External quality assessment slide schemes: pathologists' experiences and perceptions.

BACKGROUND: External quality assessment schemes (EQAS) in pathology have been established in the United Kingdom for several years with the aim of raising standards. OBJECTIVE: To determine the experiences and perceptions of pathologists undertaking EQAS. METHODS: A questionnaire was distributed to histo/cytopathologists in the south and west of England. RESULTS: A large proportion of pathologists responding felt that the EQAS was educational, and 69% said participation had encouraged them to undertake additional educational activities. Some reservations were expressed about marking schemes. Asked if EQAS using digital images (CD-ROM or web based) rather that glass slides were valid alternatives two thirds responded no, despite 75% claiming to have appropriate IT skills. CONCLUSIONS: EQAS play a valuable role in helping to maintain standards in histopathology and cytopathology. Some reservations were expressed about the marking schemes and further work is needed to establish a robust marking method. Significant barriers need to be overcome if digital EQAS are to be successfully implemented.

The NHS breast screening programme (pathology) EQA: experience in recent years relating to issues involved in individual performance appraisal.

BACKGROUND: The original role of the National Health Service breast screening programme (pathology) external quality assessment (EQA) scheme was educational; it aimed to raise standards, reinforce use of common terminology, and assess the consistency of pathology reporting of breast disease in the UK. AIMS/METHODS: To examine the performance (scores) of pathologists participating in the scheme in recent years. The scheme has evolved to help identify poor performers, reliant upon setting an acceptable cutpoint. Therefore, the effects of different cutpoint strategies were evaluated and implications discussed. RESULTS/CONCLUSIONS: Pathologists who joined the scheme improved over time, particularly those who did less well initially. There was no obvious association between performance and the number of breast cancer cases reported each year. This is not unexpected because the EQA does not measure expertise, but was established to demonstrate a common level of performance (conformity to consensus) for routine cases, rather than the ability to diagnose unusual/difficult cases. A new method of establishing cutpoints using interquartile ranges is proposed. The findings also suggest that EQA can alter a pathologist's practice: those who leave the scheme (for whatever reason) have, on average, marginally lower scores. Consequently, with the cutpoint methodology currently used (which is common to several EQA schemes) there is the potential for the cutpoint to drift upwards. In future, individuals previously deemed competent could subsequently be erroneously labelled as poor performers. Due consideration should be given to this issue with future development of schemes.

Monoclonal antibodies to the myogenic regulatory protein MyoD1: epitope mapping and diagnostic utility.

Monoclonal antibodies (MoAbs) were developed against recombinant wild-type murine MyoD1 protein. Each of 4 MoAbs was immunologically reactive with recombinant MyoD1 protein by enzyme-linked immunosorbent assay, and each specifically stained the nuclei of myogenic cells. Epitopes were mapped using fusion protein constructs with specific deletions of defined regions of the MyoD1 molecule. MoAb 5.2F recognized an epitope in the amino terminal region between amino acid residues (AAR) 3 and 56, whereas epitopes for MoAbs 1.1A, 5.4G, and 5.8A were in the carboxyl terminus (AAR 167-318) of the MyoD1 protein. The epitope for MoAb 5.8A was further delineated to AAR 170-209 by Western analysis and immunoprecipitation of in vivo transcribed and translated MyoD1 protein having specific deletions in the carboxyl terminus. The 5.8A epitope was ultimately localized to the region between AAR 180 and 189 of the protein by enzyme-linked immunosorbent assay using 10-amino acid residue synthetic peptides. This sequence is apparently unique to MyoD1 and has little homology to other myogenic regulatory proteins (myogenin, Myf5, Myf6, and MRF4). Transfection of cDNA for murine MyoD1 into a nonmuscle cell line conferred 5.8A reactivity, confirming the specificity of this reagent. MoAb 5.8A was then used to examine the expression of MyoD1 in normal and malignant human tissues. MyoD1 was not detected in any normal adult tissue but was detected in 25 of 25 histologically confirmed rhabdomyosarcomas. Staining was localized to the nucleus and showed marked heterogeneity between cells as well as differential staining within nuclei. Specific subcellular localization of 5.8A was further determined by immunoelectron microscopy, where antibody was found to localize to electron-dense areas, more frequently associated with the nuclear submembranous region. In addition to rhabdomyosarcomas, MoAb 5.8A stained 2 of 5 Wilms' tumors and one ectomesenchymoma, neoplasms known to contain myogenic elements. The 5.8A reagent was also of value in the accurate histopathological classification of 2 of 4 tumors previously diagnosed as extraosseous Ewing's sarcoma and 2 of 3 tumors diagnosed as undifferentiated sarcomas.

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.

Development and characterization of human ependymoma xenograft HxBr5.

We describe the successful heterotransplantation of a human ependymoma in CBA/CaJ mice immune deprived by infant thymectomy and whole-body irradiation. The xenograft, HxBr5, was established from a fourth ventricular ependymoma, locally recurrent in an 11-yr-old girl who had been treated with radiation therapy to the posterior fossa. HxBr5 retains histological and ultrastructural fidelity to the tumor from which it was derived as does the DNA content, as confirmed by flow cytometric analysis. The karyotype of the xenograft, which is pseudodiploid and exhibits trisomy 1q and deletion of 1p, is the first human ependymoma banded karyotype to be reported. Growth rates of the xenograft tumors are similar to the primary tumor as clinically observed with a doubling time of approximately 42 days. Cell kinetic parameters indicate that this slow-growing tumor has a relatively high growth fraction of 70.8% with a high cell loss of approximately 91%. We anticipate that HxBr5 may be useful as one component of a more complex model for studying the biology and differentiation of human ependymoma.

Characterization of cell lines derived from xenografts of childhood rhabdomyosarcoma.

Three human rhabdomyosarcoma cell lines (Rh10, Rh18, and Rh28) have been established from three independently derived xenografts. These lines have been characterized as mesenchymal in origin (reactivity to desmin and vimentin antibodies) and as expressing a human fetal muscle surface antigen recognized by monoclonal antibody 5.1 H11. Measurable levels of creatine phosphokinase have been detected in the cell lines. Rh10 and Rh28 exhibit the same chromosomal translocation and express an atypical lactate dehydrogenase isoenzyme which may be homologous to those previously reported in other tumor types. The karyotype analysis has confirmed that each cell line was derived from its respective tumor and thus provides a unique model for future investigations.

Methods to detect P-glycoprotein-associated multidrug resistance in patients' tumors: consensus recommendations.

Multidrug resistance (MDR), especially that associated with overexpression of MDR1 and its product, P-glycoprotein (Pgp), is thought to play a role in the outcome of therapy for some human tumors; however, a consensus conclusion has been difficult to reach, owing to the variable results published by different laboratories. Many factors appear to influence the detection of Pgp in clinical specimens, including its low and heterogeneous expression; conflicting definitions of detection end points; differences in methods of sample preparation, fixation, and analysis; use of immunological reagents with variable Pgp specificity and avidity and with different recognition epitopes; use of secondary reagents and chromogens; and differences in clinical end points. Also, mechanisms other than Pgp overexpression may contribute to clinical MDR. The combined effect of these factors is clearly important, especially among tumors with low expression of Pgp. Thus, a workshop was organized in Memphis, Tennessee, to promote the standardization of approaches to MDR1 and Pgp detection in clinical specimens. The 15 North American and European institutions that agreed to participate conducted three preworkshop trials with well-characterized MDR myeloma and carcinoma cell lines that expressed increasing amounts of Pgp. The intent was to establish standard materials and methods for a fourth trial, assays of Pgp and MDR1 in clinical specimens. The general conclusions emerging from these efforts led to a number of recommendations for future studies: (a) although detection of Pgp and MDR1 is at present likely to be more reliable in leukemias and lymphomas than in solid tumors, accurate measurement of low levels of Pgp expression under most conditions remains an elusive goal; (b) tissue-specific controls, antibody controls, and standardized MDR cell lines are essential for calibrating any detection method and for subsequent analyses of clinical samples; (c) use of two or more vendor-standardized anti-Pgp antibody reagents that recognize different epitopes improves the reliability of immunological detection of Pgp; (d) sample fixation and antigen preservation must be carefully controlled; (e) multiparameter analysis is useful in clinical assays of MDR1/Pgp expression; (f) immunostaining data are best reported as staining intensity and the percentage of positive cells; and (g) arbitrary minimal cutoff points for analysis compromise the reliability of conclusions. The recommendations made by workshop participants should enhance the quality of research on the role of Pgp in clinical MDR development and provide a paradigm for investigations of other drug resistance-associated proteins.

The Evi-1 zinc finger myeloid transforming gene is normally expressed in the kidney and in developing oocytes.

Activation of the Evi-1 zinc finger gene is commonly associated with the transformation of murine leukemias and is involved in some cases of human AML involving rearrangements at chromosome 3q25. To determine the normal function of the gene, we have looked for expression in a variety of cell lines and tissues. The predominant sites of expression of the gene are in the kidney and ovary. In the kidney, expression is localized to the renal tubules in the corticomedullary junction. In the ovary, high levels of the Evi-1 protein are found in the cytoplasm of developing oocytes. The latter result suggests a potential role for the Evi-1 gene product in early oocyte development.

Relationship of tumor-cell ploidy to histologic subtype and treatment outcome in children and adolescents with unresectable rhabdomyosarcoma.

Clinical and histopathologic features are often inadequate for accurate prediction of relapse or survival of individual patients with rhabdomyosarcoma (RMS). We therefore studied the cellular DNA content (ploidy) of RMS cells in relation to histology and response to therapy in 37 patients with unresectable tumors. Using flow cytometric techniques, we found that about one third of patients had diploid tumor stem lines, regardless of the histologic subtype. In the group with abnormal ploidy, a hyperdiploid classification (1.10 to 1.80 times the DNA content of normal diploid cells) was exclusively associated with embryonal histology (P = .001). By contrast, near-tetraploidy (1.80 to 2.60 times the DNA content of normal cells) was strongly associated with alveolar histology (P = .001). Thus, in these histologic subtypes of RMS, abnormal ploidy appears to arise through different mechanisms. Tumor-cell ploidy had a significant impact on survival that was especially apparent in patients with unresectable, nonmetastatic (group III) tumors. In this subgroup, hyperdiploidy conferred the best prognosis and diploidy the worst (P less than .0001). None of the eight patients with diploid tumors survived for more than 18 months. Tumor-cell ploidy was the best predictor of treatment outcome for patients with either embryonal (P less than .001; relative risk, 25.5) or alveolar (P = .073; relative risk 7.1) RMS and contributed significantly after adjustment for disease stage and anatomic site. Patients with unresectable diploid RMS have an unacceptably high risk of treatment failure, justifying new therapeutic approaches for this distinct subgroup.

Synovial sarcoma in children and adolescents: the St Jude Children's Research Hospital experience.

PURPOSE AND METHODS: We reviewed the clinical records and pathologic findings of 37 children and adolescents with synovial sarcoma treated at our institution over a 30-year period to evaluate the prognostic significance of tumor size, invasiveness, histology, and other features. RESULTS: The 20 male and 17 female patients with synovial sarcoma had a median age of 13.7 years at diagnosis. Primary tumor sites were the extremities (n = 27), trunk (n = 8), and head and neck (n = 2). Disease stage (clinical group) was as follows: group I, n = 21; group II, n = 7; group III, n = 4; and group IV, n = 5. Nineteen patients had invasive (T2) lesions, 20 had tumors more than 5 cm in diameter, and 14 had histologic grade 3 lesions. The estimated 5-year survival rate (+/- SE) for patients with group I or II disease was 80% +/- 9%, compared with 17% +/- 15% for those with group III or IV tumors (P = .0003). An exact log-rank test, adjusted for clinical group, showed that tumor invasiveness and grade independently predicted overall and progression-free survival (P < .05); tumor size was significantly correlated with progression-free survival. A borderline significant relationship with overall survival was found for both tumor size and histologic subtype (P = .09). CONCLUSION: A controlled trial of adjuvant chemotherapy is merited in children with resected synovial sarcoma (clinical group I or II) who present with unfavorable clinicopathologic features such as large, invasive, or grade 3 lesions. Children with unresected or metastatic disease fare poorly despite multimodality therapy and require novel treatment approaches.

Chemotherapy dose-intensification for pediatric patients with Ewing's family of tumors and desmoplastic small round-cell tumors: a feasibility study at St. Jude Children's Research Hospital.

PURPOSE: To evaluate the feasibility of dose-intensification for patients with Ewing's family of tumors (EFT) and desmoplastic small round-cell tumors. PATIENTS AND METHODS: From February 1992 to June 1996, we treated 53 consecutive patients on our Ewing's protocol. Induction comprised three cycles of ifosfamide/etoposide on days 1 to 3 and cyclophosphamide (CTX)/doxorubicin on day 5, followed by granulocyte colony-stimulating factor. Local control using surgery and/or radiotherapy started at week 9 along with vincristine/dactinomycin. Maintenance included four alternating cycles of ifosfamide/etoposide and doxorubicin/CTX, with randomization to one of two CTX dose levels to determine the feasibility of dose-intensification during maintenance. RESULTS: Patients had a median age of 13.4 years (range, 4.5 to 24.9 years); 34 patients were male and 43 patients were white. Nineteen patients presented with metastatic disease, 29 had tumors greater than 8 cm in diameter, and 26 had primary bone tumors. These patients received 155 induction cycles, 91% of which resulted in grade 4 neutropenia, 68% in febrile neutropenia, and 68% in grade 3 to 4 thrombocytopenia. During maintenance, grade 4 neutropenia and grade 3 to 4 thrombocytopenia occurred in 81% and 85% of cycles, respectively. Thirty-five patients (66%) completed all therapy, only 13 without significant delays; three developed secondary myeloid malignancies. The toxicity and time to therapy completion were similar in both CTX arms. Estimated 3-year survival and event-free survival were 72%+/-8% and 60%+/-9%, respectively. CONCLUSION: Although intensifying therapy seems feasible for 25% of patients on this study, toxicity was considerable. Therefore, the noninvestigational use of dose-intensification in patients with EFT should await assessment of its impact on disease-free survival.

P-glycoprotein expression at diagnosis may not be a primary mechanism of therapeutic failure in childhood rhabdomyosarcoma.

PURPOSE: To evaluate the prognostic significance of tumor cell P-glycoprotein (Pgp) expression at diagnosis in children with rhabdomyosarcoma. PATIENTS AND METHODS: A panel of three anti-Pgp monoclonal antibodies (mAb) (C219, C494, and JSB-1) that recognize different Pgp epitopes was used to measure Pgp expression in rhabdomyosarcoma specimens obtained at diagnosis from 76 patients treated at St Jude Children's Research Hospital from 1969 to 1991. Two separate experiments using different immunohistochemical methods (immune alkaline phosphatase and immunoperoxidase) were performed to evaluate Pgp expression. The immunostaining was graded using a semiquantitative scale corresponding to the percentage of tumor cells with detectable staining. The influence of Pgp expression on outcome was assessed by the Kaplan-Meier method and Cox regression analysis with stepwise selection. The relationship between Pgp expression and clinical features was assessed using the Mantel-Haenszel method. RESULTS: Pgp expression at diagnosis did not predict worse overall survival or progression-free survival when tested in either experiment with C219, C494, or JSB-1 separately. No association was shown between Pgp expression and clinical features (clinical group, primary site, or histology) or response. However, in the immune alkaline phosphatase experiment, patients whose tumors had more than 10% tumor cell staining with all three mAbs had a significantly higher rate of estimated 5-year survival (78% +/- 10%) than did all other patients (38% +/- 8%; P = .025). In this instance, Pgp expression had independent prognostic value after adjusting for clinical group. CONCLUSION: We found no strong association between Pgp expression at diagnosis and clinical features or extent of disease in pediatric rhabdomyosarcoma. Depending on the criteria used to define it, high Pgp expression at diagnosis does not predict poor outcome. Although a large prospective study is needed to provide definitive conclusions, our findings suggest that Pgp-mediated multidrug resistance may not be a primary mechanism of therapeutic failure in rhabdomyosarcoma.

Impact of intensified therapy on clinical outcome in infants and children with neuroblastoma: the St Jude Children's Research Hospital experience, 1962 to 1988.

To gauge the impact of intensified therapy on the survival of infants (younger than 1 year, n = 129) and children (greater than or equal to 1 year of age, n = 275) with neuroblastoma, we analyzed the results of eight successive clinical trials comparing various combinations of antineoplastic drugs, surgery, and radiotherapy. Changes in treatment did not affect the survival of children with involved noncontiguous lymph nodes or distant metastatic disease until the combination of cisplatin and teniposide (CDDP/VM26) was added to a basic regimen of cyclophosphamide and doxorubicin (CTX/DOX). The resulting 4-year survival was 28% +/- 5% (SE) compared with 7% +/- 2% for previous treatments (P less than .001 by the log-rank test). The 4-year survival of infants with metastatic disease was improved by administering CTX/DOX to all patients, reserving CDDP/VM26 for those whose disease was resistant to the former combination: 82% +/- 6% versus 45% +/- 8% in earlier studies; P less than .001. In the subset of infants whose tumors had disseminated to bone or bone marrow at diagnosis, this therapeutic approach increased the probability of long-term survival from 48% +/- 10% to 85% +/- 9% (P = .01). The small group of children over 1 year of age with localized unresectable tumors also fared significantly better with the switch to CTX/DOX chemotherapy (4-year survival, 93% +/- 7% v 42% +/- 13%; P = .02). Multivariate analysis indicated that young age, limited-disease stage, nonadrenal primary site, and intensified treatment were independent predictors of a more favorable outcome. We conclude that substantial advances in the treatment of neuroblastoma have occurred over the past 25 years at this institution. The current overall 4-year survival probability of 57% +/- 4% compares favorably with estimates for most other common solid tumors of childhood.

Survival after relapse in children and adolescents with rhabdomyosarcoma: A report from the Intergroup Rhabdomyosarcoma Study Group.

BACKGROUND: Despite advances in therapy, nearly 30% of children with rhabdomyosarcoma experience progressive or relapsed disease, which is often fatal. PATIENTS AND METHODS: To facilitate the development of a retrieval therapy protocol, we studied potential risk factors that were predictive of survival after first relapse in 605 children who were enrolled onto three consecutive Intergroup Rhabdomyosarcoma Study Group protocols. RESULTS: The median survival time from first recurrence was 0.8 years; the estimated percentage of patients who survived 5 years from first recurrence was 17% +/- 2% (mean +/- SD). Univariate analysis showed that tumor histology was an important predictor of 5-year survival (P <.001): the 5-year survival rate was 64% for patients with botryoid tumors (n = 19), 26% for patients with embryonal tumors (n = 313), and 5% for patients with alveolar or undifferentiated sarcoma (n = 273). Further analysis identified prognostic factors within histologic subtypes (P <.001). For patients with embryonal tumors, the estimated 5-year survival rate was 52% for patients who initially presented with stage 1 or group I disease, 20% for those with stage 2/3 or group II/III disease, and 12% for those with group IV disease. For patients with stage 1/group I disease, estimated 5-year survival rates were higher for patients with local (72%) or regional (50%) recurrence than for those with distant (30%) recurrence. Among patients with alveolar or undifferentiated sarcoma, only the disease group predicted outcome: the 5-year survival estimate was 40% for group I versus 3% for groups II through IV. We identified a "favorable risk" group (approximately 20% of patients) whose 5-year estimated survival rate was near 50%; for all other patients, the estimated survival was near 10%. CONCLUSION: This analysis demonstrates that the probability of 5-year survival after relapse for rhabdomyosarcoma is dependent on several factors at the time of initial diagnosis, including histologic subtype, disease group, and stage. These findings will form the basis of a multi-institutional risk-adapted relapse protocol for childhood rhabdomyosarcoma.

Carboplatin/ifosfamide window therapy for osteosarcoma: results of the St Jude Children's Research Hospital OS-91 trial.

PURPOSE: To determine the activity of carboplatin/ifosfamide in patients with previously untreated osteosarcoma and to estimate patient outcomes after a multiagent chemotherapy protocol that eliminated cisplatin. PATIENTS AND METHODS: Sixty-nine patients with newly diagnosed, previously untreated osteosarcoma received three cycles of carboplatin (560 mg/m(2) x 1) and ifosfamide (2.65 g/m(2)/d x 3). Assessment of response was evaluated after two (week 6) and three (week 9) chemotherapy cycles. At week 9, histologic response was assessed. Adjuvant therapy comprised two additional carboplatin/ifosfamide cycles, doxorubicin, and high-dose methotrexate. Patients were stratified at enrollment: stratum A, resectable primary tumor without metastases; stratum B, unresectable primary tumor; and stratum C, metastatic disease at diagnosis. Week 6 response was compared with that of a historic group that received only ifosfamide during the initial window evaluation. RESULTS: The clinical and radiographic response rate to three cycles of carboplatin/ifosfamide was 67.7% (95% confidence interval, 55.0% to 78.8%). Compared with the historic population who received only ifosfamide, the combination of carboplatin and ifosfamide reduced the progressive disease rate at week 6 (31.9% v 9%, P: = .003). For patients in stratum A, the 3-year event-free survival and survival were 72.3% +/- 6.7% and 76.4% +/- 6.4%, respectively. Patients who received carboplatin-based therapy had less long-term renal toxicity and ototoxicity. CONCLUSION: This pilot trial suggests that carboplatin/ifosfamide combination chemotherapy has substantial antitumor activity. In the context of a multiagent chemotherapy protocol comprising high-dose methotrexate and doxorubicin, we found that the addition of carboplatin/ifosfamide resulted in patient outcomes comparable to trials using cisplatin-based therapy with less long-term toxicity.

Are external quality assurance (EQA) slide schemes a valid tool for the performance assessment of histopathologists?

Quality assurance plays a vital role in the healthcare profession and histopathologists play a central role in the diagnosis and treatment of disease. In the past these specialists have worked in isolation and quality assurance of their work has been difficult. In recent years this has changed with the introduction of External Quality Assurance slide schemes. This paper discusses how these schemes have evolved, the problems of standard setting and their validity as a measure of pathologists performance.

Implications of a common polymorphism in intron 12 of the dystrophin gene for deletion detection by multiplex PCR.

The multiplex polymerase chain reaction (PCR) is a reliable and efficient method for detecting dystrophin gene deletions in about 65% of patients with Duchenne or Becker muscular dystrophy (DMD or BMD). The 9-plex PCR assay, which simultaneously amplifies the muscle-specific promoter and exons 3, 6, 13, 43, 47, 50, 52 and 60, is one of the multiplex PCR assays used routinely to test for DMD and BMD deletions. In this study, we describe a previously unrecognized A to G base variation in intron 12 (nt -110 from exon 13) of the dystrophin gene. This variant, located within the annealing site of the exon 13 forward primer, prevented amplification of exon 13 in the 9-plex PCR assay. Present in 56% (25 of 45) of normal Caucasian alleles and 23% (3 of 13) of normal black American alleles, it is likely encountered frequently during dystrophin deletion analysis by multiplex PCR, and may complicate test result interpretation. Therefore, we suggest two modifications for the multiplex PCR detection of dystrophin gene deletion.

The clinicopathologic spectrum of putative extrarenal rhabdoid tumors. An analysis of 42 cases studied with immunohistochemistry or electron microscopy.

The existence of extrarenal rhabdoid tumor (ERRT) as a discrete pathologic entity has been controversial despite frequent reports of its occurrence. We performed immunohistochemistry, electron microscopy, or both on 42 cases with this diagnosis sent in consultation to us. Only 12 of the 42 neoplasms had the histological findings of "classic" malignant rhabdoid tumor of the kidney; the remainder displayed a variety of neural, epithelial, myoid, mesenchymal, or ependymal patterns. Electron microscopy also showed that most possessed neural, epithelial, or ependymal features. Immunohistochemistry generally revealed marked polyphenotypia, with immunoreactivity to a wide array of antibodies against neural, epithelial, glial, and myogenic markers. A specific tissue-based diagnostic category could not be assigned in only 11 of the 42 cases, seven of which lacked material for a comprehensive ultrastructural or immunohistochemical study. We conclude that tumors currently diagnosed as ERRT represent a heterogeneous group of neoplasms that may form unique subsets of known entities within the specific site where they arise or that may defy classification into a specific alternative category. Our findings lead us to believe that the term ERRT is not valid as representing a specific diagnostic entity and to prefer the term "poorly differentiated neoplasm with rhabdoid features" for undifferentiated tumors.

Infectious mononucleosis. The spectrum of morphologic changes simulating lymphoma in lymph nodes and tonsils.

Lymph-node and tonsillar biopsies occasionally are obtained from patients with the infectious mononucleosis syndrome secondary to Epstein-Barr viral infection, particularly if the clinical presentation is atypical and a viral etiology is not suspected. The presence of Reed-Sternberg-like cells in infectious mononucleosis resulting in confusion with Hodgkin's disease is well-known; however, similar difficulty in excluding a non-Hodgkin's lymphoma can be encountered. Eleven cases of reactive lymphoid hyperplasia with the morphologic features of infectious mononucleosis are reported, nine of which had documented Epstein-Barr viral infection. The spectrum of morphologic changes associated with Epstein-Barr viral infection is discussed, with emphasis on the features that permit their distinction from non-Hodgkin's lymphoma. Morphologic features mimicking lymphoma included extensive immunoblastic proliferations in sheets and nodules and marked cytologic atypia. Hodgkin's disease was simulated by the tendency in some cases for the atypical Reed-Sternberg-like cells to cluster about necrotic foci and to show pronounced cytologic atypia. Features permitting the distinction from non-Hodgkin's lymphoma included persistent reactive foci with the classic features of infectious mononucleosis, a polymorphous background of transformed lymphocytes rather than irregular or twisted lymphoid cells as seen in non-Hodgkin's lymphoma, and preservation of underlying reticulin architecture rather than destruction, even in cases with extensive immunoblastic proliferation. Hodgkin's disease was excluded by requiring strict criteria for Reed-Sternberg cells and noting the reactive background as inconsistent with Hodgkin's disease. Immunoperoxidase staining of seven of the cases with anti-Leu-M1 failed to demonstrate immunoreactivity of the Reed-Sternberg-like cells with this monoclonal antibody.

Immunohistochemical detection of FLI-1 protein expression: a study of 132 round cell tumors with emphasis on CD99-positive mimics of Ewing's sarcoma/primitive neuroectodermal tumor.

The histologic and immunohistochemical differentiation of Ewing' s sarcoma/primitive neuroectodermal tumor (ES/PNET) from other small, blue, round cell tumors may be difficult. Despite initial promise, CD99 (MIC2) has not proven to be a specific marker. Approximately 90% of ES/PNET have a specific t(11; 22)(q24;q12) that results in fusion of the EWS and FLI-1 genes, and overexpression of FLI-1 protein. A recent study has shown immunohistochemical FLI-1 expression in five of seven of the ES/PNET cases tested. We evaluated FLI-1 expression in 132 well-characterized small, blue, round cell tumors. All tumors were immunostained for FLI-1 (1:40, Sc 356 polyclonal, Santa Cruz Biotechnology) using steam heat for epitope retrieval. Only nuclear staining was accepted as positive. Endothelial cells were strongly positive in all cases and served as an internal control. In many cases, a subset of lymphocytes also stained positive. No staining was seen in any other normal tissue. FLI-1 expression was seen in 29 of 41 (71%) ES/PNET, 7 of 8 (88%) lymphoblastic lymphomas, 0 of 8 poorly differentiated synovial sarcomas (PDSS), 0 of 32 rhabdomyosarcoma (RMS), 0 of 30 neuroblastomas, 0 of 8 esthesioneuroblastomas, 0 of 3 Wilms' tumors, 0 of 1 mesenchymal chondrosarcoma, and in 1 of 1 desmoplastic round cell tumor. This last case was known to have an EWS/WT-1 fusion. Although the EWS/FLI-1 fusion gene is specific for ES/PNET, FLI-1 protein expression is not. Significantly, the great majority of lymphoblastic lymphomas (also CD99-positive) are strongly FLI-1-positive. Immunohistochemical detection of FLI-1 may be valuable in confirming the diagnosis of ES/ PNET in cases in which molecular genetic evaluation is not feasible. FLI-1 protein expression is also helpful in distinguishing ES/PNET from other tumors that may be CD99-positive, such as PDSS and RMS. It is not surprising that some ES/ PNET are FLI-1-negative, because not all ES/PNET have the classic EWS/FLI-1, and some cases of ES/PNET may produce either low levels of protein or idiotypically different protein.