Samuel Navarro

Quality Assessment of Genetic Markers Used for Therapy Stratification

PURPOSE Therapy stratification based on genetic markers is becoming increasingly important, which makes commitment to the highest possible reliability of the involved markers mandatory. In neuroblastic tumors, amplification of the MYCN gene is an unequivocal marker that indicates aggressive tumor behavior and is consequently used for therapy stratification. To guarantee reliable and standardized quality of genetic features, a quality-assessment study was initiated by the European Neuroblastoma Quality Assessment (ENQUA; connected to International Society of Pediatric Oncology) Group. MATERIALS AND METHODS One hundred thirty-seven coded specimens from 17 tumors were analyzed in 11 European national/regional reference laboratories using molecular techniques, in situ hybridization, and flow and image cytometry. Tumor samples with divergent results were re-evaluated. RESULTS Three hundred fifty-two investigations were performed, which resulted in 23 divergent findings, 17 of which were judged as errors after re-evaluation. MYCN analyses determined by Southern blot and in situ hybridization led to 3.7% and 4% of errors, respectively. Tumor cell content was not indicated in 32% of the samples, and 11% of seemingly correct MYCN results were based on the investigation of normal cells (eg, Schwann cells). Thirty-eight investigations were considered nonassessable. CONCLUSION This study demonstrated the importance of revealing the difficulties and limitations for each technique and problems in interpreting results, which are crucial for therapeutic decisions. Moreover, it led to the formulation of guidelines that are applicable to all kinds of tumors and that contain the standardization of techniques, including the exact determination of the tumor cell content. Finally, the group has developed a common terminology for molecular-genetic results.

HIF-2 alpha maintains an undifferentiated state in neural crest-like human neuroblastoma tumor-initiating cells

High hypoxia-inducible factor-2α (HIF-2α) protein levels predict poor outcome in neuroblastoma, and hypoxia dedifferentiates cultured neuroblastoma cells toward a neural crest-like phenotype. Here, we identify HIF-2α as a marker of normoxic neural crest-like neuroblastoma tumor-initiating/stem cells (TICs) isolated from patient bone marrows. Knockdown of HIF-2α reduced VEGF expression and induced partial sympathetic neuronal differentiation when these TICs were grown in vitro under stem cell-promoting conditions. Xenograft tumors of HIF-2α-silenced cells were widely necrotic, poorly vascularized, and resembled the bulk of tumor cells in clinical neuroblastomas by expressing additional sympathetic neuronal markers, whereas control tumors were immature, well-vascularized, and stroma-rich. Thus, HIF-2α maintains an undifferentiated state of neuroblastoma TICs. Because low differentiation is associated with poor outcome and angiogenesis is crucial for tumor growth, HIF-2α is an attractive target for neuroblastoma therapy.

Histological heterogeneity of Ewing’s sarcoma/PNET: an immunohistochemical analysis of 415 genetically confirmed cases with clinical support

Ewing’s sarcoma (ES)/peripheral neuroectodermal tumor (PNET) are malignant neoplasms affecting children and young adults. We performed a study to typify the histological diversity and evaluate antibodies that may offer diagnostic/prognostic support. In total, 415 cases of genetically confirmed paraffin-embedded ES/PNET were analyzed on whole sections and in tissue microarrays. This study confirms the structural heterogeneity of ES/PNET, distinguishing three major subtypes: conventional ES (280 cases); PNET (53 cases); and atypical ES/PNET (80), including large cells, vascular-like patterns, spindle pattern, and adamantinoma-like configuration. All cases presented positivity for at least three of the four tested antibodies (CD99, FLI1, HNK1, and CAV1). CAV1 appeared as a diagnostic immunomarker of ES/PNET being positive in CD99-negative cases. Hence, the immunohistochemical analysis confirmed the diagnostic value of all four antibodies, which together cover more than 99% of the tumors, independently of the histological variety. The univariate analysis for survival revealed atypical ES as the only histological parameter apparently associated with less favorable clinical outcome, particularly in the subgroup of patients treated with surgery. In conclusion, the diagnosis of atypical ES is a challenge for the pathologist and needs support from molecular techniques to perform an optimal differential diagnosis with other small round cell tumors.

Preoperative chemoradiation may not always be needed for patients with T3 and T2N+ rectal cancer.

Preoperative chemoradiation is becoming the standard treatment for patients with locally advanced rectal cancer. However, since the introduction of total mesorectal excision (TME), local recurrence rates have been reduced significantly, and some patients can be spared from potentially toxic over treatment. The current study was designed to assess the factors that predict recurrence in an institutional series of patients with rectal cancer who had clinical T2 lymph node‐positive (cT2N+) tumors or cT3N0/N+ tumors and underwent radical surgery without receiving preoperative chemoradiation.

Characterization of a Novel Type of Serine/Threonine Kinase That Specifically Phosphorylates the Human Goodpasture Antigen

Goodpasture disease is an autoimmune disorder that occurs naturally only in humans. Also exclusive to humans is the phosphorylation process that targets the unique N-terminal region of the Goodpasture antigen. Here we report the molecular cloning of GPBP (Goodpasture antigen-bindingprotein), a previously unknown 624-residue polypeptide. Although the predicted sequence does not meet the conventional structural requirements for a protein kinase, its recombinant counterpart specifically binds to and phosphorylates the exclusive N-terminal region of the human Goodpasture antigen in vitro. This novel kinase is widely expressed in human tissues but shows preferential expression in the histological structures that are targets of common autoimmune responses. The work presented in this report highlights a novel gene to be explored in human autoimmunity.

Immunohistochemical detection of EWS and FLI-1 proteinss in Ewing sarcoma and primitive neuroectodermal tumors: comparative analysis with CD99 (MIC-2) expression.

The molecular analysis of the t(11;22) rearrangement involving EWS/ FLI-1 genes is likely to be of diagnostic value in Ewing sarcoma (ES) and primitive neuroectodermal tumors (PNET). The objective of the current study was to analyze the immunohistochemical expression of the EWS and FLI-1 proteins in a group of small round-cell tumors (SRCT) to determine their specificity and relevance in their differential diagnosis. Forty-eight cases—10 conventional ES, 4 large-cell ES, 5 PNET, 9 neuroblastomas (NB), 6 undifferentiated synovial sarcomas (SS), 5 rhabdomyosarcomas (RB), 5 non-Hodgkin lymphomas (NHL), 1 round-cell liposarcoma, and 3 mesenchymal chondrosarcomas—were analyzed. Immunocytochemistry was performed on paraffin sections after the LSAB method and antigen retrieval using ethylenediaminetetraacetic acid buffer (pH 6). Primary antibodies included FLI-1 (C-19), EWS (N-18), EWS (C-19), and CD99 (MIC-2). As expected, CD-99 expression was found in 100% of ES/PNET cases, in 2 cases of RB, 2 SS, and 1 NHL. FLI-1 protein was observed as nuclear staining in 16 cases of ES/PNET (84%) and in 4 cases of NHL, 2 NB, and 3 SS. Normal endothelial cells and lymphocytes also were positive. EWS expression (both proteins N-18 and C-19) was detected not only in 95% of ES/PNET cases but also in more than 50% of cases from the other tumoral types (4 of 9 and 7 of 9 NB, 5 of 6 and 6 of 6 SS, 3 of 5 and 5 of 5 RB, and 2 of 5 and 3 of 5 NHL, respectively). Whereas EWS expression does not appear specific for ES/PNET, analysis of FLI-1 expression together with CD-99 is a powerful marker for ES/PNET and important factors in the differential diagnosis of SRCT.

HIF-1{alpha} and HIF-2{alpha} Are Differentially Regulated In vivo in Neuroblastoma: High HIF-1{alpha} Correlates Negatively to Advanced Clinical Stage and Tumor Vascularization.

Purpose: Hypoxia is considered to be a major driving force behind tumor angiogenesis. The stabilization and activation at hypoxia of the hypoxia-inducible factors HIF-1α and HIF-2α and the concomitant induction of expression of vascular endothelial growth factor (VEGF) and other proangiogenic factors provide a molecular frame for hypoxia-driven tumor angiogenesis. This study has investigated how HIF and VEGF protein levels relate to each other with regard to vascularization, tumor stage, and overall survival in neuroblastoma. Experimental Design: Tissue cores taken from tumor specimens representing 93 children with neuroblastoma were arranged on a microarray and stained for HIF-1α, HIF-2α, VEGF, and CD31 proteins. Both fraction of positive cells and staining intensity were evaluated and protein levels were correlated with each other and with clinical variables. Results: Although high levels of both HIF-1α (P < 0.001) and HIF-2α (P < 0.001) correlated positively to VEGF expression, they did not fully correlate with each other. Moreover, HIF-1α (P = 0.002) and VEGF (P < 0.001), but not HIF-2α, correlated negatively to vascularization as determined by CD31 staining abundance. VEGF expression or degree of vascularization did not correlate with tumor stage or overall survival. High HIF-1α levels correlated with low tumor stage (P < 0.001) and were associated with a favorable patient prognosis (P = 0.08). Conclusions: The discordant results on expression of HIF-1α and HIF-2α suggest that these two proteins are differentially regulated in vivo, thus reflecting distinctive protein expression/stabilization mechanisms. The association between HIF-1α and favorable outcome stresses the importance of discriminating HIF-2α from HIF-1α expression and has implications for using HIFs as treatment targets. (Clin Cancer Res 2009;15(23):7130–6)

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.

Macroscopic Assessment of Mesorectal Excision in Rectal Cancer: A Useful Tool for Improving Quality Control in a Multidisciplinary Team

High quality of surgical technique and the use of descriptive measures to assess and report surgical proficiency have been shown to influence locoregional tumor control in patients with rectal cancer. In this study, the authors have aimed to audit the implementation of a macroscopic assessment of mesorectal excision (MAME) and to investigate factors that influenced surgical quality and disease recurrence.

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 Ewings 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.