John W. Gerwels

Preservation of RNA for functional genomic studies: A multidisciplinary tumor bank protocol

Few human tumors are collected such that RNA is preserved for molecular analysis. Completion of the Human Genome Project will soon result in the identification of more than 100,000 new genes. Consequently, increasing attention is being diverted to identifying the function of these newly described genes. Here we describe a multidisciplinary tumor bank procurement protocol that preserves both the integrity of tissue for pathologic diagnosis, and the RNA for molecular analyses. Freshly excised normal skin was obtained from five patients undergoing wound reconstruction following Mohs micrographic surgery for cutaneous neoplasia. Tissues treated for 24 hours with RNAlater™ were compared histologically and immunohistochemically to tissues not treated with RNAlater. Immunohistochemical stains studied included: CD45, CEA, cytokeratin AE1/3, vimentin, S-100, and CD34 on formalin-fixed, paraffin embedded tissue and CD45 staining of frozen tissue. Slides were blinded and evaluated independently by three pathologists. The histologic and immunohistochemical parameters of tissue stored in RNAlater were indistinguishable from tissue processed in standard fashion with the exception of S-100 stain which failed to identify melanocytes or Langerhans cells within the epidermis in any of the RNAlater -treated tissues. Interestingly, nerve trunks within the dermis stained appropriately for S-100. Multiple non-cutaneous autopsy tissues were treated with RNAlater, formalin, liquid nitrogen (LN2), and TRIzol Reagent®. The pathologists were unable to distinguish between tissues treated with RNAlater, formalin, or frozen in LN2, but could easily distinguish tissues treated with TRIzol Reagent because of extensive cytolysis. RNA was isolated from a portion of the tissue treated with RNAlater and used for molecular studies including Northern blotting and microarray analysis. RNA was adequate for Northern blot analysis and mRNA purified from RNAlater -treated tissues consistently provided excellent templates for reverse transcription and subsequent microarray analysis. We conclude that tissues treated with RNAlater before routine processing are indistinguishable histologically and immunohistochemically from tissues processed in routine fashion and that the RNA isolated from these tissues is of high quality and can be used for molecular studies. Based on this study, we developed a multidisciplinary tumor bank procurement protocol in which fresh tissue from resection specimens are routinely stored in RNAlater at the time of preliminary dissection. Thus, precious human tissue can be utilized for functional genomic studies without compromising the tissues diagnostic and prognostic qualities.

Cutaneous malignant melanoma and oculodermal melanocytosis (nevus of Ota): Report of a case and review of the literature

A 29-year-old white man, with oculodermal melanocytosis, had a rapidly enlarging, erythematous, painful nodule over his left brow, within the nevus. The lesion was excised and diagnosed as a malignant melanoma. Systemic evaluation showed no evidence of distant disease. This is the tenth case reported of a cutaneous melanoma developing in a nevus of Ota. Melanoma arising in the choroid, brain, orbit, iris, ciliary body, or optic nerve in association with a nevus of Ota is well documented. Careful observation is necessary in patients with a nevus of Ota, particularly in white patients, in whom malignant degeneration seems to occur with a disproportionate frequency.

Basal cell carcinomas are populated by melanocytes and Langerhan's cells

Several reports have documented the coexistence of basal cell carcinoma (BCC) with other lesions, including melanoma. This study was performed to determine whether nests of BCC contain benign melanocytes and Langerhans cells. Ten cases of BCC were investigated to determine whether benign melanocytes and Langerhans cells populate tumor nests. The BCCs were stained with antibodies to cytokeratin AE1/AE3, S-100, HMB-45, Melan-A, and CD1a proteins. We report that all 10 BCCs were populated by dendritic melanocytes distributed at the periphery (5/10 cases) or evenly throughout tumor nests (5/10 cases). Clusters of melanocytes were not identified in any of the BCCs. A total of 9 of 10 tumors showed staining of dendritic Langerhans cells with CD1a. A total of 8 of 10 tumors stained with cytokeratin AE1/AE3; in 6 of the 8 tumors, the staining was focal. We compared these findings with a single example of a BCC and melanoma in situ (MIS) collision tumor in which the cytokeratin AE1/AE3–positive epithelial nests of BCC were populated by a high density of malignant melanocytes that stained with S-100 and HMB-45. Melanocytes were disposed singly and in clusters of two or more cells within BCC tumor nests. We conclude from this study that BCCs are regularly populated by benign melanocytes and Langerhans cells. Furthermore, when BCC is infiltrated with malignant melanocytes of MIS, the melanocyte density is higher and clusters of melanocytes can be observed. The significance of these two findings is unclear, as additional cases of BCC MIS collision tumor need to be studied.

Atypical lymphoid hyperplasia of the eyelids manifesting as xanthelasma-like lesions

We describe a patient who had bilateral, yellow papules of the upper eyelids. This proved to be the clinical manifestation of an atypical lymphoid hyperplasia of the orbits. We describe this clinical presentation as a new sign of this condition. This finding serves to broaden the differential diagnosis of yellow papules on the eyelids. Atypical lymphoid hyperplasia of the orbits poses a challenging problem. Their benign or malignant nature cannot usually be determined by clinical and radiologic criteria. Most of these infiltrates result in extraocular lymphoma. We describe a patient with bilateral, yellow papules of the upper eyelids that proved to be a manifestation of an atypical lymphoid hyperplasia of the orbits.