Formalin-fixed, paraffin-embedded tissue is the most widely available material for retrospective clinical studies. In combination with the potential of genomics, these tissues represent an invaluable resource for the elucidation of disease mechanisms and validation of differentially expressed genes as novel therapeutic targets or prognostic indicators. We describe here an approach that, in combination with laser-assisted microdissection allows quantitative gene expression analysis in formalin-fixed, paraffin-embedded archival tissue. Using an optimized RNA microscale extraction procedure in conjunction with real-time quantitative reverse transcriptase-polymerase chain reaction based on fluorogenic TaqMan methodology, we analyzed the expression of a panel of cancer-relevant genes, EGF-R, HER-2/neu, FGF-R4, p21/WAF1/Cip1, MDM2, and HPRT and PGK as controls. We demonstrate that expression level determinations from formalin-fixed, paraffin-embedded tissues are accurate and reproducible. Measurements were comparable to those obtained with matching fresh-frozen tissue and neither fixation grade nor time significantly affected the results. Laser microdissection studies with 5-microm thick sections and defined numbers of tumor cells demonstrated that reproducible quantitation of specific mRNAs can be achieved with only 50 cells. We applied our approach to HER-2/neu quantitative gene expression analysis in 54 microdissected tumor and nonneoplastic archival samples from patients with Barrett's esophageal adenocarcinoma and showed that the results matched those obtained in parallel by fluorescence in situ hybridization and immunohistochemistry. Thus, the combination of laser-assisted microdissection and real-time TaqMan reverse transcriptase-polymerase chain reaction opens new avenues for the investigation and clinical validation of gene expression changes in archival tissue specimens.

Rhabdomyosarcoma (RMS) isMedicine (Baltimore) 66, 98 -113), and they are present in the corresponding murine models. RMS in Ptch mutant mice consistently contain elevated levels of the tumor growth-promoting insulin-like growth factor 2 (Igf2). We have investigated the mechanism of Igf2 overexpression and its significance in medulloblastoma and RMS tumorigenesis. Here we report that Igf2 is indispensable for the formation of medulloblastoma and RMS in Ptch mutants. Overexpression of Igf2 in RMS in these mice does not involve loss of imprinting, uniparental disomy, amplification of the Igf2 locus, or polyploidy. Since Igf2 is also overexpressed in non-tumor tissue deficient in Ptch, these observations suggest that Ptch regulates Igf2 levels through a transcriptional mechanism. They also identify Igf2 as a potential target for medulloblastoma and RMS.Somatic or inherited deficiency in the tumor suppressor PTCH leads to the formation of several tumor in man, including basal cell carcinoma, fibroma, medulloblastoma, and RMS

Merkel cell carcinoma (MCC) has a small cell variant, indistinguishable in hematoxylin-eosin sections from metastatic small cell carcinoma of the lung (SCCL). To investigate whether intermediate filament expression is helpful in this distinction, 17 MCCs of the small cell type were examined for cytokeratin, as well as neurofilament protein immunostaining, and compared with 59 intermediate-type MCCs and 22 SCCL. With a pan-cytokeratin cocktail (cytokeratin 1-8, 10, 13-16, 19), most (39 of 55) intermediate-type tumors and, more important, 11 of 16 cases of the small cell variant exhibited focal paranuclear staining with dot-like positivity, crescentic positivity, or both. A combined focal (dot-like/crescentic) and diffuse cytoplasmic pan-cytokeratin staining was seen in additional 8 of 55 intermediate and 4 of 16 small cell MCCs. Cytokeratin 20 also evoked focal cytoplasmic staining and occasionally focal and diffuse positivity in the MCCs, irrespective of the subtype. Exclusively diffuse cytokeratin 20 patterns did not occur. Conversely, most SCCL showed a diffuse expression of pancytokeratin, and all cases remained cytokeratin 20 negative. When neurofilament protein was applied, approximately half of the MCCs (25 of 40), including 7 of 11 of the small cell variant, were positive, whereas all SCCL were negative. In conclusion, the cytokeratin and neurofilament protein patterns of small cell MCCs are identical to the pattern of intermediate MCCs but differ from the profile of SCCL, which may help in the differential diagnosis.

Based on a phase I study in 1986, 22 patients have been entered in a phase II study of high-dose human tumor necrosis factor (rH-TNF) since May 1987. Of these patients, 18 are evaluable at present, 2 are still under investigation, and 2 have dropped out. All had advanced stages of cancer (9 soft-tissue sarcomas, 3 melanomas, 5 hypernephromas) and inclusion in the study was ethically acceptable (informed consent). The daily dose of rH-TNF was 15 x 10(5) units/m2, escalated to 21 x 10(5) units/m2 (683-956 micrograms/m2 every week; range 1-6 cycles). Additional prophylactic ketoprofen administration was carried out. Of the 18 evaluable patients, 4 responded with no change (2/4, clinical improvement) and 14 showed progressive disease. The main toxicities observed were hypotension (decrease in systolic blood pressure, 21-60 Torr), leukocytosis, increases in ALAT/ASAT (WHO grade 0-4), fever (WHO grade 1-2), chills (mild to moderate), neurotoxicity (WHO grade 0-2), and nausea/vomiting (WHO grade 0-3).

In this comparative medical decision analysis, it was shown that ferucarbotran-enhanced MRI has the potential to improve medical management and save health care costs.