Defining Ewing and Ewing-like small round cell tumors (SRCT): The need for molecular techniques in their categorization and differential diagnosis. A study of 200 cases.

BACKGROUND: Differentiation of Ewing sarcoma family of tumors (ESFT) and Ewing-like tumors remains problematic. Certain ESFT with morphological and immunohistochemical (IHC) profiles lack the EWSR1-ETS transcript. To improve diagnostic accuracy we investigated the presence of several specific transcripts in 200 small round cell tumors (SRCT) displaying ESFT morphology and immunophenotype in which EWSR1 FISH analysis was non-informative or negative. DESIGN: 200 tumors (formalin-fixed, paraffin-embedded) were analyzed by RT-PCR. All tumors were tested for EWSR1-ETS, EWSR1/WT1, PAX3/7-FOX01 or SYT/SSX transcripts, and the negative tumors were subsequently analyzed for CIC/DUX4, BCOR/CCNB3 and CIC/FOX04 transcripts. RESULTS: 133 (66.5%) ESFT displayed one of the above EWSR1-ETS translocations. Three cases (1.5%) revealed the SYT-SSX transcript for Synovial sarcoma, and one (0.5%) a EWSR1-WT1 transcript for Desmoplastic Small Round Cell tumor. The CIC-DUX4 translocation was found in six Ewing-like tumors (3%) with CD99 positivity. The BCOR-CCNB3 gene fusion was observed in 5 tumors (2.5%) displaying round or spindle cells with strong CCNB3 IHC expression in 3 tumors. Moreover, RT-PCR failed to detect any gene fusion transcripts in 19 tumors (9.5%) and were considered "undifferentiated small round cell sarcoma" (SRCS). Molecular biology results were non-informative in 33 SRCTs (16.5%) due to RNA degradation through inadequate fixation and/or decalcification. CONCLUSION: Our analysis of 200 SRCTs confirms the molecular heterogeneity of neoplasms with ESFT morphology and highlight that molecular studies with RT-PCR including new emerging gene fusion transcripts are mandatory for the diagnosis when EWSR1 FISH is negative or non-informative. The incidence of CIC-DUX4, BCOR-CCNB3 and CIC-FOX04 transcripts was relatively low. A small group of Ewing-like sarcomas or undifferentiated SRCS remains unclassified. Adopting appropriate tissue fixation and processing protocols is important to avoid degradation of fixed/embedded tissue when no frozen tumor is available.

Ewing-like sarcoma with CIC-DUX4 gene fusion in a patient with neurofibromatosis type 1. A hitherto unreported association.

Sarcoma with CIC-DUX4 gene fusion is emerging as the most prevalent subset of Ewing-like undifferentiated small round cell sarcomas with around 50 cases published. We report hereby the case of a 40-year-old male who presented a CIC-DUX4 sarcoma in deep soft tissues in his thigh. He had been diagnosed with neurofibromatosis type 1 at age 19 and over the years underwent resection of multiple neural neoplasms, including two malignant peripheral nerve sheath tumors with classical spindle-cell histopathology. The CIC-DUX4 sarcoma was treated with surgical resection, radiation and chemotherapy, but lung and brain metastases developed and the patient died from the disease 14 months after diagnosis. This is the first case of sarcoma with CIC-DUX4 gene fusion reported in a patient with NF1. Whether this association is coincidental or CIC-DUX4 sarcomas could be related to NF1 remains to be clarified. Study of alternative molecular alterations in EWSR1-negative undifferentiated small round cell sarcomas is clinically relevant, since CIC-DUX4 sarcomas seem to be a very aggressive subset with poor response to the presently used therapeutic regimens.

Deregulation of glyceraldehyde-3-phosphate dehydrogenase expression during tumor progression of human cutaneous melanoma.

BACKGROUND/AIM: Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a highly abundant housekeeping gene. GAPDH overexpression has been reported in diverse types of human cancers including cutaneous melanoma. Our goal was to quantify GAPDH mRNA and protein expression in the whole spectrum of primary and metastatic melanomas in the search for a specific role for this ubiquitous molecule during tumor progression. MATERIALS AND METHODS: Intratumoral GAPDH mRNA expression was quantified by real-time PCR in 71 cases, including 29 primary melanomas and 42 metastatic cases. Relative expression levels in thin (≤1 mm) and thick (>1 mm) primary tumors and 'in-transit', lymph node and distant metastases were compared. Similarly, protein expression was investigated by means of immunohistochemistry. Specific exons of GAPDH were analyzed by DNA sequencing. RESULTS: GAPDH mRNA expression was significantly up-regulated in thick melanomas when compared to primary thin melanomas. Similar differences were also encountered between metastatic melanomas when compared to lymph-node metastatic melanomas. Interestingly, GAPDH protein immunoexpression was higher in thick melanomas and distant metastases than in thin tumors and lymph node metastases, respectively. However, no specific point-mutations in GAPDH-specific exons were found in any patient. CONCLUSION: Deregulation of GAPDH during melanoma progression was demonstrated in our series by mRNA and protein expression studies.

CCL27-CCR10 and CXCL12-CXCR4 chemokine ligand-receptor mRNA expression ratio: new predictive factors of tumor progression in cutaneous malignant melanoma.

CXCR4, CCR7 and CCR10 chemokine receptors are known to be involved in melanoma metastasis. Our goal was to compare the relative intratumoral mRNA expression of these receptors with that of their corresponding chemokine ligands, CXCL12, CCL19, CCL21, and CCL27 across the full spectrum of human melanoma progression: thin and thick primary melanomas, as well as "in transit", lymph node, and distant metastases. Expression was quantified by real-time RT-PCR in 103 melanoma samples: 51 primary tumors and 52 metastases. Particular emphasis was focused on chemokine ligand-receptor expression ratios. Immunohistochemistry was performed to identify the cell types expressing these molecules. CXCL12-CXCR4 and CCL27-CCR10 ratios were higher in thin than in thick primary melanomas, and all four chemokine-receptor ratios were higher in primary tumors than in melanoma metastases. CCL27-CCR10 and CXCL12-CXCR4 expression ratios in primary tumors were inversely associated with the development of distant metastases, and improved the predictive value of tumor thickness for distant metastasis, which is important since chemokine ligand-receptor ratios are not affected by the endogenous gene employed for normalizing mRNA expression. Both receptor and ligand immunolabeling were detected in neoplastic cells suggesting autocrine mechanisms. Our results support the concept that low CCL27/CCR10 and CXCL12/CXCR4 intratumoral mRNA ratios are associated with melanoma progression, and in combination with Breslow thickness, are the best predictive factors for the development of distant metastases in primary cutaneous melanoma.

Characterization of a new human cell line (CH-3573) derived from a grade II chondrosarcoma with matrix production.

Chondrosarcomas are malignant cartilage-forming tumors that represent the third most common malignant solid tumor of bone. In patients with grades II and III, local recurrence, increasing tumor size and dedifferentiation have been associated with lower survival rates. These biologically poorly-understood neoplasms vary considerably in clinical presentation and biological behavior. Cytogenetic studies have shown that heterogeneity is related to karyotypic complexity; moreover, alterations in the 9p21 locus and TP53 gene are related to disease progression. Despite the relatively high frequency of chondrosarcoma only a limited number of cell lines exist in the scientific community, limiting the possibility to study hypothesis-derived research or primary drug interaction necessary for pre-clinical studies. We report a chondrosarcoma cell line, CH-3573, derived from a primary tumor that may serve as a useful tool for both in vitro and in vivo models to study the molecular pathogenesis. In addition, xenograft passages in nude mice were studied to characterize the genetic stability over the course of tumor progression. In contrary to other reported cell lines, an important feature of our established cell line was the retained matrix production, a characteristic feature of a conventional grade II chondrosarcoma. The cell line (CH-3573) was characterized by pathological, immunohistochemical and molecular genetic methods.

Dermatofibrosarcoma protuberans: a clinicopathological, immunohistochemical, genetic (COL1A1-PDGFB), and therapeutic study of low-grade versus high-grade (fibrosarcomatous) tumors.

BACKGROUND: Dermatofibrosarcoma protuberans (DFSP) is an uncommon cutaneous tumor, usually low grade, except for the fibrosarcomatous variant (DFSP-FS). OBJECTIVES: We sought to compare the clinicopathological, immunohistochemical, genetic, and therapeutic features between DFSP and DFSP-FS. METHODS: The clinicopathological features were reviewed in 63 DFSP and 12 DFSP-FS. Immunohistochemistry and multiplex reverse transcriptase-polymerase chain reaction were carried out using formalin-fixed, paraffin-embedded tissue, using specific primers for collagen type I alpha 1 (COL1A1) and platelet-derived growth factor beta (PDGFB). RESULTS: DFSP-FS was associated with tumor history longer than 5 years (P = .009), tumor size greater than 4 cm (P = .001), more stages of modified Mohs micrographic surgery (P = .005), expansive subcutaneous infiltration (P = .005), muscular invasion (P = .0001), absence of CD34 staining (P = .018), p53 positivity (P = .006), and increased proliferative activity (P = .004) compared with DFSP. The COL1A1-PDGFB fusion transcript was found in 100% DFSP-FS and 72% DFSP. No association was found between the different COL1A1-PDGFB fusion transcripts and the different histologic subtypes. Wide local excision (2 cm) was performed in 47% of cases and modified Mohs micrographic surgery in 53%. After a mean follow-up of 73 months (range 21-235), 6 patients had local recurrence (5 DFSP, 1 DFSP-FS) and one died of disease (DFSP-FS). The only factor related to local recurrence was the type of surgery (17% wide local excision vs 0% modified Mohs micrographic surgery) (P = .006). LIMITATIONS: Our study is retrospective. Prospective studies are necessary to confirm our results. CONCLUSIONS: DFSP-FS reflects tumor progression in DFSP, with larger size, particular invasive patterns, p53 expression, and increased proliferative activity. However, as in low-grade DFSP, appropriate surgery permits a tumor-free excision. COL1A1-PDGFB is a useful tool for diagnosis of DFSP and particularly for DFSP-FS.

Evaluation of prognostic factors and their capacity to predict biological behavior in gastrointestinal stromal tumors.

Gastrointestinal stromal tumors (GISTs) are c-KIT-signaling-driven mesenchymal tumors of the human digestive tract, many of which have c-KIT or PDGFRα activating mutations. The authors studied the immunohistochemical markers, c-KIT and PDGFRα mutations, in GISTs and their association with the clinicopathological and clinical follow-up in 145 GISTs. Tumors were located mainly in the stomach, the median tumor size being 7.5 cm. The mitotic index was ≤5 mitoses per 50 high-power fields in 61% of cases, 96% expressed CD117, and c-KIT or PDGFRα mutations were detected in 68% of cases. The median follow-up of the series was 52 months (range = 1 to 244.9 months). Tumor size, cell morphology, mitotic index, incomplete resection, Fletcher's risk classification, Ki-67 overexpression, and c-KIT mutations were associated with progression-free survival. Incomplete resection and mitotic activity also provide information about overall survival. In conclusion, complete clinicopathological, immunohistochemical, and genetic descriptions are necessary to characterize this disease and optimize its clinical management.

The epithelial mesenchymal transition process in wilms tumor: a study based on a xenograft model.

BACKGROUND: Until now, only a few mouse-transplanted human tumors or experimental Wilms tumor (WT) cell lines have been described. The aim of this study was to show the biological behavior, including histology, immunohistochemistry (IHC), and molecular biology, of a WT including the original tumor and metastasis transferred into nude mice and followed for successive generations in xenografts. METHODS: A WT metastasis was xenotransplanted into nude mice and the mice was monitored for 7 passages over a period of 29 months; the original neoplasm was comparatively studied. The morphology was evaluated by optical and electron microscopy. The protein expression was analyzed by immunohistochemistry in whole sections and in tissue microarray. The molecular studies were carried out by multiplex ligation-dependent probe amplification and polymerase chain reaction analysis. RESULTS: The histology changed markedly between the fourth and fifth transfer. The tumor exhibited an increased epithelial component (>40%) together with a slowing in the growth rate (8 mo). An epithelial-mesenchymal transition seemed to take place in the fourth passage and increased thereafter. The genetic studies also showed a WT5 deletion and a MYCN gain in all the tumor samples in passage 4 and beyond, but did not show E-cadherin, ß-catenin, and APC mutations. CONCLUSIONS: An epithelial pattern was associated with slow tumor growth, whereas the predominance of mesenchymal spindle cells with striated muscle cell differentiation was related with a high growth rate. The in vivo reorganization of the tumor components (blastemal, epithelial, and mesenchymal) does not seem to be related with the Wnt and EMT pathways.

Clinicopathological significance of cell cycle regulation markers in a large series of genetically confirmed Ewing's sarcoma family of tumors.

More than 90% of all Ewing's Sarcoma Family of Tumors (ESFT) exhibit specific chromosomal rearrangements between the EWS gene on chromosome 22 and various members of the ETS gene family of transcription factors. The gene fusion type and other secondary genetic alterations, mainly involving cell cycle regulators, have been shown to be of prognostic relevance in ESFT. However, no conclusive results have been reported. We analyzed the clinicopathological significance of relevant cell cycle regulators in genetically confirmed ESFT. A total of 324 cases were analyzed for the immunohistochemical expression of p53, p21(Waf1/Cip1) , p27(Kip1) and Ki67 and the chromosomal alterations of the p53 and 9p21 locus by fluorescent in situ hybridization. We observed that expression of p53 (p = 0.025), p21(Waf1/Cip1) (p = 0.015) and p27(Kip1) (p = 0.013) was higher in disseminated than in localized disease. Furthermore, a cohort of 217 patients with localized disease was considered for studying the prognosis involvement of these factors on patient follow-up. The median follow-up was 39 months (range: 0.17-452) with an overall survival (OS) of 55%. Ki67 was expressed in 34% of cases and constituted an independent prognostic factor for progression free survival and OS independently of the type of treatment [hazard ratio of 2.0 (95% CI: 1.3-3.1; p = 0.003) and 1.9 (95% IC: 1.3-2.9; p = 0.007) for progression free survival and OS, respectively, being especially relevant in the group of patients which incorporated radiotherapy in their regimen schedules. In conclusion, this study demonstrates that Ki67 expression constitutes a valuable indicator of poor prognosis in localized ESFT.

Molecular diagnosis of Ewing sarcoma family of tumors: a comparative analysis of 560 cases with FISH and RT-PCR.

AIMS: To compare the sensitivity and specificity of fluorescence in situ hybridization (FISH) with reverse transcription polymerase chain reaction (RT-PCR) in the diagnosis of Ewing sarcoma family of tumors (ESFTs) and other small round-cell tumors (SRCTs) in formalin-fixed paraffin-embedded tissue assembled in tissue microarrays (TMAs). The second objective is to confirm the value of molecular methods and immunohistochemical (IHC) assays, to perform a differential diagnosis between ESFTs and SRCTs with similar or overlapping morphology. MATERIALS AND METHODS: A total of 560 cases were selected for the present study out the 806 cases collected from the PROgnosis and THerapeutic Targets in the Ewing's Family of TumorS project. Case selection bias included only the cases with enough material to enable the TMA construction, as FISH analysis and the majority of IHC studies were performed in TMAs. Histopathologic, IHC, and molecular assays were carried out. RESULTS: Of the 560 total cases, 411 (73.4%) were considered informative (with results by FISH and/or RT-PCR assays). From the informative cases, 382 (92.9%) were diagnosed as ESFT, 23 cases (5.6%) as non-ESFT but with specific diagnosis for another established entity, and 6 cases (1.5%) as small round cell tumors not otherwise specified. Sensitivity and specificity for the FISH assays was 96.3% and 95.2%, respectively, whereas RT-PCR presented a sensitivity of 97.5% and specificity of 92.9%. In concordant cases, both methods showed a sensitivity and specificity of 99.2% and 100%, respectively. Twenty-nine cases (7.1%) initially interpreted at morphologic level as atypical ESFTs were finally reclassified, with the support of molecular methods and IHC, as either non-ESFT with another specific histologic type or as small round cell tumors not otherwise specified. CONCLUSIONS: FISH and RT-PCR are ancillary techniques possessing high sensitivity in the diagnosis of ESFT; nevertheless, FISH is more specific than RT-PCR in the diagnosis of formalin-fixed paraffin-embedded tissue. Both methods in combination displayed the highest sensitivity and specificity. The combination of histopathologic, IHC, and molecular findings is the method of choice for the diagnosis of ESFT, as well as for the differential diagnosis with other SRCTs.

New approaches in the diagnosis of small-cell round tumors of bone and soft tissue.

The diagnosis of 17 small-cell round tumors of the bone and soft tissue, which were histologically similar to Ewing's sarcoma (EWS), was verified on paraffin sections, by using tissue microarray (TMA) technology, immunohistochemistry, cytogenetic (FISH) and molecular biological (RT-PCR) methods. Classical EWS was found to be in 8 patients, large-cell EWS in 1 patient; atypical EWS in 1, and endothelial ESW in 1. Two patients were diagnosed as having primitive neuroectodermal tumor (PNET), synovial sarcoma was present in 1 patient, embryonic rhabdomyosarcoma in 1, high-grade undifferentiated sarcoma in 1 and diffuse B-cell large-cell lymphoma in 1. TMA makes it possible to perform a number of diagnostic procedures on the same block containing a copious number of tumor samples and to assess the results of their use. It is emphasized that the diagnosis of small-cell round tumors requires the use of a package of the currently available methods providing the qualitative characteristics of each neoplasm.

Inflammatory fibroid polyp of the small bowel with a mutation in exon 12 of PDGFR alpha.

Inflammatory fibroid polyp (IFP) is a benign reactive uncommon submucosal lesion of the gastrointestinal tract, the small intestine being the most common site of origin. Histologically, IFPs are characterized by spindle cells, a heavy inflammatory infiltrate including eosinophils and onion-sheet-like formation of lesional cells around blood vessels. We present a case report of an IFP harboring an activation mutation in the PDGFR alpha gene. The lesion was positive for CD34, PDGFR alpha, and p-PDGFR alpha immunostaining but was negative for c-KIT and desmin. After a sequencing analysis of KIT and PDGFR alpha, a mutation consisting of an in-frame deletion of codons 567-571 and a missense mutation in codon 566 (S566R) of PDGFR alpha was observed. This mutation could activate key cellular pathways with involvement in the pathogenesis of this entity. We concluded that more studies are necessary in order to clarify if this finding is a biologically distinct behavior or, on the contrary, represents a specific feature of the IFP.

Tissue microarrays: applications in study of p16 and p53 alterations in Ewing's cell lines.

BACKGROUND: Tissue microarrays (TMAs) are used to study genomics and proteomics in several tumour tissue samples. Cell lines (CC) are of great importance in the study of the genetic changes in tumours, and some reveal several aspects of tumour oncogenesis. There are few published reports on Ewing's tumours with TMAs including original tumours (OT) and corresponding CC. METHODS: We have performed four TMAs, from 3 OT and the corresponding CC of successive in vivo and in vitro tumour passages. Xenotransplant CC in nude mice from OT (XT/OT) was made. Subsequently multiple XT were performed and in vitro XT cell line (CC/XT) was obtained. In vivo re-inoculation of CC/XT (XT/CC) was planned. TMAs with the successive tumour passages that grew in nude mice (XT/OT and XT/CC) were analyzed by morphologic pattern (Hematoxilin/eosin), immunohistochemical staining (CD99, FLI1, p16, p53, ki-67), fluorescent in situ hybridization-FISH-(EWSR1 break apart, p16 and p53 status) and gene fusion types. RESULTS: Heterogeneous results of the p16, p53 and ki67 in OT, XT/OT, CC/XT and XT/CC were observed. The three cell lines revealed EWS/FLI1 rearrangements. p16 gene was deleted only in one case. The deletion was detected by FISH and confirmed by PCR assays. A p53 alteration was found in the second case with monosomy and subsequently polysomic status of chromosome 17 during the evolution of CC. The PCR study revealed p53 mutation. The third case showed hypermethylation in the promoter of p16. The growth of the tumour in nude mice was more accelerated when the inoculation was performed from the CC/XT, increasing progressively over the passages. The third case did not reveal tumour growth in nude mice after the re-inoculation of CC/XT. CONCLUSION: The study of several cores from original tumours and successive tumour passages in TMAs facilitated the analysis of the genetic alteration and protein expression in Ewing's tumours.

Evaluation of genetic stability of the SYT gene rearrangement by break-apart FISH in primary and xenotransplanted synovial sarcomas.

Synovial sarcomas (SS) are infrequent and morphologically heterogeneous soft tissue sarcomas. The t(X;18)(p11.2;q11.2), which results in fusion of the SYT gene at 18q11 with the SSX1, SSX2, or (rarely) SSX4 gene is a primary genetic event in 90% of SS. To determine whether the t(X;18) present in the original tumor is maintained in its passages, a dual-color break-apart FISH assay for SYT gene disruption was performed in two tissue microarrays (TMA) comprising eight molecularly confirmed primary SSs and their xenografts, which were followed for several generations. A simplified scoring system was applied to the FISH results of the primary and xenotransplanted SS to classify the FISH data into distinct groups. SYT disruption was identified in all eight primary SS and in all their passages without any significant differences among them, despite wide variations in xenotransplantation time between the primary tumors and their xenografts. The TMA-based FISH assay demonstrated genetic stability related to SYT gene rearrangement in primary and xenografted SS.

Tissue microarray profiling of primary and xenotransplanted synovial sarcomas demonstrates the immunophenotypic similarities existing between SYT-SSX fusion gene confirmed, biphasic, and monophasic fibrous variants.

This paper discusses the diversity of synovial sarcomas (SSs) [biphasic (BSS), monophasic fibrous (MFSS), and poorly differentiated (PDSS)] and tissue microarray (TMA) evaluation of the immunophenotypic and histological progression of SSs in nude mice using three TMAs comprising 11 primary SSs (8 MFSSs, 2 BSSs, and 1 PDSS) and their xenografts. BSS and MFSS progressively transformed to a similar undifferentiated phenotype with loss of glandular component in the xenografts. Epidermal growth factor receptor and SALL2 were expressed in primary tumors and xenografts. Enhanced bcl-2 and bax expression were noted in xenografts. Ki-67 overexpression in xenografts correlated with high mitotic index. Epithelial membrane antigen (EMA) and cytokeratin AE1/AE3 were detected in all original and xenografted SSs. Hierarchical clustering differentiated original MFSS and BSS, but their xenografts clustered together due to similar immunoexpression profile. Our study demonstrates definite phenotypic variability of BSS and MFSS in the xenografts. Differences in immunoexpression for various markers existed between primary tumor and xenografts but not between subtypes. Hierarchical clustering grouped TMA immunostaining data and confirmed immunophenotypic variability; however, it failed to reveal any immunophenotypic differences between SYT-SSX1 and SYT-SSX2 type tumors. Nonetheless, reverse-transcriptase-polymerase chain reaction detected SYT-SSX transcripts in all primary SSs and their xenografts, thereby demonstrating their genetic stability.

[Analysis of the COL1A1-PDGFB fusion gene in a case of dermatofibrosarcoma protuberans with a fibrosarcoma component]. / Análisis del gen de fusión COL1A1-PDGFB en un caso de dermatofibrosarcoma protuberans con componente de fibrosarcoma.

Dermatofibrosarcoma protuberans (DFSP) is an infrequent tumor of intermediate malignancy, with little tendency to develop metastases but with a high rate of local recurrence. Cytogenetically, DFSP is characterized by a reciprocal translocation, t(17;22)(q22;q13), which is a conditioning factor in the fusion of the collagen type I alpha I gene (COL1A1) in chromosome 17q with the platelet-derived growth factor beta chain gene (PDGFB) in chromosome 22q. The fusion of these genes is variable, involving one of the 51 exons of the COL1A1 gene and exon 2 of the PDGFB gene. We present the case of a 37-year-old woman with a tumor on the arm whose histology showed a neoplastic infiltration of the subcutaneous cellular tissue made up of fusiform cells with an elongated nucleus in a storiform pattern and other more pleomorphic cells in a herringbone pattern, compatible with DFSP with a fibrosarcoma component. The molecular biology study with RT-PCR analysis of paraffin-embedded material and later sequencing showed a new fusion of exon 19 of the COL1A1 gene and exon 2 of PDGFB, supporting a diagnosis of DFSP. A study of the COL1A1-PDGFB fusion products is useful in cases where histology and immunohistochemistry are insufficient for the differential diagnosis of DFSP versus other sarcomas. It also justifies the use of new avenues of treatment with tyrosine kinase inhibitors.

Prognostic value of the International Neuroblastoma Pathology Classification in Neuroblastoma (Schwannian stroma-poor) and comparison with other prognostic factors: a study of 182 cases from the Spanish Neuroblastoma Registry.

In addition to clinical and biological factors, further valuable prognostic information in neuroblastoma (Schwannian stroma-poor) (NB) patients is provided by the histopathologic analysis and the application of the International Neuroblastoma Pathology Classification (INPC) system. The objective of this study was to assess the prognostic impact of the INPC classification in a series of NB (Schwannian stroma-poor) and its relation with other prognostic factors. One hundred eighty-two cases of NB were collected from the files of the Spanish Neuroblastoma Registry. Slides were reviewed, and NB cases were grouped into favorable and unfavorable categories according to INPC criteria, taking into account morphological features (mitosis-karyorrhexis index, histological subtype) and patient's age at diagnosis. Other pathological [presence of calcifications, tissular components, and number of mitotic cells per 10 high-power field (HPF)], immunohistochemical (P-glycoprotein and Ki-67 protein expression) and genetic (MYCN amplification and chromosome 1p deletion) features were also studied. Statistical analyses of overall survival with Kaplan-Meier curves and a multivariate study using Cox regression were performed (40.3% of NBs were considered favorable and 59.7% unfavorable). Unfavorable NB showed a mean survival time of 57 months compared with 89 months in favorable cases. Advanced stage, more than ten mitoses per 10 HPF, Ki-67 expression in more than 30% of tumor cells, MYCN oncogene amplification and chromosome 1p deletion were observed more frequently in unfavorable NB. The Cox regression analysis demonstrated that clinical stage (International Neuroblastoma Staging System stage 4) and histological subtype (undifferentiated NB) were the most important factors that influence the overall survival (p<0.001). INPC classification results are major prognostic indicators in NB and should be considered in the therapeutic stratification of NB patients.

Molecular and immunohistochemical analysis of the prognostic value of cell-cycle regulators in urothelial neoplasms of the bladder.

OBJECTIVE: To evaluate the prognostic and predictive value of molecular and immunohistochemical markers related to cell-cycle control in terms of recurrence, progression, and survival in urothelial neoplasms of the bladder (UNB). PATIENTS AND METHODS: Clinical and pathological findings of 84 patients with UNB were assessed. Homozygous deletion (HD) and promoter methylation of p14ARF, p15INK4B, p16INK4A, loss of heterozygosity of the locus 9p21, p53 mutations, and immunohistochemical expression of p53, p16, p14, p21, p27, pRb, Ki67, MDM2, and cyclin D1 proteins were evaluated in relation to overall survival (OS), recurrence-free survival (RFS), and progression-free survival (PFS). RESULTS: In the univariate analysis, RFS was shorter in cases with p14ARF (p=0.006), p15INK4B (p=0.003), p16INK4A (p=0.03) HD, low p14 immunoreactivity index (IRI) (p=0.01) and high Ki67 IRI (p=0.04); HD of the 9p21 locus genes and p14 IRI remained as independent prognostic factors for early UNB recurrence (p=0.006) whereas tumour stage (p=0.00001) and cyclin D1 IRI (p=0.049) were related to worse PFS in the multivariate analysis. In the univariate analysis, IRI for Ki67 (p=0.002), cyclin D1 (p=0.06), p53 (p=0.00008), p16 (p=0.02), p27 (p=0.0005) MDM2 (p=0.01) and p53 mutations (p=0.03) were related to poor OS, and only the Ki67 IRI retained their independent value in the multivariate analysis. CONCLUSION: 9p21 HD and p14 IRI constitute independent predictive factors for UNB recurrence and cyclin D1 IRI and tumour stage for progression. In addition, Ki67 IRI and tumour stage are independent prognostic factors for overall survival in UNB.