The frozen section yesterday and today: pediatric solid tumors--crucial issues.

This article is the offshoot of a Pediatric Oncology Group (POG) seminar presented at the Adams Mark Hotel, Denver, Colorado, Friday, May 21, 1999, titled "The Frozen Section in Pediatric Solid Tumors--Crucial Issues." There were eight presenters who spoke on a wide range of topics that included historical perspectives of the frozen section and discussion of the following systems: brain, renal, germ cell, bone, soft tissue, and lymph nodes. To complement these presentations, a pediatric surgeon explained his concern and philosophy regarding the use of frozen sections, and a lawyer tackled the issues and risks in rendering a frozen section diagnosis. We think that this review covers all the important aspects of the frozen section in our current practice of pediatric pathology.

Inhibition of the interferon-gamma/signal transducers and activators of transcription (STAT) pathway by hypermethylation at a STAT-binding site in the p21WAF1 promoter region.

Expression of the cyclin-dependent kinase inhibitor p21WAF1 can be up-regulated by activation of signal transducers and activators of transcription (STAT) proteins in response to IFN-gamma. In this study, we examined CpG methylation at the p21WAF1 promoter region in rhabdomyosarcomas (RMSs) using Southern blot analysis with the methylation-sensitive restriction enzyme HpaII. Sis-inducible element (SIE)-1, a STAT-responsive element located upstream of the p21 WAF1 CpG island, was completely methylated at an internal CpG in 13 of 26 (50%) primary RMS tumors and 2 of 5 RMS cell lines. In contrast, all normal tissues examined showed a partial methylation pattern at SIE-1. Complete methylation within SIE-1 strongly correlated with decreased p21WAF1 mRNA expression in RMS. We further studied the effects of SIE-1 hypermethylation on p21WAF1 induction by STAT activation. CpG methylation within SIE-1 significantly inhibited binding of activated STAT1 in electrophoretic mobility shift assays and abrogated STAT-mediated transcription activation in response to IFN-gamma in luciferase reporter gene assays. Activation of STAT1 in response to IFN-gamma resulted in increased p21WAF1 expression and growth suppression in RMS cells containing unmethylated SIE-1 but failed to induce p21WAF1 or growth inhibition in RD and A673 cells, both of which were completely methylated within SIE-1. However, demethylation at SIE-1, induced by a demethylating agent 5-aza-2'-deoxycytidine, reactivated p21WAF1 expression and restored the responsiveness to IFN-gamma in RD cells. Our results indicate a mechanism by which altered DNA methylation in the p21 WAF1 promoter region, by precluding STAT1 binding to SIE-1, directly inhibits the p21WAF1 induction and cell growth regulation through the IFN-gamma/STAT signaling pathway in RMS cells.

Increased DNA methyltransferase expression in rhabdomyosarcomas.

In normal somatic cells, the methylation pattern of DNA is stably maintained by DNA (cytosine-5-)-methyltransferase (DNA methyltransferase). Increased expression of DNA methyltransferase has been detected in many types of human cancer and has been thought to play an important role in tumorigenesis. In our study, we developed a standardized reverse transcription-polymerase chain reaction (RT-PCR) assay to determine the mRNA levels of DNA methyltransferase in rhabdomyosarcoma, the most common soft tissue cancer in children. Using this assay, expression of DNA methyltransferase was analyzed for 32 rhabdomyosarcomas and 12 normal skeletal muscle samples. All tumor samples, of which 18 were embryonal and 14 were alveolar subtype, showed increased expression of DNA methyltransferase after normalization to beta-actin. Compared to normal skeletal muscle, the average increase of DNA methyltransferase expression was 6.7-fold (6.7 +/-()0.96) in the embryonal tumors and 3.7-fold (3.7 +/- 0.46) in the alveolar rhabdomyosarcomas. The difference in the average increase of the DNA methyltransferase expression was statistically significant in the 2 rhabdomyosarcoma subtypes, which have distinct etiologies and clinical behaviors. Our results are consistent with previous reports that an increase in DNA methyltransferase activity is associated with neoplastic transformation; however, the role of increased DNA methyltransferase expression in the development and progression of rhabdomyosarcoma needs to be investigated in future studies.

Methylation alterations of the MyoD1 upstream region are predictive of subclassification of human rhabdomyosarcomas.

MyoD1 expression is a distinguishing characteristic of rhabdomyosarcoma. In this study, distinct methylation alterations were identified in the 5' flanking region of the MyoD1 gene from the two major subtypes, ie, alveolar and embryonal rhabdomyosarcoma. The MyoD1 methylation patterns of 26 rhabdomyosarcomas were compared with that of normal skeletal muscle and nonmuscle specimens by Southern blot analysis using methylation-sensitive restriction enzymes HhaI and HpaII. A 5-kb region immediately upstream of the MyoD1 coding sequence was found to be methylated in adult muscle and all nonmuscle tissues tested. The MyoD1 upstream region was unmethylated in the majority of the alveolar rhabdomyosarcomas (13 of 15, 87%) examined in this study. In contrast, 10 of 11 (91%) embryonal rhabdomyosarcomas showed a methylation pattern that was also observed in fetal muscle cells, in which the CpG sites in the MyoD1 upstream region were partially methylated. Our data suggest that the methylation status of the MyoD1 upstream CpG sites may be related to rhabdomyosarcoma tumorigenesis and may have valuable implications for its differential diagnosis.

Bcl-2 expression in Langerhans' cell histiocytosis.

Langerhans' cell histiocytosis (LCH) is an abnormal accumulation of dendritic histiocytes of unknown pathogenesis. It has recently been shown to be a clonal process. Bcl-2 is a proto-oncogene whose protein product is known to inhibit apoptosis. The overexpression of bcl-2 has been demonstrated in a number of neoplasms, presumably prolonging the survival of the neoplastic cells. We examined the expression of bcl-2 in normal Langerhans' cells in the skin and in LCH by immunohistochemistry for protein and in situ hybridization for mRNA to see if it could be implicated in the pathogenesis of this disorder. Additionally, we performed Southern analysis to determine if genomic rearrangement of the bcl-2 gene occurs in cases of LCH. Bcl-2 was not detected in normal skin Langerhans' cells. Eleven of thirteen cases of LCH demonstrated bcl-2 protein expression in the cytoplasm of the Langerhans' cells by immunohistochemistry, while 12 of 13 cases had evidence of bcl-2 mRNA by in situ hybridization. Southern analysis revealed a germline configuration of the bcl-2 gene in the five cases studied. These findings suggest that bcl-2 expression is present and up-regulated in pathologic Langerhans' cells, however, this overexpression does not appear to be due to genomic rearrangement.

Mucinous tumors of the ovary: interobserver diagnostic variability and utility of sectioning protocols.

OBJECTIVES: To determine the interobserver variability of the subclassification of ovarian mucinous tumors and the utility of different sectioning protocols. METHODS: Six pathologists retrospectively reclassified 73 mucinous tumors (30 adenomas, 22 low malignant potential tumors, and 21 carcinomas). Using probabilities, the accuracy of limited sectioning protocols was compared with that of a one section per centimeter protocol. RESULTS: The mean kappa statistic was 0.56, indicating only good agreement. Although a limited sectioning protocol would result in misdiagnosing cases of stage IA carcinoma as a low malignant potential tumor, the overall prognosis of patients with low malignant potential tumors would not markedly change. The prognosis of a patient with a low malignant potential tumor using limited sectioning was within the prognostic range owing to interobserver variability alone. CONCLUSIONS: We conclude that extensive sectioning of ovarian mucinous tumors has limited benefit.

CD44 expression in neuroblastoma and related tumors.

PURPOSE: A retrospective study was conducted to investigate the relationship between CD44 expression in neuroblastoma and related tumors and other known prognostic indicators. MATERIALS AND METHODS: Immunostaining of CD44 was done on surgical specimens of 55 cases (42 patients) of neuroblastoma (NB) and ganglioneuroblastoma (GNB) and nine cases of ganglioneuroma. The percentage of positive tumor cells was scored semiquantitatively (0-4+) by two observers. CD44 expression was then correlated with survival, age, stage, and N-myc amplification. RESULTS: Fifty-seven percent of the patients with NB or GNB had heterogeneous positive staining (2-4+) on their diagnostic specimens. Twenty-four percent of the patients had no staining for CD44, and 19% had 1+ staining. In the 17 cases with N-myc analysis, an inverse relationship was demonstrated between N-myc and CD44 expression by univariate analysis. Lack of expression of CD44 was highly associated with poor survival (p = 0.0002). When assessing the joint effects of age, stage, and CD44 in multivariate analysis, the effect of CD44 remains significant (p = 0.028) and appears to be independent of age and stage. CONCLUSION: Our data suggest a relationship between CD44 and N-myc amplification. Absence or low expression of CD44 correlates with poor survival and may be a biologic marker of tumor aggressiveness. CD44 appears to be an independent prognostic marker and deserves continued investigation in prospective studies of neuroblastoma.

Low-grade transitional cell carcinoma of the urinary bladder: application of select cytologic criteria to improve diagnostic accuracy [corrected].

The cytologic diagnosis of low-grade transitional cell carcinoma of the bladder is difficult, and the reported sensitivity of a positive diagnosis ranges from 0 to 73%. Using regression analysis, our laboratory previously reported the criteria of increased nuclear/cytoplasmic ratios, irregular nuclear membranes, and cytoplasmic homogeneity as indicative of low-grade transitional cell carcinoma. To examine the validity of these criteria, six observers examined 88 bladder-wash specimens (39 transitional cell carcinomas and 49 benign) and, using the selected criteria, graded each wash for the probability of malignancy. Diagnostic accuracy was measured using the receiver operating characteristic curve and the likelihood ratio. Overall observer accuracy was 76%, the sensitivity of a definitive negative diagnosis was 82%, and the specificity of a definitive positive diagnosis was 96%. We conclude that key cytologic criteria can be learned and effectively applied with high accuracy. Observer variation in diagnostic categories might reflect different confidence levels and probabilities of transitional cell carcinoma.

Clinical and pathological features of pediatric dermatofibrosarcoma protuberans.

Dermatofibrosarcoma protuberans (DFSP) is an uncommon malignant mesenchymal tumor characterized by local invasion and recurrence. Fewer than 50 cases have been reported in the pediatric population. We reviewed our experience in the treatment of children with DFSP to define clinical and pathological characteristics. Seven pediatric patients were included in the study (mean age, 11.7 yr). Clinically, the tumors were described as firm nodules fixed to the skin but mobile over the deep fascia, with slow, progressive growth. Diagnosis was made by excisional biopsy in 6 patients and punch biopsy in 1 patient. Six of 7 patients had positive margins after the diagnostic procedure. Pathologically, diagnosis was based on histology, with confirmation by CD34 staining. Definitive surgical therapy consisted of wide local excision (1-3 cm margins) in 5 patients and Moh's micrographic resection in 2 patients. There have been no local recurrences or distant metastases, with a mean follow-up of 15.1 months. Pathological and clinical diagnostic criteria for the pediatric population are reviewed, and treatment options are discussed.