Basal cell carcinoma vs basaloid squamous cell carcinoma of the skin: an immunohistochemical reappraisal.

Typical cutaneous basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) are morphologically dissimilar. It is well known, however, that poorly differentiated SCC may assume a basaloid phenotype, complicating the histologic distinction between these 2 neoplasms. Selected immunohistochemical stains have been used in the past to aid in that differential diagnosis. In the current study, additional markers were evaluated to determine whether they would be helpful in that regard. Twenty-nine cases of metatypical (squamoid) BCC (MBCC) and 25 examples of basaloid SCC (BSCC) were studied using the antibodies Ber-EP4 and MOC-31 as well as a plant lectin preparation from Ulex europaeus I (UEA-1). The resulting immunostains were interpreted independently by 3 pathologists, and the results showed that MBCCs demonstrated strong and diffuse staining for Ber-EP4 (25/29) and MOC-31 (29/29). In contrast, BSCCs tended to be only sporadically reactive for both markers (4/25 and 1/25 cases, respectively). Labeling for UEA-1 was observed in almost all BSCCs (24/25), but only 6 of 29 cases of MBCC showed limited, focal staining with that lectin. These data suggest that MOC-31 is a useful marker in the specified differential diagnosis, especially when used together with UEA-1.

A case of spontaneous systemic immunity to melanoma associated with cure after amputation for extensive regional recurrence.

PURPOSE: Survival after amputation for melanoma is short; however, rare long-term survivors are reported. The mechanism for durable systemic tumor control in patients with regional failure is not known. To explore whether systemic tumor immunity may be implicated, tumor and circulating immune responses were examined in a patient who survived disease-free 14 years after hip disarticulation. METHODS: A 71-year-old female with extensive regional metastases of melanoma in the left lower extremity underwent amputation for palliative reasons. Tumor was collected at surgery, and blood was collected during follow-up. Tumor sections were evaluated for lymphocytic infiltration and NY-ESO-1 expression by immunohistochemistry. Cellular immune responses to defined tumor antigens were evaluated by ELISPOT assay, and antibody responses to a panel of tumor antigens were assayed by ELISA. RESULTS: The patient's tumor had minimal lymphocytic infiltrate (immunotype A). NY-ESO-1 was strongly expressed by the melanoma cells. Circulating T-cell responses to NY-ESO-1 peptides were observed 6 and 12 years postoperatively, and antibodies to NY-ESO-1 were detected 2-6 years after surgery. CONCLUSION: The patient described in this report experienced relentless regional tumor progression, with intravascular metastases, and then 14-year systemic disease-free survival after palliative resection, without evidence of melanoma recurrence before death from other causes. Her immune response to NY-ESO-1 likely failed to control established regional metastases because T cells were unable to infiltrate them. It is possible, however, that among other factors, the host immune response may have contributed to systemic protection.

The vaccine-site microenvironment induced by injection of incomplete Freund's adjuvant, with or without melanoma peptides.

Cancer vaccines have not been optimized. They depend on adjuvants to create an immunogenic microenvironment for antigen presentation. However, remarkably little is understood about cellular and molecular changes induced by these adjuvants in the vaccine microenvironment. We hypothesized that vaccination induces dendritic cell (DC) activation in the dermal vaccination microenvironment but that regulatory processes may also limit the effectiveness of repeated vaccination. We evaluated biopsies from immunization sites in 2 clinical trials of melanoma patients. In 1 study (Mel38), patients received 1 injection with an adjuvant mixture alone, composed of incomplete Freund's adjuvant (IFA) plus granulocyte-macrophage colony stimulating factor (GM-CSF). In a second study, patients received multiple vaccinations with melanoma peptide antigens plus IFA. Single injections with adjuvant alone induced dermal inflammatory infiltrates consisting of B cells, T cells, mature DCs, and vessels resembling high endothelial venules (HEVs). These cellular aggregates usually lacked organization and were transient. In contrast, multiple repeated vaccinations with peptides in adjuvant induced more organized and persistent lymphoid aggregates containing separate B and T cell areas, mature DCs, HEV-like vessels, and lymphoid chemokines. Within these structures, there are proliferating CD4and CD8 T lymphocytes, as well as FoxP3CD4 lymphocytes, suggesting a complex interplay of lymphoid expansion and regulation within the dermal immunization microenvironment. Further study of the physiology of the vaccine site microenvironment promises to identify opportunities for enhancing cancer vaccine efficacy by modulating immune activation and regulation at the site of vaccination.

Analysis of MYB expression and MYB-NFIB gene fusions in adenoid cystic carcinoma and other salivary neoplasms.

Recent studies have shown that the recurrent t(6;9)(q22-23;p23-24) translocation in adenoid cystic carcinoma results in a novel fusion of the MYB proto-oncogene with the transcription factor gene NFIB. To determine the frequency of this finding, we used RT-PCR assays of the MYB and MYB-NFIB fusion transcripts, and immunohistochemistry for the MYB protein, to study adenoid cystic carcinomas and other epithelial tumors of the salivary glands, and head and neck region. MYB-NFIB fusion transcript was detected in 25 of 29 (86%) frozen adenoid cystic carcinoma tumor samples, and in 14 of 32 (44%) formalin-fixed paraffin-embedded adenoid cystic carcinoma tumor specimens. In contrast, the MYB-NFIB fusion was not expressed in non-adenoid cystic carcinoma neoplasms of the head and neck, confirming the high specificity of the MYB-NFIB fusion. Adenoid cystic carcinomas from various anatomic sites, including salivary gland, sinonasal cavity, tracheobronchial tree, larynx, breast, and vulva were repeatedly fusion-positive, indicating that adenoid cystic carcinomas located in different anatomic sites not only have important morphologic features in common, but also probably evolve through activation of the same molecular pathways. Studies of the expression of MYB revealed that 89% of the tumors, including both fusion-positive and fusion-negative cases, overexpressed MYB RNA. Similarly, 82% of adenoid cystic carcinomas stained positive for MYB protein, compared with 14% of non-adenoid cystic carcinoma neoplasms, indicating that MYB immunostaining may be useful for the diagnosis of adenoid cystic carcinoma, but that neoplasms sometimes in the differential diagnosis are also labeled. The latter are, however, fusion-negative. In summary, our studies show that MYB activation through gene fusion or other mechanisms is a major oncogenic event in adenoid cystic carcinoma occurring at various anatomic sites. In addition to being a diagnostically useful biomarker for adenoid cystic carcinoma, MYB and its downstream effectors are also novel potential therapeutic targets.

CD10, p63 and CD99 expression in the differential diagnosis of atypical fibroxanthoma, spindle cell squamous cell carcinoma and desmoplastic melanoma.

BACKGROUND: Atypical fibroxanthoma (AFX) is a pleomorphic spindle cell lesion of the skin; it is considered in the differential diagnosis with spindle cell malignant melanoma (MM) and sarcomatoid carcinoma/spindle cell squamous cell carcinoma (SCC). An optimum approach has yet to fully emerge with respect to the immunohistochemical discrimination of these lesions. METHODS: Departmental archives from 1978 onwards were searched for clinicopathologically confirmed cases of AFX, MM and SCC. Immunostains for CD10, CD99 and p63 were performed in each case. Scored staining results were analyzed using Fisher's Exact Test. RESULTS: Twenty-seven of 31 cases of AFX were positive for CD10, as compared with 3 of 22 SCCs and 0 of 20 MMs. CD10 positivity was preferentially associated with the diagnosis of AFX (p < 0.001). p63 reactivity was observed in 15/22 cases of SCCs, 5/31 AFXs and 1/20 MMs. CD99 reactivity was observed in 3/31 cases of AFX, 2/22 SCCs and 3/20 MMs. CONCLUSION: CD10 positivity is relatively specific in this context for the diagnosis of AFX. Its utility is enhanced when only strong, diffuse membranocytoplasmic staining is considered as a positive result. In contrast to prior reports, p63 was not found to be highly sensitive for SCC. Similarly, CD99 showed no preferential staining of any single diagnostic group of lesions.

Preanalytical variables in measurement of free (ionized) calcium in lithium heparin-containing blood collection tubes.

BACKGROUND: Measurements of free (ionized) calcium (iCa) are increasingly requested in patient care locations where immediate analysis is unavailable. Evacuated blood collection tubes containing lithium heparin and gel separator material are widely used in clinical laboratories, but little information is available on the effects of these tubes or of delay prior to analysis on the concentration or stability of iCa. METHODS: We collected blood from volunteers into lithium-heparin tubes (PST, Vacutainer PST, BD Pre-Analytic Systems) of multiple lots and into electrolyte-balanced heparin syringes (Portex Dry Heparin, Smiths Medical). iCa was measured (Siemens 1265 blood gas analyzers) immediately and, in PST, at 0-7 h with or without transportation of the tubes from remote sites. RESULTS: The mean difference of free calcium results in the PST tubes and electrolyte-balanced syringes was -0.08 (95% confidence interval -0.17 to 0.012) mmol/l, and the SD of the residuals (Sy, x) of the regression was 0.03 mmol/l. There was no detectable lot-to-lot variation in results. Free calcium was stable in tubes at room temperature and at 4 degrees C for at least 7 h with or without transportation. CONCLUSION: iCa measured in the examined blood collection tubes is stable and unaffected by lot-to-lot variation of tubes, but results are slightly lower than with special blood gas syringes.

Dependence on electron transport chain function and intracellular signaling of genomic responses in SH-SY5Y cells to the mitochondrial neurotoxin MPP(+).

SH-SY5Y neuroblastoma cells exposed to the complex I inhibitor/parkinsonian neurotoxin methylpyridinium ion (MPP(+)) activate both survival and death-promoting signaling pathways and undergo MEK/ERK-dependent, phosphatidylinositol-3 kinase-dependent, and c-Jun kinase-dependent cell death. Because genomic responses to MPP(+) are not extensively characterized, we used nylon cDNA arrays to measure gene expression following exposure to an apoptosis-producing [MPP(+)]. Many changes occurred within 5 min, and all gene expression changes appeared before biochemical and morphological markers of apoptosis. The majority of gene expression changes in SY5Y were not found in rho(0) cells, indicating dependence of these changes on intact electron transport activity. rho(0) cells exposed to MPP(+) produced different expression profiles, indicating the potential for responses independent of complex I inhibition. MPP(+)-induced gene expression patterns in normal SY5Y cells were sensitive to inhibitors of MEK/ERK (UO 126) or phosphatidylinositol-3 kinase (LY 294002), demonstrating regulation of gene expression by these survival-promoting signaling pathways. The primary signaling molecules mediating these MPP(+)-induced gene expression changes are unknown but ultimately utilize MEK/ERK and phosphatidylinositol-3 kinase signaling. Genes suppressed by UO 126 or LY 294002 during MPP(+) exposure may mediate cell survival; those expressed in the presence of UO 126 or LY 294002 may mediate cell death in this in vitro model of Parkinson's disease.

Stability of gene expression in postmortem brain revealed by cDNA gene array analysis.

We utilized nylon arrays to measure gene expression in mouse brains after various postmortem intervals (PMIs). Gene expression after overnight refrigeration or 4 h at room temperature then overnight refrigeration correlated highly and approximately equivalently to that in brains processed immediately. After 8-24 h at room temperature and overnight refrigeration gene expression correlation and equivalency declined, but 90-95% of detected genes were within +/-40% of baseline levels. Brain homogenate pH did not change with PMI.