lymphocytes and histiocytes. Regression could be divided into four more or less identifiable stages, associated with different subsets of lymphocytes and monocyte-macrophage lineage cells. Stage I (preregression): nests of unremarkable nevus cells were surrounded by a moderate number of T lymphocytes (relatively small percentage of helper inducer T cells), occasional B cells and macrophages. Stage II (early regression): large number of T lymphocytes and FXIIIa-positive cells were in close contact with nevus cell clusters which showed ragged edges. Lysozyme-positive cells and epidermal Langerhans cells were mildly increased. Stage III (late regression): single nevomelanocytes showing mild atypia were present. Numerous T lymphocytes and macrophages positive for lysozyme, KP1 and/or FXIIIa were interspersed between the nevus cells. Increased numbers of epidermal Langerhans cells were present. Stage IV (complete regression): no nevus cells were observed and moderate numbers of T lymphocytes only remained. These results suggest that T cells, especially T-suppressor cells, and different subsets of macrophages participate in the regression of the nevi.