Michael Piepkorn

Generation of T-Cell Immunity to the HER-2/neu Protein After Active Immunization With HER-2/neu Peptide–Based Vaccines

PURPOSE The HER-2/neu protein is a nonmutated tumor antigen that is overexpressed in a variety of human malignancies, including breast and ovarian cancer. Many tumor antigens, such as MAGE and gp100, are self-proteins; therefore, effective vaccine strategies must circumvent tolerance. We hypothesized that immunizing patients with subdominant peptide epitopes derived from HER-2/neu, using an adjuvant known to recruit professional antigen-presenting cells, granulocyte-macrophage colony-stimulating factor, would result in the generation of T-cell immunity specific for the HER-2/neu protein. PATIENTS AND METHODS Sixty-four patients with HER-2/neu-overexpressing breast, ovarian, or non-small-cell lung cancers were enrolled. Vaccines were composed of peptides derived from potential T-helper epitopes of the HER-2/neu protein admixed with granulocyte-macrophage colony-stimulating factor and administered intradermally. Peripheral-blood mononuclear cells were evaluated at baseline, before vaccination, and after vaccination for antigen-specific T-cell immunity. Immunologic response data are presented on the 38 subjects who completed six vaccinations. Toxicity data are presented on all 64 patients enrolled. RESULTS Ninety-two percent of patients developed T-cell immunity to HER-2/neu peptides (stimulation index, 2.1 to 59) and 68% to a HER-2/neu protein domain (stimulation index range, 2 to 31). Epitope spreading was observed in 84% of patients and significantly correlated with the generation of a HER-2/neu protein-specific T-cell immunity (P =.03). At 1-year follow-up, immunity to the HER-2/neu protein persisted in 38% of patients. CONCLUSION The majority of patients with HER-2/neu-overexpressing cancers can develop immunity to both HER-2/neu peptides and protein. In addition, the generation of protein-specific immunity, after peptide immunization, was associated with epitope spreading, reflecting the initiation of an endogenous immune response. Finally, immunity can persist after active immunizations have ended.

Atypical Spitz nevi/tumors : Lack of consensus for diagnosis, discrimination from melanoma, and prediction of outcome

The biological nature of Spitz nevi/tumors and their diagnostic distinction from, or relationship to, melanoma remain unresolved issues. In this report, a series of 30 melanocytic lesions removed from 28 patients, including atypical Spitz nevi/tumors and metastasizing Spitzoid tumors/melanomas, were evaluated by a panel of dermatopathologists to evaluate interobserver diagnostic concordance and to assess the prognostic power of histological criteria. For inclusion in the study, each lesion had to display some criteria for the Spitz nevus, and in addition one of the following was required: (1) definitive clinical outcome such as metastasis or death of disease, or (2) long-term follow-up if the patient remained disease free. Each lesion was reviewed independently and blinded as to the clinical data by 10 pathologists, who categorized them as (1) typical Spitz nevus/tumor, (2) atypical Spitz nevus/tumor, (3) melanoma, (4) tumor with unknown biological potential, or (5) other melanocytic lesion. There was limited discussion of criteria before the review. Evaluation of 17 Spitzoid lesions yielded no clear consensus as to diagnosis; in only one case did six or more pathologists agree on a single category, regardless of clinical outcome. Notably, however, some lesions that proved fatal were categorized by most observers as either Spitz nevi or atypical Spitz tumors. Conversely, seven or more pathologists scored 13 lesions as melanoma. These results illustrate (1) substantial diagnostic difficulties posed by many Spitz tumors, especially those with atypical features, even among experts, and (2) the lack of objective criteria for their distinction from melanoma and for gauging their malignant potential. Nevertheless, our observations do suggest that a biological relationship exists between the Spitz nevus/tumor and melanoma.

Frequent Detection of Kaposi's Sarcoma-Associated Herpesvirus (Human Herpesvirus 8) DNA in Saliva of Human Immunodeficiency Virus-Infected Men: Clinical and Immunologic Correlates

The prevalence, quantity, temporal pattern, and clinical and immunologic correlates of shedding of Kaposis sarcoma (KS)-associated herpesvirus (KSHV; or human herpesvirus [HHV]-8) DNA in saliva were studied. KSHV DNA was detected in saliva from 18 (75%) of 24 human immunodeficiency virus (HIV)-positive patients with KS and from 1 of 1 HIV-negative patient with KS, 3 (15%) of 20 HIV-positive patients without KS, and none of 24 controls. KSHV DNA levels ranged from 10(2.4) to 10(6) copies/mL and were lower than levels for Epstein-Barr virus but comparable to those for HHV-6. Detection of KSHV DNA in saliva was not associated with oral KS or decreased peripheral blood CD4 cell counts. KSHV DNA was not detected in semen. Resistance of KSHV DNA from saliva to DNase treatment was consistent with the presence of virions. These data suggest that KSHV can replicate in the oropharynx and that salivary contact could contribute to KSHV transmission.

The dysplastic melanocytic nevus: A prevalent lesion that correlates poorly with clinical phenotype

We estimated the prevalence of persons with histologic dysplasia in at least one of two nevi examined by biopsy to be 53% in Utahs caucasians. This apparently high prevalence indicates that such lesions may represent a normal variant of a melanocytic nevus, perhaps those in the process of active proliferation. Regardless of the apparent ubiquity of these lesions, examination of biopsy specimens led to a grading scheme of histologic dysplasia that may reflect chronologic stages in the neoplastic development of melanocytic nevi. Comparison of these histologic findings with the clinical examination yielded the unexpected result that dysplasia and lesion size are independent of each other. Lesions 3 mm in diameter or smaller were as likely to be dysplastic as those much larger. There was, however, a statistically significant relationship between histologic dysplasia of a nevus examined by biopsy and the persons total number of melanocytic lesions. This finding indicates that the pathology grading scheme may be useful. The high prevalence of dysplastic nevi dilutes the clinical significance of a dysplastic nevus as an isolated finding and thereby lessens the importance of pathologic findings in the diagnosis of dysplastic nervus syndrome.

A multiobserver, population-based analysis of histologic dysplasia in melanocytic nevi*

BACKGROUND Nevi that are clinically atypical and histologically dysplastic have been associated with increased melanoma risk. There are few reproducibility studies or population-based studies of nevus histology. OBJECTIVE Our purpose was to quantify concordance in histologic diagnosis of melanocytic lesions among a diverse group of pathologists, to assess intraobserver concordance by comparing readings of the same slide as well as of adjacent recuts from the same block, to correlate histology with nevus appearance and melanoma risk, and to estimate the range of prevalence of histologic dysplasia. METHODS Histologic slides were prepared from 149 tissue blocks of pigmented lesions from melanoma cases, relatives, and controls. Six dermatopathologists independently evaluated the lesions for histologic dysplasia, without prior agreement on criteria. RESULTS According to kappa statistics, intraobserver reproducibility was substantial, and interobserver concordance was fair, despite differences in criteria. The estimated prevalences of histologic dysplasia for the six pathologists ranged from 7% to 32%. Histologic dysplasia was correlated with nevus size for most observers, confounding the observed correlation between nevus appearance and histology. CONCLUSION Although experienced dermatopathologists use different diagnostic criteria for histologic dysplasia, their usage is consistent. Histologic changes ascribed to melanocytic dysplasia are prevalent in the white population for all pathologists. The term nevus with histologic dysplasia should be used in preference to dysplastic nevus.

Immunohistochemical characterization of dermatofibrosarcoma protuberans with practical applications for diagnosis and treatment

Dermatofibrosarcoma protuberans (DFSP) is a rare, clinically challenging, soft tissue tumor. The main histologic differential of DFSP is usually a dermatofibroma. In 1990, the first report appeared demonstrating that cells of DFSP express the human progenitor antigen CD34 on their surface. Since then, there have been increasing reports of the usefulness of immunohistochemical staining with CD34 to differentiate DFSP from dermatofibroma and other soft tissue tumors. This literature is reviewed with special emphasis on the insights studies have provided into the histogenesis of DFSP. The literature demonstrating the practical applications of CD34 staining in the diagnosis and treatment of DFSP is also discussed.

The female, red Duroc pig as an animal model of hypertrophic scarring and the potential role of the cones of skin

Hypertrophic scarring occurs after deep dermal wounds. Our understanding of the etiology is poor; one reason is the lack of an animal model. In 1972, Silverstein described scarring in the Duroc pig but the model was never confirmed nor disproved. Another reason, as we previously suggested, is that hypertrophic scarring only occurs within regions of human skin that contain cones and the cones have not been studied in relation to hypertrophic scarring. We, therefore (i) explored healing in the female, red Duroc model for similarities to human hypertrophic scarring, studying wound thickness, appearance, healing status at 3 weeks, histology, and immunocytochemical localization of decorin, versican, TGFbeta1 and IGF-1; and (ii) examined Duroc skin for cones. We found that healing after deep wounds in Duroc pigs is similar, but not identical, to human hypertrophic scarring. We also found that Duroc skin contains cones. Healing in the female, red Duroc pig is sufficiently similar to human hypertrophic scarring to warrant further study so that it can be accepted or rejected as a model of human hypertrophic scarring. In addition, the relationship of the cones to hypertrophic scarring needs further detail and can be studied in this model.

Evidence that the p75 neurotrophin receptor mediates perineural spread of desmoplastic melanoma

BACKGROUND The desmoplastic variant of melanoma has a striking propensity for neurotropism. This neurotropism may reflect Schwannian differentiation. The migration of Schwann cells along embryonic nerves is reportedly regulated by the 75 kd neurotrophin receptor (p75NTR) and one of its cognate ligands, nerve growth factor (NGF). OBJECTIVE The purpose of this study was to test the hypothesis that the mechanism of perineural spread in desmoplastic melanomas is analogous to that of neurotropism in Schwann cells, which apparently depends on expression of p75 neurotrophin receptor and its ligands. METHODS Immunolabeling of p75NTR in histologic sections of spindle cell and desmoplastic melanomas was compared and contrasted with that of epithelioid melanomas. RESULTS The histologic material consisted of spindle cell melanoma specimens from 11 patients, of which seven exhibited features of desmoplastic melanoma. All spindle cell melanoma specimens expressed the p75NTR in at least 10% of the cells, and most expressed p75NTR in more than 50% of cells. In contrast, 10 of 11 control melanomas of conventional epithelioid phenotype expressed lower levels of p75NTR (0% to 10% of cells). CONCLUSION There is a strong correlation between expression of p75NTR and the desmoplastic phenotype, supporting the hypothesis that the propensity of these melanomas for neurotropism involves p75NTR and its ligands. Immunolabeling for p75NTR may be a useful marker for the neurotropic phenotype, as well as for detecting perineural spread in histologic sections of melanomas.

Proliferation-differentiation relationships in the expression of heparin-binding epidermal growth factor-related factors and erbB receptors by normal and psoriatic human keratinocytes

The topological relationships between erbB receptors and ligands of the epidermal growth factor family were characterized by immunocytochemistry in normal and psoriatic epidermis and in proliferating and differentiating human keratinocytes in culture. Spatial colocalization of receptors and ligands was assessed by dual immunostaining. Expression of epidermal growth factor receptor (EGFr), erbB2, and erbB3, but not erbB4, was detected throughout the epidermis, although labeling for erbB2 and erbB3 was accentuated in the upper spinous layers, and EGFr was more strongly labeled in basal cells. Of the tested growth factors, heparin-binding epidermal growth factor (HB-EGF) was diffusely expressed throughout normal and psoriatic epidermis and sparsely colocalized with EGFr in all viable epidermal layers, with increased colocalization in psoriatic epidermis. In contrast, betacellulin and heregulin/neu differentiation factor (NDF) α were largely restricted in their distribution to the upper spinous and granular layers. Betacellulin was downregulated in psoriatic keratinocytes. Although heregulin/NDF-β was undetectable in normal epidermis, it was upregulated in psoriasis. Betacellulin and heregulin/NDF-α strikingly colocalized with EGFr and erbB3 receptors in the granular layer and in a declining gradient from the granular zone to the basal layer, respectively. Similar patterns were observed in cultured keratinocytes under proliferative conditions and upon differentiation in high-calcium medium. These morphological data collectively suggest divergent functions for members of the growth factor family, and in particular, we propose that betacellulin and heregulin/NDF-α are involved in epidermal morphogenesis and/or in maintenance of the differentiated phenotype.

Analysis of microvascular changes in frostbite injury

Reported here is the characterization of the hairless mouse ear as a model system for defining the microvascular effects of minimal frostbite injury by means of gross, in vivo microscopic, histologic, and electron microscopic analysis. Initial efforts, using controlled temperatures and time of freezing, defined the minimum conditions necessary to produce consistent tissue necrosis to be -4 degrees C for 3 min. In vivo observation, after rapid thaw, showed a return of blood flow to apparent normal prefreeze rates, followed by a gradual sludging of blood 15 to 20 min post-thaw and eventually in cessation of blood flow. No vascular spasm was observed. Histologic and electron microscopic examination, unexpectedly, did not reveal evidence of early platelet or fibrin thrombi. However, marked vasodilatation and circulatory congestion began 2-3 hr after thawing. Subsequently, prominent discontinuities between damaged endothelial cells and breaks in the microvascular basement membranes were found at the light and ultrastructural level. Interstitial edema and extravasated erythrocytes occurred shortly thereafter, preceding gross tissue necrosis evident at post-freeze Day 3. We conclude that in this animal system thrombus formation is not an initial event, but rather that vascular injury in the form of endothelial cell damage predominates in early frostbite injury.