Short, full-dose adjuvant chemotherapy (CT) in high-risk adult soft tissue sarcomas (STS): long-term follow-up of a randomized clinical trial from the Italian Sarcoma Group and the Spanish Sarcoma Group.

BACKGROUND: To report on long-term results of a phase 3 trial comparing three versus five cycles of adjuvant chemotherapy (CT) with full-dose epirubicin+ifosfamide in high-risk soft tissue sarcomas (STS). METHODS: Patients (pts) were randomized to receive three preoperative cycles of epirubicin 120 mg/m2 and ifosfamide 9 g/m2 (Arm A) or to receive the same three preoperative cycles plus two postoperative cycles (Arm B). Radiotherapy could be either delivered in the preoperative or in the postoperative setting. Non-inferiority of the primary end point, OS, was assessed by the confidence interval of the hazard ratio (HR; Arm A/Arm B) derived from Cox model. RESULTS: Between January 2002 and April 2007, 164 pts were assigned to arm A and 164 to arm B. At a median follow-up (FU) of 117 months (IQ range 103-135 months), 123 deaths were recorded: 58 in Arm A and 65 in Arm B. Ten-year OS was 61% for the entire group of patients: 64% in Arm A and 59% in Arm B. The intention-to-treat analysis confirmed that three cycles were not inferior to five cycles (one-sided 95% upper confidence limit was 1.24). A per protocol analysis was consistent with these results. Pts with leiomyosarcoma and undifferentiated pleomorphic sarcoma (UPS) had the lowest, and the highest response rates, respectively. Consistently, Leiomyosarcoma and UPS had the worse and the best prognosis, respectively. CONCLUSIONS: At a longer FU, the non-inferiority of three cycles of a full-dose conventional CT in comparison to five is confirmed. Response to therapy is also confirmed to be associated with better survival. This regimen is currently tested within an ongoing international trial against three cycles of a neoadjuvant histology-tailored CT (ClinicalTrials.gov Identifier: NCT01710176).

The GIST paradigm: how to establish diagnostic and prognostic criteria.

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal neoplasms of the gastrointestinal tract. The present review evaluates the most important parameters in GIST: epidemiology, principal clinical presentations, histopathological patterns of GIST with differential diagnosis and Fletcher's and Miettinen's risk classification, immunohistochemistry, prognostic factors, c-KIT and PDGFRalpha mutations and treatment of this tumor. The most frequent site for GISTs is the stomach, followed by the duodenum and small intestine; spindle cell morphology is described in 70% with CD17 positivity in around 95% of the cases; exon 11 of c-KIT is the most mutated, with Gleevec being the principal treatment. In GISTs, high mitotic rate, large tumor size, incomplete resection, extragastrointestinal site, duplication 502-503 in exon 9 of c-KIT, and secondary mutations are negative prognostic factors.

Prognostic time dependence of deletions affecting codons 557 and/or 558 of KIT gene for relapse-free survival (RFS) in localized GIST: a Spanish Group for Sarcoma Research (GEIS) Study.

BACKGROUND: To assess whether deletions involving codons 557 and/or 558 (critical deletions) of exon 11 of KIT are relevant in the prognosis of relapse-free survival (RFS) in gastrointestinal stromal tumor (GIST) patients with a long follow-up. PATIENTS AND METHODS: A univariate and multivariate analysis for RFS were carried out on 162 localized GIST patients over the entire follow-up period and over the intervals 0-4 years and >4 years. Factors assessed among others were Fletcher/National Institutes of Health and Miettinen-Lasota/Armed Forces Institute of Pathology (M-L/AFIP) risk categories, critical deletions and non-deletion-type mutation (NDTM) within exon 11 of KIT. RESULTS: Multivariate analyses revealed that M-L/AFIP [relative risk (RR) 11.45, confidence interval (CI) 4.40-29.76, for the high-risk subgroup and RR 5.97, CI 2.09-17.06, for the intermediate subgroup] and critical deletions (RR 3.05, CI 1.59-5.85) were independent prognostic factors for RFS for the first 4 years and for the entire follow-up period. Beyond 4 years, the high-risk M-L/AFIP subgroup (RR 8.12, CI 1.48-44.4) and NDTM (RR 6.42, CI 1.17-35.12) were independent prognostic factors for RFS. The median follow-up was 84 months. CONCLUSION: Critical deletions represent a time-dependent prognostic factor limited to the first 4 years after surgery, which could help identify a subset with higher and earlier risk for relapse in GIST patients.

Low levels of WWOX protein immunoexpression correlate with tumour grade and a less favourable outcome in patients with urinary bladder tumours.

AIMS: To correlate the immunohistochemical detection of WWOX with histological measures and disease progression within the whole spectrum of urothelial bladder neoplasms. METHODS AND RESULTS: One hundred and one patients with primary bladder tumours were retrospectively analysed. Immunohistochemically, a polyclonal antibody was utilized and the level of WWOX protein expression was analysed by using a combined score system based on intensity of the reaction and percentage of immunoreactive tumour cells. WWOX protein expression was consistently expressed in non-neoplastic urothelium, whereas a progressive loss of immunoreactivity was observed as tumour grade and stage increased (P < 0.05). Principal component analysis showed that reduced WWOX immunoexpression was significantly associated with high histological grades (P = 0.001), advanced stage (P = 0.002), tumour size (P = 0.04) and cancer progression (P = 0.028). Invasive urothelial carcinomas of the bladder with squamous metaplasia presented the lowest levels of WWOX protein. Kaplan-Meier analyses demonstrated a significant correlation between loss of WWOX expression and a shorter progression-free survival (P = 0.042), whereas the prediction of overall survival achieved borderline significance (P = 0.053). CONCLUSION: Loss of WWOX immunoexpression strongly correlates with classical clinicopathological factors and appears to be a potential predictive marker of progressive disease.

Distribution of Vascular Patterns in Different Subtypes of Renal Cell Carcinoma. A Morphometric Study in Two Distinct Types of Blood Vessels.

To analyze the presence of mature and immature vessels as a prognostic factor in patients with renal cell carcinoma and propose a classification of renal cancer tumor blood vessels according to morphometric parameters. Tissue samples were obtained from 121 renal cell carcinoma patients who underwent radical nephrectomy. Staining with CD31 and CD34 was used to differentiate between immature (CD31+) and mature (CD34+) blood vessels. We quantified the microvascular density, microvascular area and different morphometric parameters: maximum diameter, minimum diameter, major axis, minor axis, perimeter, radius ratio and roundness. We found that the microvascular density was higher in CD31+ than CD34+ vessels, but CD34+ vessels were larger than CD31+ vessels, as well as being strongly correlated with the ISUP tumor grade. We also identified four vascular patterns: pseudoacinar, fascicular, reticular and diffuse. Pseudoacinar and fascicular patterns were more frequent in clear cell renal cell carcinoma (37.62 and 35.64% respectively), followed by reticular pattern (21.78%), while in chromophobe tumors the reticular pattern predominated (90%). The isolated pattern was present in all papillary tumors (100%). In healthy renal tissue, the pseudoacinar and isolated patterns were differentially found in the renal cortex and medulla respectively. We defined four distinct vascular patterns significantly related with the ISUP tumor grade in renal cell carcinomas. Further studies in larger series are needed in order to validate these results. Analysis of both mature and immature vessels (CD34+ and CD31+) provides additional information when evaluating microvascular density.

CXCR3 chemokine receptor immunoreactivity in primary cutaneous malignant melanoma: correlation with clinicopathological prognostic factors.

BACKGROUND: A role for CXCR3, the receptor for chemokines Mig, IP-10 and interferon-inducible T cell alpha-chemoattractant, in tumour cell migration during melanoma progression has been proposed. AIMS: To analyse CXCR3 expression in primary cutaneous malignant melanomas and its comparison with clinicopathological and prognostic factors. METHODS: A retrospective immunohistochemical study was carried out on formalin-fixed paraffin-wax-embedded sections from 82 patients with primary invasive cutaneous melanomas, with a monoclonal antibody to CXCR3 (clone 49801.111; R&D Systems). Immunoreactivity was semiquantitatively evaluated: labelling intensity (0, absent; 1, weak; 2, moderate; 3, strong) multiplied by the percentage of cells in each of the four intensity categories. A positive staining was considered when the score was >100. Melanomas were categorised by age, sex, primary site, tumour thickness, growth phase, ulceration, lymphocytic infiltration, recurrence, lymph node and distant metastasis, and survival. Univariate and multivariate statistical analyses were carried out. RESULTS: Of the 82 patients, a positive CXCR3 staining was found in 26 (31.7%) patients, whereas 56 (68.3%) were negative. In univariate analysis, a significant association of CXCR3-positive tumour cell immunostaining with tumour thickness >1 mm (p = 0.003), absence of lymphocytic infiltration (p = 0.04) and the presence of distant metastasis (p = 0.048) was found. Multivariate analysis found tumour thickness as the only independent factor with considerable association with distant metastases. CONCLUSIONS: Our findings of a positive correlation of CXCR3 tumour cell immunoreactivity in human primary cutaneous melanoma with tumour thickness >1 mm and absence of intratumoral lymphocytic infiltration support the biological implication of CXCR3 in the tumour progression of cutaneous malignant melanoma.

Immunophenotypic profile of biomarkers related to anti-apoptotic and neural development pathways in the Ewing's family of tumors (EFT) and their therapeutic implications.

BACKGROUND: Ewing's family of tumors (EFT) comprises a broad spectrum of tumors composed of primitive committed cells with neuroectodermal capacity. The degree of neural differentiation within EFT, as measured with morphological features and expression of neural markers, delimits two members: Ewing's sarcoma (ES) and peripheral primitive neuroectodermal tumor (pPNET). Molecules such as c-kit and its ligand (Stem cell factor, SCF), CD95 (FAS), CD95L (FASL), IGF-IR, protect EFT cells from apoptosis, whereas c-erb-B2, erythropoietin (EPO) and its receptor (EPO-R) participate in the maturation of primitive committed neuroectodermal cells and in the normal embryonal brain development. The aim of the present study was to analyse the expression of these molecules in paraffin-embedded material from a series of EFT. MATERIALS AND METHODS: Forty-five cases of EFT (23 typical ES, 4 atypical and 18 pPNET) were analysed following the immunohistochemical LSAB method, with antigen retrieval heating using an autoclave, citrate buffer pH 6.0 and the following primary antibodies: FAS (APO-CD 95), FAS-L, c-kit, SCF, IGF-IR and c-erbB2. The expression was evaluated independently by three of the authors and the final score (0 to 3+) was based on the intensity and percentage of positively stained cells. In a second cooperative analysis, tissues from 30 cases of EFT (15 typical, 3 atypical and 12 PNET) were immunostained with EPO and EPO-R. RESULTS: High expression of c-kit/SCF (2+, 3+) was detected in 28/45 cases of EFT (62.2%), whereas FAS-FAS-L and IGF-IR were observed in 16/45 (37.7%) and 9/45 (20%), respectively. Regarding the neuroectodermal pathway, membranous and cytoplasmic expression of c-erb-B2 was observed in 9/45 (20%) EFT, regardless of the morphological and immunohistochemical expression of conventional neural markers. High expression of EPO and EPO-R was observed in 20/30 EFT (66.6%). CONCLUSION: C-kit/SCF and EPO/EPO-R seem to participate in the pathway of anti-apoptotic and proliferative advantage, while c-erb-B2 does not play an important role in the neuroectodermal differentiation pathway in EFT cells.

TuBaFrost 4: access rules and incentives for a European tumour bank.

When designing infrastructure for a networked virtual tumour bank (samples remain at the collector institutes and sample data are collected in a searchable central database), it is apparent that this can only function properly after developing an adequate set of rules for use and access. These rules must include sufficient incentives for the tissue sample collectors to remain active within the network and maintain sufficient sample levels in the local bank. These requirements resulted in a key TuBaFrost rule, stating that the custodianship of the samples remains under the authority of the local collector. As a consequence, the samples and the decision to issue the samples to a requestor are not transferred to a large organisation but instead remain with the collector, thus allowing autonomous negotiation between collector and requestor, potential co-authorship in publications or compensation for collection and processing costs. Furthermore, it realises a streamlined cost effective network, ensuring tissue visibility and accessibility thereby improving the availability of large amounts of samples of highly specific or rare tumour types as well as providing contact opportunities for collaboration between scientists with cutting edge technology and tissue collectors. With this general purpose in mind, the rules and responsibilities for collectors, requestors and central office were generated.

TuBaFrost 3: regulatory and ethical issues on the exchange of residual tissue for research across Europe.

The regulatory regimes for research with residual tissue and accompanying data differ widely between countries in the European Union (EU): from specific consent to opt-out or even no consent at all. This could greatly hamper research where the exchange of tissue and accompanying data has become the gold standard, like in TubaFrost. Instead of adhering to international guidelines, which have a democratic deficit, or an attempt for a new set of possible harmonising rules, TubaFrost chose to create a coordinating rule: if tissue may legitimately be used for a certain kind of research in the country where it was taken and under whose jurisdiction the patient falls, it may also be used for such research in the country where it is sent to in the context of a scientific program even if in that other country other regulations would apply for research with residual tissue taken from patients under their jurisdiction. This coordinating rule has a sound basis in EU law in general and will solve the problems related to diverging national regulatory regimes in the case of cross national research with residual tissue.

TuBaFrost 6: virtual microscopy in virtual tumour banking.

Many systems have already been designed and successfully used for sharing histology images over large distances, without transfer of the original glass slides. Rapid evolution was seen when digital images could be transferred over the Internet. Nowadays, sophisticated Virtual Microscope systems can be acquired, with the capability to quickly scan large batches of glass slides at high magnification and compress and store the large images on disc, which subsequently can be consulted through the Internet. The images are stored on an image server, which can give simple, easy to transfer pictures to the user specifying a certain magnification on any position in the scan. This offers new opportunities in histology review, overcoming the necessity of the dynamic telepathology systems to have compatible software systems and microscopes and in addition, an adequate connection of sufficient bandwidth. Consulting the images now only requires an Internet connection and a computer with a high quality monitor. A system of complete pathology review supporting bio-repositories is described, based on the implementation of this technique in the European Human Frozen Tumor Tissue Bank (TuBaFrost).

TuBaFrost 1: Uniting local frozen tumour banks into a European network: an overview.

TuBaFrost is the consortium responsible for the creation of a virtual European human frozen tumour tissue bank: a collection of high quality frozen residual, accurately classified tumour tissue samples, which are stored in European cancer centres and universities. This virtual tissue bank, searchable on the internet, has rules for access and use, and a code of conduct to comply with the various legal and ethical regulations in European countries. The easy accessibility and the European scale of the bank will result in the availability of a large number of samples even of rarer tumour types. Standardisation of collection, storage and quality control throughout the network is achieved minimising inter-institutional variability. A website providing access to upload, search and request samples is a key tool of the tissue bank. The search engine makes use of virtual microscopy. An overview of the development of the European virtual frozen tissue bank infrastructure is described in this paper. The various key aspects are described in more detail in a series of articles to appear in this Journal.

TuBaFrost 2: Standardising tissue collection and quality control procedures for a European virtual frozen tissue bank network.

Tumour Bank Networking presents a great challenge for oncological research as in order to carry out large-scale, multi-centre studies with minimal intrinsic bias, each tumour bank in the network must have some fundamental similarities and be using the same standardised and validated procedures. The European Human Frozen Tumour Tissue Bank (TuBaFrost) has responded to this need by the promotion of an integrated platform of tumour banks in Europe. The operational framework for TuBaFrost has drawn upon the best practice of standard workflows and operating procedures employed by members of the TuBaFrost project and key initiatives worldwide.

TuBaFrost 5: multifunctional central database application for a European tumor bank.

Developing a tissue bank database has become more than just logically arranging data in tables combined with a search engine. Current demand for high quality samples and data, and the ever-changing legal and ethical regulations mean that the application must reflect TuBaFrost rules and protocols for the collection, exchange and use of tissue. To ensure continuation and extension of the TuBaFrost European tissue bank, the custodianship of the samples, and hence the decision over whether to issue samples to requestors, remains with the local collecting centre. The database application described in this article has been developed to facilitate this open structure virtual tissue bank model serving a large group. It encompasses many key tasks, without the requirement for personnel, hence minimising operational costs. The Internet-accessible database application enables search, selection and request submission for requestors, whereas collectors can upload and edit their collection. Communication between requestor and involved collectors is started with automatically generated e-mails.

TuBaFrost: European virtual tumor tissue banking.

TuBaFrost is a consortium responsible for the task to create a virtual European human frozen tumor tissue bank, composed of high quality frozen tumor tissue collections with corresponding accurate diagnosis stored in European cancer centers and universities, searchable on the Internet, providing rules for access and use and a code of conduct to comply with the various legal and ethical regulations in European countries. Such infrastructure would enlarge tissue availability and accessibility in large amounts of specified or even rare tumor samples. Design of an infrastructure for European residual tissue banking with the described characteristics, clear focus points emerge that can be broken down in dedicated subjects: (1) standardization and quality assurance (QA) to avoid inter-institute quality variation; (2) law and ethics enabling exchange of tissue samples possible between institutes in the different European countries, where law and ethics are characterized by a strong variability; (3) rules for access, with sufficient incentives for collectors; (4) central database application containing innovations on search and selection procedures; (5) support when needed with histology images; and (6) Internet access to search and upload, with in addition a solid website giving proper information on the procedures, intentions and activities not only to the scientific community, but also to the general public. One consortium decision, part of the incentives for collectors, had major impact on the infrastructure; custodianship over the tissues as well as the tissues stay with the collector institute. Resulting in specimens that are not given to an organization, taking decisions on participation of requests, but instead the local collected tissues stay very easy to access by the collector and allows autonomous negotiation between collector and requestor on cooperation, coauthorship in publication or compensation in costs. Thereby, improving availability of large amounts of high quality samples of a highly specified or rare tumor types and contact opportunities for cooperation with other institutes.

Virtual microscopy in virtual tumor banking.

Many systems have already been designed and successfully used for sharing histology images over large distances, without transfer of the original glass slides. Rapid evolution was seen when digital images could be transferred over the Internet. Nowadays, sophisticated virtual microscope systems can be acquired, with the capability to quickly scan large batches of glass slides at high magnification and compress and store the large images on disc, which subsequently can be consulted through the Internet. The images are stored on an image server, which can give simple, easy to transfer pictures to the user specifying a certain magnification on any position in the scan. This offers new opportunities in histology review, overcoming the necessity of the dynamic telepathology systems to have compatible software systems and microscopes and in addition, an adequate connection of sufficient bandwidth. Consulting the images now only requires an Internet connection and a computer with a high quality monitor. A system of complete pathology review supporting biorepositories is described, based on the implementation of this technique in the European Human Frozen Tumor Tissue Bank (TuBaFrost).

[Genomic profile in high risk neuroblastoma by comparative genomic hybridization]. / Perfil genómico del neuroblastoma de alto riesgo mediante hibridación genómica comparada.

Different subtypes of neuroblastoma (NB) carry associated genetic aberrations that predict their clinical course. Whole chromosome gains are usually associated with early clinical stages and good prognosis, while 1p deletion, 17q gain and MYCN amplification (MNA) are related to advanced stages and poor prognosis. High-risk neuroblastomas (NB-HR) include NB in children aged more than 1 year old, either stage 4 or any stages showing MNA except stage 1. The prognosis of NB-HR patients remains poor, despite aggressive therapy. Only MNA confers poor prognosis. Between January 2000 and February 2005, tumoral specimens from 60 patients with NB-HR were sent to the Spanish Reference Center for NB biological studies. In all cases, MYCN together with 1p36 status was analyzed by fluorescence in situ hybridization (FISH). Comparative genomic hybridization (CGH) was performed in 24 cases. Using FISH we detected 31 MNA cases including 29 with 1p36 deletion; there were 21 cases without MYCN amplification (MNNA) but 7 of these had 1p36 deletion; 8 cases showed MYCN gain (MNG) but 6 of these had 1p36 deletion. CGH showed other chromosomal alterations. Of 11 MNA cases, none had 11q loss and all of them showed 17q gain or 17 disomy. Of the 7 MNNA cases, there were 4 with 11q loss including 2 with 3p loss and all presented 17q gain or 17 disomy. The 6 MNG cases included 4 cases with 11q loss and 5 cases with 17q gain or 17 disomy. Genomic profiling by CGH in NB-HR confirms the interaction among genetic alterations, the prognostic significance of which should be evaluated to establish new treatment criteria.

[Usefulness of bcl-2 expression as a new basal cell marker in prostatic pathology]. / Utilidad de bcl-2 como nuevo marcador de expresión de células basales en patología prostática.

INTRODUCTION AND OBJECTIVES: The diagnosis of invasive adenocarcinoma of the prostate is often difficult in needle prostatic cores, where, additionally, the assessment of the presence of basal cells has demonstrated to be of paramount importance. Currently, the immunohistochemical expression of 34betaE12 antigen and p63 protein are the most utilized markers. In our study, we analyzed comparatively the expression of 34betaE12, p63, bcl-2 and alpha-methylacyl-CoA racemase in order to evaluate the usefulness of bcl-2 as a new marker of the basal cells in prostatic pathology. METHODS AND RESULTS: This study comprises radical prostatectomy specimens from 48 patients which were studied in order to determine the lack of staining of basal cells in invasive tumor areas together with the expression of racemase. Likewise, the presence of basal cells in areas of atrophy, hyperplasia, adenosis, and high-grade prostatic intraepithelial neoplasia (PIN) was also examined. Within the areas of adenosis and PIN a discontinuous pattern of basal cell expression was found in some cases. In 2 out of 48 cases (4,2%) of invasive carcinoma a weak bcl-2 expression without a basal cell distribution was found. Moreover, the expression of bcl-2 in the stromal lymphocytes appeared to be essential as an internal positive control of the technique. CONCLUSIONS: In addition to classical markers, we demonstrated the diagnostic value of bcl-2 as a new basal cell marker.

Angiogenesis in neuroblastoma: relationship to survival and other prognostic factors in a cohort of neuroblastoma patients.

PURPOSE: To study angiogenesis in neuroblastoma, using morphometric and computerized image analysis, and correlate the results with survival and other prognostic factors. PATIENTS AND METHODS: Sixty-nine patients from the Spanish Cooperative Study for Neuroblastoma were studied. Tumoral angiogenesis was studied using an avidin-biotin immunoperoxidase technique with an anti-CD34 antibody. Vascular parameters (VPs) were analyzed by a computerized system. Statistical analysis was also performed. RESULTS: Sixty-six samples had adequate tumoral tissue, and their tumoral vessels were counted. Endothelial cells were more prominent in pure neuroblastomas than in maturing and more mature tumors. VPs showed no statistical difference between the groups of patients as defined by the levels of the other prognostic factors in neuroblastoma: age, stage, histopathology, TRK-A, P-glycoprotein expression, or MYCN copy number. In patients who relapsed, tumors did not show statistically significant difference in VPs when compared with tumors from patients who did not relapse. There was also no difference in VPs in tumors from living patients when compared with tumors from deceased patients. Overall survival was 75%, and event-free survival was 55% at 50 months. CONCLUSION: VPs could be adequately determined by a computerized system in neuroblastoma; however, VPs were not predictive of survival for our patients. In our patients, neither disseminated nor local relapses were influenced by the angiogenic characteristics of the tumors.

H7, an inhibitor of protein kinase C, inhibits tumour cell division in mice bearing ascitic Ehrlich's carcinoma.

We have previously shown that H7, an inhibitor of protein kinase C (PKC), inhibits proliferation of several cell lines as well as of primary cultured cells from human tumours. The aim of this work was to assess whether H7 is able to prevent the division of tumour cells in mice bearing Ehrlich's ascitic carcinoma. The LD50 of H7 injected intravenously was 61 mg/kg and 94 mg/kg for starved and fed mice, respectively. Acute intraperitoneal injection of 100 mg/kg of H7 decreased the number of mitoses in tumoral cells from ascitic fluid of mice bearing the carcinoma. The reduction was maximal (approximately 50%) after 90 min and then the number of mitosis rose due to a decrease in H7. Continuous delivery of H7 from mini-osmotic pumps implanted on the backs of the mice reduced the number of mitoses by approximately 65%, and the effect was maintained for approximately 24 h. The effect cannot be maintained for longer because H7 is unstable at body temperature. These results indicate that inhibition of PKC can block division of tumour cells in carcinoma-bearing animals, and support the idea that inhibitors of PKC could be useful for the clinical control of proliferation of certain tumours.

EWS/FLI-1 rearrangement in small round cell sarcomas of bone and soft tissue detected by reverse transcriptase polymerase chain reaction amplification.

Recent cloning of the t(11;22) region has led to the detection of a number of sequences involved in the breakpoints by substituting a sequence which encodes a putative RNA binding domain for that of the DNA binding domain of the human homologue of murine FLI-1. Several tumours display consistent translocation at t(11;22) (q24;q12), a finding that suggests these fusion transcripts could be expressed and detected by reverse transcriptase polymerase chain reaction amplification. To date, only a small number of Ewing's sarcomas (Es) and peripheral neuroectodermal tumours (pPNET) of bone have been tested with this novel molecular biology approach. In this study, we confirmed the presence of the three putative chimaeric transcripts on 7 cases of Es and pPNET sarcomas of bone and soft tissue, providing 100% positivity for the tested tumours. For comparative purposes, a number of other neuroectodermal tumours were analysed with negative results: esthesioneuroblastoma, retinoblastoma, Schwannoma. A primitive soft tissue sarcoma (ectomesenchymoma) with a 22 chromosome rearrangement did not express any transcript, nor did a number of non-neuroectodermal small round cell sarcomas of soft tissue (rhabdomyosarcomas) and bone (microcellular osteosarcoma), conventional bone sarcomas, leiomyosarcomas, malignant fibrous histiocytomas and synovial sarcomas. These results reinforce the value of molecular biology techniques for the correct assessment of histology difficult evaluable neoplasms, such as the group of small round cell tumours within the Es family.