EGFR amplification in gliomas is commonly defined by an EGFR/CEP7 ratio of ≥2. In testing performed at a major reference laboratory, a small subset of patients had ≥5 copies of both EGFR and CEP7 yet were not amplified by the EGFR/CEP7 ratio and were designated high polysomy cases. To determine whether these tumors are more closely related to traditionally defined EGFR-amplified or nonamplified gliomas, a retrospective search identified 22 out of 1143 (1.9%) gliomas with an average of ≥5 copies/cell of EGFR and CEP7 with an EGFR/CEP7 ratio of <2 displaying high polysomy. Of these cases, 4 had insufficient clinicopathologic data to include in additional analysis, 15 were glioblastomas, 2 were IDH-mutant astrocytomas, and 1 was a high-grade glial neoplasm, NOS. Next-generation sequencing available on 3 cases demonstrated one with a TERT promoter mutation, TP53 mutations in all cases, and no EGFR mutations or amplifications, which most closely matched the nonamplified cases. The median overall survival times were 42.86, 66.07, and 41.14 weeks for amplified, highly polysomic, and nonamplified, respectively, and were not significantly different (p =  0.3410). High chromosome 7 polysomic gliomas are rare but our data suggest that they may be biologically similar to nonamplified gliomas.

IDH wild-type (wt) grade 2/3 astrocytomas are a heterogenous group of tumors with disparate clinical and molecular profiles. cIMPACT-NOW recommendations incorporated in the new 2021 World Health Organization (WHO) Classification of Central Nervous System (CNS) Tumors urge minimal molecular criteria to identify a subset that has an aggressive clinical course similar to IDH -wt glioblastomas (GBMs). This paper describes the use of a panel of molecular markers to reclassify IDH -wt grade 2/3 diffuse astrocytic gliomas (DAGs) and study median overall survival concerning for to IDH -wt GBMs in the Indian cohort. IDH -wt astrocytic gliomas (grades 2, 3, and 4) confirmed by IDHR132H immunohistochemistry and IDH1/2 gene sequencing, 1p/19q non-codeleted with no H3F3A mutations were included. TERT promoter mutation by Sanger sequencing, epidermal growth factor receptor amplification, and whole chromosome 7 gain and chromosome 10 loss by fluorescence in situ hybridization was assessed and findings correlated with clinical and demographic profiles. The molecular profile of 53 IDH -wt DAGs (grade 2: 31, grade 3: 22) was analyzed. Eleven cases (grade 2: 8, grade 3: 3) (20.75%) were reclassified as IDH -wt GBMs, WHO grade 4 ( TERT promoter mutation in 17%, epidermal growth factor receptor amplification in 5.5%, and whole chromosome 7 gain and chromosome 10 loss in 2%). Molecular GBMs were predominantly frontal (54.5%) with a mean age of 36 years and median overall survival equivalent to IDH -wt GBMs (18 vs. 19 mo; P =0.235). Among grade 2/3 DAGs not harboring these alterations, significantly better survival was observed for grade 2 versus grade 3 DAGs (25 vs. 16 mo; P =0.002). Through the incorporation of a panel of molecular markers, a subset of IDH -wt grade 2 DAGs can be stratified into molecular grade 4 tumors with prognostic and therapeutic implications. However, IDH -wt grade 3 DAGs behave like GBMs irrespective of molecular profile.

Update 3 of the Consortium to Inform Molecular and Practical Approaches to CNS Tumor Taxonomy (cIMPACT-NOW) recognizes amplification of epidermal growth factor receptor (EGFR) as one important aberration in diffuse gliomas (World Health Organization [WHO] grade II/III). While these recommendations endorse testing, a cost-effective, clinically relevant testing paradigm is currently lacking. Here, we use real-world clinical data to propose a financially effective diagnostic test algorithm in the context of new guidelines.

To determine the prevalence, distribution, neuroradiographic features (Visually Accessible REMBRANDT Images [VASARI]), and prognostic relevance of EGFR amplification in lower-grade gliomas, we assembled a consecutive series of diffuse gliomas. For validation we included publicly available data from The Cancer Genome Atlas. For a cost-utility analysis we compared combined EGFR and isocitrate dehydrogenase (IDH) testing, EGFR testing based on IDH results, and no EGFR testing.

In n = 71 WHO grade II/III gliomas, we identified EGFR amplification in 28.2%. With one exception, all EGFR amplifications occurred in IDH-wildtype gliomas. Comparison of overall survival showed that EGFR amplification denotes a significantly more aggressive subset of tumors (P < 0.0001, log-rank). The radiologic phenotype in the EGFR-amplified tumors includes diffusion restriction (15%, P = 0.02), >5% tumor contrast enhancement (75%, P = 0.016), and mild (not avid) enhancement (P = 0.016). The proposed testing algorithm reserves EGFR fluorescence in situ hybridization (FISH) testing for IDH-wildtype cases. Implementation would result in ~37.9% cost reduction at our institution, or about $1.3-4 million nationally.

EGFR-amplified diffuse gliomas are "glioblastoma-like" in their behavior and may represent undersampled glioblastomas, or subsets of IDH-wildtype diffuse gliomas with inherently aggressive biology. EGFR FISH after IDH testing is a financially effective and clinically relevant test algorithm for routine clinical practice.

We have previously demonstrated a significant correlative relationship between PTEN deletion and ERG rearrangement, both in the development of clinically localized prostate cancers and metastases. Herein, we evaluate the cooperative role of ERG and PTEN in oncological outcomes after radical prostatectomy for clinically localized prostate cancer. We evaluated ERG and PTEN status using three previously described cohorts. The first cohort included 235 clinically localized prostate cancer cases represented on tissue microarrays (TMA), evaluated using previously validated FISH assays for ERG and PTEN. The second cohort included 167 cases of clinically localized prostate cancer on TMAs evaluated for PTEN by FISH, and for PTEN and ERG by dual IHC. The third cohort comprised 59 clinically localized prostate cancer cases assessed by array comparative genomic hybridization (aCGH). Kaplan-Meir plots and long rank tests were used to assess the association of ERG and PTEN status with biochemical recurrence after radical prostatectomy for clinically localized prostate cancer. Of the 317 cases eligible for analyses with evaluable ERG and PTEN status, 88 (27.8%) patients developed biochemical recurrence over a median follow-up of 5.7 years. Overall, 45% (142/317) of cases demonstrated ERG rearrangement and 20% (62/317) of cases demonstrated PTEN loss. Hemizygous and homozygous deletion of PTEN was seen in 10% (18/175) and 3% (5/175) of ERG-negative cases, respectively. In contrast, hemizygous and homozygous deletion of PTEN was seen in 11% (15/142) and 17% (24/123) of ERG-positive cases, respectively. PTEN loss (heterozygous or homozygous) was significantly associated with shorter time to biochemical recurrence compared to no PTEN loss (p < 0.001). However, ERG rearrangement versus no rearrangement was not associated with time to PSA recurrence (p = 0.15). Patients who exhibited ERG rearrangement and loss of PTEN had no significant difference in time to recurrence compared to patients with wild-type ERG and loss of PTEN (p = 0.30). Our findings confirm a mutual cooperative role of ERG and PTEN in the pathogenesis of prostate cancer, particularly for homozygous PTEN deletion. ERG did not stratify outcome either alone or in combination with PTEN in this cohort.

Immunohistochemical staining for phosphatase and tensin homolog (PTEN) does not have either an acceptable standard protocol or concordance of scoring between pathologists. Evaluation of PTEN mRNA with a unique and verified sequence probe may offer a realistic alternative providing a robust and reproducible protocol. In this study, we have evaluated an in situ hybridization (ISH) protocol for PTEN mRNA using RNAScope technology and compared it with a standard protocol for PTEN immunohistochemistry (IHC). PTEN mRNA expression by ISH was consistently more sensitive than PTEN IHC, with 56% of samples on a mixed-tumor tissue microarray (TMA) showing high expression by ISH compared with 42% by IHC. On a prostate TMA, 49% of cases showed high expression by ISH compared with 43% by IHC. Variations in PTEN mRNA expression within malignant epithelium were quantifiable using image analysis on the prostate TMAs. Within tumors, clear overexpression of PTEN mRNA on malignant epithelium compared with benign epithelium was frequently observed and quantified. The use of SpotStudio software in the mixed-tumor TMA allowed for clear demonstration of varying levels of PTEN mRNA between tumor samples by the mRNA methodology. This was evident by the quantifiable differences between distinct oropharyngeal tumors (up to 3-fold increase in average number of spots per cell between 2 cases). mRNA detection of PTEN or other biomarkers, for which optimal or standardized immunohistochemical techniques are not available, represents a means by which heterogeneity of expression within focal regions of tumor can be explored with more confidence.

Pediatric glioblastoma (pedGBM) is amongst the most common malignant brain tumors of childhood and carries a dismal prognosis. In contrast to adult GBM, few molecular prognostic markers for the pediatric counterpart have been established. We, therefore, investigated the prognostic significance of genomic and epigenetic alterations through molecular analysis of 202 pedGBM (1-18 years) with comprehensive clinical annotation. Routinely prepared formalin-fixed paraffin-embedded tumor samples were assessed for genome-wide DNA methylation profiles, with known candidate genes screened for alterations via direct sequencing or FISH. Unexpectedly, a subset of histologically diagnosed GBM (n = 40, 20 %) displayed methylation profiles similar to those of either low-grade gliomas or pleomorphic xanthoastrocytomas (PXA). These tumors showed a markedly better prognosis, with molecularly PXA-like tumors frequently harboring BRAF V600E mutations and 9p21 (CDKN2A) homozygous deletion. The remaining 162 tumors with pedGBM molecular signatures comprised four subgroups: H3.3 G34-mutant (15 %), H3.3/H3.1 K27-mutant (43 %), IDH1-mutant (6 %), and H3/IDH wild-type (wt) GBM (36 %). These subgroups were associated with specific cytogenetic aberrations, MGMT methylation patterns and clinical outcomes. Analysis of follow-up data identified a set of biomarkers feasible for use in risk stratification: pedGBM with any oncogene amplification and/or K27M mutation (n = 124) represents a particularly unfavorable group, with 3-year overall survival (OS) of 5 %, whereas tumors without these markers (n = 38) define a more favorable group (3-year OS ~70 %).Combined with the lower grade-like lesions, almost 40 % of pedGBM cases had distinct molecular features associated with a more favorable outcome. This refined prognostication method for pedGBM using a molecular risk algorithm may allow for improved therapeutic choices and better planning of clinical trial stratification for this otherwise devastating disease.

Recent advances in molecular diagnostics have led to better understanding of glioma tumorigenesis and biology. Numerous glioma biomarkers with diagnostic, prognostic, and predictive value have been identified. Although some of these markers are already part of the routine clinical management of glioma patients, data regarding others are limited and difficult to apply routinely. In addition, multiple methods for molecular subclassification have been proposed either together with or as an alternative to the current morphologic classification and grading scheme. This article reviews the literature regarding glioma biomarkers and offers a few practical suggestions.

The prognostic significance of activity biomarkers within the phosphatidylinositol 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) signaling pathway was assessed in two independent cohorts of malignant pleural mesothelioma (MPM) patients uniformly treated with a multimodal approach. We specifically assessed expression signatures in a unique set of pre- and postchemotherapy tumor samples.

Biomarker expression was assessed in samples of two independent cohorts of 107 (cohort 1) and 46 (cohort 2) MPM cases uniformly treated with platinum-based induction chemotherapy followed by extrapleural pneumonectomy from two different institutions, assembled on tissue microarrays. Expression levels of phosphatase and tensin homologue (PTEN), phospho-mTOR, and p-S6 in addition to marker of proliferation (Ki-67) and apoptosis (cleaved caspase-3) were evaluated by immunohistochemistry and correlated with overall survival (OAS) and progression-free survival (PFS). To assess PTEN genomic status, fluorescence in situ hybridization was performed.

Survival analysis showed that high p-S6 and Ki-67 expression in samples of treatment naïve patients of cohort 1 was associated with shorter PFS (p = 0.02 and p = 0.04, respectively). High Ki-67 expression after chemotherapy remained associated with shorter PFS (p = 0.03) and OAS (p = 0.02). Paired comparison of marker expression in samples before and after induction chemotherapy of cohort 1 revealed that decreased cytoplasmic PTEN and increased phospho-mTOR expression was associated with a worse OAS (p = 0.04 and p = 0.03, respectively).

These novel data reveal a prognostic significance of expression changes of PI3K/mTOR pathway components during induction chemotherapy if confirmed in other patient cohorts and support the growing evidence to target the PI3K/mTOR pathway in the treatment of MPM.

Overexpressions of EGFR and HER2 are thought to be prognostic factors of cholangiocarcinoma (CCA). The SOX4 transcription factor is involved in the development and cell fate decision. Although up-regulation of SOX4 has been described in multiple human malignancies, the prognostic value of SOX4 and its relationship to EGFR/HER2 in CCA remain unclear. In the current study, we showed that SOX4 and EGFR were overexpressed in 17 (29.3%), and 13 (22.4%) of the 58 intrahepatic cholangiocarcinomas (IHCCs), as well as 28 (29.8%), and 33 (35.1%) of the 94 extrahepatic cholangiocarcinomas (EHCCs), respectively. Overexpression of HER2 was exclusively identified in EHCCs, with the rate being 4.4% (4/90). In all, amplification of EGFR was identified in 1.8% (1/52) of IHCC cases, and in 2% (3/82) of EHCC cases. By contrast, HER2 amplification was present only in 3.5% (3/94) of the EHCC cases. Notably, Kaplan-Meier survival analysis suggested that SOX4 expression is a significant prognostic factor for poor prognosis in IHCC patients. Importantly, our findings suggested significant association of SOX4 and EGFR expression both in IHCC (P<0.001) and EHCC (P=0.014). SOX4 may modulate expression of EGFR, and SOX4+/EGFR+ defines a subset of CCA patients with poor prognosis. Finally, in vitro data indicated that SOX4 inhibits cellular migratory capacity and promotes epithelial-mesenchymal transition (EMT) process of CCA cells. Collectively, our results define an important role for SOX4 in CCA by orchestrating EMT and modulation on EGFR expression. SOX4 expression may serve as a prognostic marker for patients with IHCC.

Recently, ETS-related gene (ERG) gene rearrangements, phosphatase tensin homologue (PTEN) deletions and EGFR family aberrations were characterized as potential biomarkers for prostate cancer (PCa) patient management. Although ERG gene rearrangement has been identified in approximately 50% of localized prostate cancers in western countries, the prognostic significance of this critical molecular event remains unknown in Chinese patients. Using fluorescence in situ hybridization (FISH) and immunohistochemistry, we evaluated ERG, PTEN and EGFR family aberrations in a cohort of 224 Chinese prostate cancer patients diagnosed in transurethral resection of the prostate (TUR-P). Overall, ERG rearrangement was detected in 23.2% (44/190) cases, of which 54.5% (24/44) showed deletion of the 5'end of ERG. PTEN deletion was identified in 10.8% (19/176) cases. Amplification of EGFR and HER2 genes was present in 1.1% (2/178) and 5.8% (10/173) of cases, respectively. Significant correlation between ERG rearrangement and PTEN deletion was identified in this cohort. EGFR and HER2 aberrations occurred more frequently in PCas without ERG rearrangement than in those with ERG rearrangement, although this did not reach statistical significance. Overall, ERG rearrangement was associated with pre-operative PSA values (P = 0.038) and cancer-related death (P = 0.02), but not with the age, clinical T stage, Gleason score, or Ki-67 labeling index (LI). Notably, multivariate analysis including known prognostic markers revealed ERG rearrangement was an independent prognostic factor (P = 0.022). Additionally, ERG rearrangement status was helpful to identify patients with poor prognosis from PCa group with low Ki-67 LI. In summary, we reported that ERG rearrangement was associated with cancer-related death in Chinese PCa patients. Determination of ERG rearrangement status allows stratification of PCa patients into different survival categories.

There is a lack of standardization of a best practice protocol for Phosphatase and Tensin Homolog (PTEN) assessment by immunohistochemistry in anatomic pathology routine practice. We performed immunohistochemistry for 19 antibodies against PTEN, eleven of which were excluded during the standardization step. Immunohistochemistry of the remaining eight antibodies was performed on a Tissue Microarray containing 55 prostate and 40 renal carcinoma samples. Fluorescent in situ hybridization (FISH) was used as reference standard for immunohistochemistry specificity evaluation. Concerning nuclear staining, polyclonal (Cat#22034-1-AP); 6H2.1 mMAb (Cat#ABM-2052), Y184 RabMAb (Cat#NB110-57441) and 217702 mMAb antibodies presented the highest agreement with fluorescent in situ hybridization (p<0.001 for all) and with regard to cytoplasmic staining, Y184 RabMAb (Cat#NB110-57441); polyclonal (Cat#22034-1-AP) and 217702 mMAb presented the highest agreement (p<0.001 for all). Our results indicate that several commercially available antibodies do not show reliability of sensitivity and specificity for PTEN evaluation and we propose 6H2.1 mMAb (Cat#ABM-2052) as the antibody of choice for laboratory standardization and best practice in clinical routine, which demonstrated excellent sensitivity for both nuclear and cytoplasmic staining, specificity for PTEN by Western blot and good correlation with PTEN status by FISH with regard to nuclear staining.

The SOX4 transcription factor is involved in the development and cell fate decision. Although upregulation of SOX4 has been described in human prostate cancer (PCa), the prognostic value of SOX4 and its exact role in PCa progression remain unclear.

Three tissue microarrays were constructed from 241 Chinese PCa patients who underwent TURP. Immunohistochemistry (IHC) was used to detect the expression of SOX4. Genetic aberrations of epidermal growth factor receptor and HER2 were detected by fluorescence in situ hybridization. The effect of SOX4 on proliferation was evaluated by MTT (methyl thiazolyl tetrazelium), and cell migration and invasion were evaluated by transwell and wound-healing assays. The distribution of cell-cycle phase was analyzed by flow cytometry. Real-time PCR and western blot were used to study transcript and protein levels.

Using tissue microarray, we found that SOX4 was overexpressed in 33.0% (76/230) Chinese PCa patients by IHC. SOX4 overexpression was significantly associated with high Gleason scores (P=0.009) and the presence of distant metastasis (P=0.023). Additionally, SOX4 overexpression was significantly correlated with high Ki67 labeling index (P=0.005) and tended to associate with amplification of HER2 (P=0.052) in our cohort. Notably, SOX4 was correlated with cancer-specific mortality of PCa patients by Kaplan-Meier analysis (P=0.001). Multivariate Cox regression analysis indicated that SOX4 was an unfavorable independent prognostic factor in Chinese PCas (P=0.017). SOX4 overexpression enhanced proliferation of Vcap cells and siRNA knockdown of SOX4 significantly decreased Vcap cell migration and invasion, suggesting a role of SOX4 in cancer metastasis. Additionally, flow cytometry DNA analysis revealed that siRNA SOX4 leads to significant accumulation of cells in the S phase and marked decrease of cells in the G2/M phase. Further in vitro study revealed that SOX4 silencing could inhibit TGF-β (transforming growth factor-β)-induced epithelial-mesenchymal transition (EMT) in Vcap cells. Overexpression of SOX4 could promote the EMT phenotype in Vcap cells.

Our results define an important role for SOX4 in the progression of PCa by orchestrating EMT and may serve as a prognostic marker for PCa patients.

Prostatic adenocarcinoma is an epithelial malignancy characterized by marked histological heterogeneity. It most often has a multifocal distribution within the gland, and different Gleason grades may be present within different foci. Data from our group and others have shown that the genomic deletion of the phosphatase and tensin homolog deleted on chromosome 10 (PTEN) tumor suppressor gene and the disruption of the ETS gene family have a central role in prostate cancer and are likely to be associated with Gleason grade. In this study, prostate cancer samples were systematically analyzed to determine whether there was concordance between PTEN losses and TMPRSS2-ERG fusion rearrangements, within or between foci in multifocal disease, using well-annotated tissue microarrays (TMAs) consisting of 724 cores derived from 142 radical prostatectomy specimens. Three-color fluorescence in situ hybridization analysis of both the PTEN deletion and the TMPRSS2-ERG fusion was used to precisely map genetic heterogeneity, both within and between tumor foci represented on the TMA. PTEN deletion was observed in 56 of 134 (42%) patients (hemizygous=42 and homozygous=14). TMPRSS2-ERG fusion was observed in 63 of 139 (45%) patients. When analyzed by Gleason pattern for a given TMA core, PTEN deletions were significantly associated with Gleason grades 4 or 5 over grade 3 (P<0.001). Although TMPRSS2-ERG fusions showed a strong relationship with PTEN deletions (P=0.007), TMPRSS2-ERG fusions did not show correlation with Gleason grade. The pattern of genetic heterogeneity of PTEN deletion was more diverse than that observed for TMPRSS2-ERG fusions in multifocal disease. However, the marked interfocal discordance for both TMPRSS2-ERG fusions and PTEN deletions was consistent with the concept that multiple foci of prostate cancer arise independently within the same prostate, and that individual tumor foci can have distinct patterns of genetic rearrangements.

Phosphatase and tensin homolog (PTEN) has been established as a tumor suppressor gene with an important role in regulating the phosphatidylinositol-3-kinase/AKT antiapoptotic and survival pathway. The prognostic role of PTEN in non-small-cell lung carcinoma has not been evaluated completely in the context of other molecular information.

Tissue microarrays containing 152 resected non-small-cell lung cancer specimens were used to investigate PTEN and p53 by immunohistochemistry and PTEN by fluorescence in situ hybridization. DNA was isolated and subjected to mutational profiling using the Sequenom Oncocarta v1.0 panel. Clinicopathological features were correlated with PTEN expression, gene copy number, and mutation status.

PTEN staining was absent in 63 (41.4%) of the cases. Significantly more squamous cell carcinomas compared with adenocarcinomas demonstrated loss of (negative) PTEN staining (26 of 44 [59%] versus 32 of 94 [34%]; p = 0.009). PTEN gene copy deletion was present in only seven of 124 evaluable cases (5.6%); all deleted cases were immunohistochemistry negative. In univariate and multivariate (MV) analyses adjusted for sex, age, histology, and stage, loss of PTEN protein expression was associated with significantly shorter disease-free survival (MV hazard ratio: 1.78, 95% confidence interval: 1.01-3.14, p = 0.048), whereas no significant associations were seen with p53 or KRAS and epidermal growth factor receptor (EGFR) mutation status. Importantly, the prognostic value of absent PTEN staining was limited to adenocarcinomas, with MV disease-free survival hazard ratio of 2.68 (95% confidence interval: 1.35-5.32, p = 0.005), whereas no such association was seen in squamous cell carcinomas.

Absence of PTEN protein expression is an independent prognostic marker in early-stage resected lung adenocarcinoma.

Cell cycle regulator genes are major target of mutation in many human malignancies including glioblastomas (GBMs). CDKN2A is one such tumor suppressor gene which encodes p16INK4a protein and serves as an inhibitor of cell cycle progression. Very few studies are available regarding the association of CDKN2A deletion with p16 protein expression in GBMs. There is limited data on the frequency of CDKN2A deletion in different age groups. The aim of the present study was to analyze the frequency of CDKN2A gene deletions in GBM and correlate CDKN2A deletional status with (i) age of the patient (ii) p16 protein expression and (iii) other genetic alterations, namely EGFR amplification and TP53 mutation. A combined retrospective and prospective study was conducted. Sixty seven cases were included. The patients were grouped into pediatric (≤ 18 years), young adults (19-40 years) and older adults (>40 years). CDKN2A and EGFR status were assessed by Fluorescence in situ Hybridization.TP53 mutation was analyzed by PCR based method. p16 expression was assessed using immunohistochemistry. CDKN2A deletion was noted in 40.3% cases of GBM with majority being homozygous deletion (74%). It was commoner in primary GBMs (65.8%) and cases with EGFR amplification (50%). A variable frequency of CDKN2A was observed in older adults (42.3%), young adults (44%), and pediatric patients (31.25%). The difference however was not statistically significant. There was statistically significant association between CDKN2A deletion and p16 immunonegativity with a high negative predictive value of immunohistochemistry.

Despite advances in therapy, gliomas remain associated with poor prognosis. Clinical advances will be achieved through molecularly targeted biological therapies, for which knowledge of molecular genetic and gene expression characteristics in relation to histopathology and in vivo imaging are essential. Recent research supports the molecular classification of gliomas based on genetic alterations or gene expression profiles, and imaging data supports the concept that molecular subtypes of glioma may be distinguished through non-invasive anatomical, physiological and metabolic imaging techniques, suggesting differences in the baseline biology of genetic subtypes of infiltrating glioma. Furthermore, MRI signatures are now being associated with complex gene expression profiles and cellular signalling pathways through genome-wide microarray studies using samples obtained by image guidance which may be co-registered with clinical imaging. In this review we describe the pathobiology, molecular pathogenesis, stem cells and imaging characteristics of gliomas with emphasis on astrocytomas and oligodendroglial neoplasms.

The microscope - the classical tool for the investigation of cells and tissues - remains the basis for the classification of tumors throughout the body. Nowhere has this been more true than in the grading of astrocytomas. In spite of the fact that our parents warned us not to judge a book by its cover, we have continued to assume that adult and pediatric malignant gliomas that look the same, will have the same mutations, and thus respond to the same therapy. Rapid advances in molecular biology have permitted us the opportunity to go inside the cell and characterize the genetic events that underlie the true molecular heterogeneity of adult and pediatric brain tumors. In this paper, we will discuss some of the important clinical differences between pediatric and adult gliomas, with a focus on the molecular analysis of these different age groups.

The median survival time of patients with glioblastoma multiforme (GBM) is 12 months, and only 3-5% of patients survive longer than 3 years. We performed histomorphological and detailed molecular analyses of seven long-term survivors of GBM to identify any prognostic factors that potentially contribute to survival. Morphology and immunohistochemistry for p53, phosphatase and tensin homologue (PTEN) and epidermal growth factor receptor (EGFR) protein expression were investigated. EGFR amplification and 1p/19q deletion were assessed by fluorescent in situ hybridization. The O6-methylguanine-DNA methyltransferase (MGMT) gene methylation status was evaluated by performing methylation-specific polymerase chain reaction assays. All tumors were classical GBMs and no significant oligodendroglial differentiation was noted. The majority showed EGFR amplification (4/7), PTEN protein expression (6/7) and MGMT promoter methylation (5/6). Immunopositivity for p53 was noted in three of seven patients. Deletion of chromosome 1p/19q, either isolated or combined, was not identified in any of the se patients. All patients were treated by gross total resection followed by radiotherapy; six patients received additional temozolomide treatment. A relatively young age of onset (48 years), with a high MGMT promoter methylation and PTEN protein expression were favorable factors for long-term survival. The presence of EGFR amplification indicates that more than a single factor determines survival in GBM.

Fluorescence in situ hybridization (FISH) is a powerful, morphology-based technique to assess targeted copy number alterations or gene rearrangements in formalin-fixed, paraffin-embedded tissues. It has a wide range of applications in routine clinical contexts to identify cytogenetic biomarkers for more accurate diagnosis and prognostic stratification. This review and update addresses practical uses of FISH as a molecular diagnostic tool in the setting of brain tumors, including gliomas, embryonal neoplasms, ependymomas and meningiomas, focusing on key genetic biomarkers, such as 1p19q codeletion, epidermal growth factor receptor (EGFR) gene amplification, BRAF rearrangement and many others. Also discussed are lessons learned over the past decade, including common technical issues to consider when implementing and interpreting FISH results in a clinical setting.

Tumors from a prospective cohort of adult patients with newly diagnosed glioblastoma (n=73), treated uniformly with radiochemotherapy, were examined for 10q23/PTEN deletion by fluorescence in situ hybridization (FISH). Statistical methods were employed to evaluate the degree of association between 10q23/PTEN deletion status and patient age. Survival analysis was performed using Kaplan-Meier log-rank test and multivariable Cox models to assess the prognostic value of 10q23/PTEN deletion. Interestingly, 10q23/PTEN homozygous deletion was frequent in patients >45 years of age (P=0.034) and the median age of patients harboring PTEN homozygous deletions was significantly higher than those with the retained status (P=0.019). 10q23/PTEN homozygous deletion was associated with shorter survival in the entire cohort as well in patients >45 years (P<0.05), indicating that loss of 10q23/PTEN showed clinical importance in elderly patients. Our study highlights the independent prognostic/predictive value of 10q23/PTEN deletion status as identified by FISH, particularly in glioblastoma patients aged >45 years.

Inhibitors of the mammalian target of rapamycin (mTOR) are emerging as promising therapies for metastatic renal cell carcinoma (RCC). Because rational treatment strategies require understanding the activation status of the underlying signaling pathway being targeted at the desired stage of disease, the authors examined the activation status of different components of the mTOR pathway in RCC metastases and matched primary tumors.

The authors immunostained metastatic RCC samples from 132 patients and a subset of 25 matched primary RCCs with antibodies against phosphatidylinositol 3'-kinase, PTEN, phospho-Akt, phospho-mTOR, and p70S6. PTEN genomic status was assessed by fluorescent in situ hybridization. Marker expression was correlated to clinicopathologic variables and to survival.

The mTOR pathway showed widespread activation in RCC metastases of various sites with strong correlation between different components of this signaling cascade (P<.0001), but without significant PTEN genomic deletion. Only cytoplasmic phospho-mTOR showed independent prognostic significance (P = .029) and fidelity between primary RCCs and their matched metastases (P = .004).

Activation of various components of the mTOR signaling pathway in metastatic RCC lesions across various tumor histologies, nuclear grades, and metastatic sites suggests the potential for vertical blockade of multiple steps of this pathway. Patient selection may be improved by mTOR immunostaining of primary RCC.

Glioblastomas (GBM) are rare in children, but reportedly have more varied outcome which suggests differences in tumor etiology compared to typical GBM of adults. To investigate this we performed high resolution array comparative genomic hybridization (aCGH) analysis on three pediatric infratentorial GBM, ages 3.5, 7 and 14 years. Two of these tumors occurred in the brainstem and one in the spinal cord. While histologically typical, one brainstem tumor showed mainly pleomorphic astrocytic cells, whereas the other brainstem and spinal tumors showed a GFAP positive small cell component. Whole chromosomal gains (#1 and #2) and loss (#20) were seen only in the pleomorphic brainstem GBM, which also showed a high level of segmental genomic copy number changes. Segmental loss involving chromosome 8 was seen in all three tumors (Chr8;133039446-136869494, Chr8;pter-3581577, and Chr8;pter-30480019 respectively), whereas loss involving chromosome 16 was seen in only 2 cases with small cell components (Chr16;31827239-qter and Chr16;pter-29754532). Segmental gain of chromosome 7 was shared only between the 2 brainstem cases (Chr7;17187166-qter and Chr7;69824947-qter). Chromosome 17 showed segmental gain of 17q in the backdrop of loss of 17p only in case 1. Segmental gain of chromosome 1q was seen only in case 2. The spinal GBM showed a relatively stable karyotype with a unique loss of Chr19;32848902-qter. None of the frequent losses, gains and amplifications known to occur in adult GBM were identified, suggesting that pediatric infratentorial glioblastomas show a molecular karyotype that was more characteristic of pediatric embryonal tumors than adult GBM.

The link between ERG rearrangement and PTEN (phosphatase and tensin homolog deleted on chromosome 10) deletion is unclear in prostate cancer progression. Using fluorescence in situ hybridization, we systematically validated the frequency and distribution of ERG and PTEN aberrations in a cohort of 73 benign prostate tissues, 59 high-grade prostatic intraepithelial neoplasia (HGPIN) foci, 281 localized prostate cancer and 47 androgen-independent metastatic prostate cancer patients. Overall, ERG rearrangement was present in 15% (5/33) of HGPIN, 45% (121/267) of localized cancers and 35% (15/43) of metastases. By contrast, PTEN deletion was identified in 9% (3/33) of HGPIN, 17% (42/251) of localized cancers and 54% (22/41) of metastases, of which 0%, 40% (17/42) and 45% (10/22) were homozygous, respectively. Concomitance of ERG rearrangement and PTEN deletion was observed in a subset of HGPIN. Significantly, association between PTEN deletion and ERG rearrangement was present both in localized cancers (P=0.0008) and metastases (P=0.02). Further, immunohistochemistry revealed significant correlation of decreased PTEN protein expression with PTEN genomic deletion both in localized and metastatic cancer. Of note, ERG aberration, but not PTEN deletion, was consistently identical both in localized cancer and adjacent HGPIN. Similarly, whereas all metastases (41/41, 100%) shared the same ERG status across multiple sites from the same patient, 5% (2/41) of cases showed discordance for PTEN deletion status across multiple sites. Collectively, our data support PTEN deletion as a late genetic event in human prostate cancer, presumably a 'second hit' after ERG rearrangement. PTEN deletion and ERG rearrangement may cooperate, but contribute at different stages, in prostate cancer progression.

To estimate the maximum-tolerated dose (MTD) of erlotinib administered during and after radiotherapy, and to describe the pharmacokinetics of erlotinib and its metabolite OSI-420 in patients between 3 and 25 years with newly diagnosed high-grade glioma who did not require enzyme-inducing anticonvulsants.

Five dosage levels (70, 90, 120, 160, and 200 mg/m(2) per day) were planned in this phase I study. Dose-limiting toxicities (DLT) were evaluated during first 8 weeks of therapy. Local radiotherapy (dose between 54 and 59.4 Gy) and erlotinib started preferentially on the same day. Erlotinib was administered once daily for a maximum of 3 years. Pharmacokinetic studies were obtained after first dose and on day 8 of therapy. Mutational analysis of EGFR kinase domain, PIK3CA, and PTEN was done in tumor tissue.

Median age at diagnosis of 23 patients was 10.7 years (range, 3.7-22.5 years). MTD of erlotinib was 120 mg/m(2) per day. Skin rash and diarrhea were generally well controlled with supportive care. Dose-limiting toxicities were diarrhea (n = 1), increase in serum lipase (n = 1), and rash with pruritus (n = 1). The pharmacokinetic variables of erlotinib and OSI-420 in children were similar to those described in adults. However, there was no relationship between erlotinib dosage and drug exposure. No EGFR kinase domain mutations were observed. Two patients with glioblastoma harbored mutations in PIK3CA (n = 1) or PTEN (n = 1).

Although the MTD of erlotinib in children with newly diagnosed high-grade glioma was 120 mg/m(2) per day, pharmacokinetic studies showed wide interpatient variability in drug exposure.

TMPRSS2:ERG gene fusions and PTEN deletions are the most common genomic aberrations in prostate cancer. Recent work has suggested that the TMPRSS2:ERG fusion is associated with a more aggressive phenotype. Similarly, PTEN deletion has been associated with biochemical recurrence and lymph node metastasis. To date, there has been no systematic analysis of the combined influence of genomic PTEN deletion with TMPRSS2:ERG gene fusions on clinical parameters of prostate cancer progression. We carried out a retrospective analysis of 125 prostate cancers with known clinical outcome using interphase fluorescence in situ hybridization to detect the relative prevalence of TMPRSS2:ERG rearrangements and/or PTEN genomic deletions. TMPRSS2:ERG rearrangement was found in 60 of 125 (48%) prostate cancers. Duplication of TMPRSS2:ERG fusion was observed in seven (6%) tumors. Gleason grade (P=0.0002)/score (P=0.001), median tumor volume (P=0.0024), preoperative PSA (P=0.001) and perineural invasion (P=0.0304) were significantly associated with biochemical recurrence by univariate analysis with TMPRSS2:ERG approaching significance (P=0.0523). By multivariate analysis, relevant factors associated with recurrence were Gleason scores 7 (P=0.001) and 8-10 (P=0.015), PTEN homozygous deletion (P=0.013) and concurrent TMPRSS2:ERG fusion and PTEN deletion (P=0.036). Kaplan-Meier analysis indicated that the presence of TMPRSS2:ERG fusion was marginally less favorable in comparison to no fusion. Duplication of fusion gene showed worse prognosis. It was possible to determine the relative frequencies of PTEN deletion and/or TMPRSS2:ERG fusions in 82 of 125 prostate cancers. With biochemical recurrence as an endpoint, the genomic biomarkers identified three patient groups: (1) 'poor genomic grade' characterized by both PTEN deletion and TMPRSS2:ERG fusions (23/82, 28%); (2) 'intermediate genomic grade' with either PTEN deletion or TMPRSS2:ERG fusion (35/82, 43%) and (3) 'favorable genomic grade' in which neither rearrangement was present (24/82, 29%). Kaplan-Meier and multivariate analysis indicate that TMPRSS2:ERG fusion and PTEN loss together are a predictor of earlier biochemical recurrence of disease.

An intratumoral or peritumoral microbial intracranial abscess is an infrequent diagnosis. The development of this complication may not be preceded by apparent local or general infection in all cases. To identify this diagnosis by radiological (MRI) or laboratory investigations is very intricate. Nevertheless, the recommended life-saving strategy is early surgery with resolution of both the tumor and infection. If subsequent oncological treatment is required, it has to be adjusted for prevention of re-inflammation. The described patient suffered from an intracranial abscess superimposed on a Glioblastoma Multiforme. The confirmed etiological agent was Staphylococcus aureus. The suspected route of microbial migration and colonization in this tumor was bacteremia via agents from thrombophlebitis. The patient is in a good condition following surgery, antimicrobial treatment, and radiotherapy.

Glioblastoma multiforme (GBM) occurs rarely in children. Relatively few studies have been performed on molecular properties of pediatric GBMs. Our objective in this study was to evaluate the genetic alterations in pediatric GBM (age < or = 18 years) with special reference to p53, p16, and p27 protein expression, alterations of the epidermal growth factor receptor (EGFR), and deletion of the phosphate and tensin homolog gene (PTEN). Thirty cases of childhood GBMs reported between January 2002 and June 2007 were selected, and slides stained with hematoxylin and eosin were reviewed. Immunohistochemical staining was performed for EGFR, p53, p16, and p27, and tumor proliferation was assessed by calculating the MIB-1 labeling index (LI). Fluorescence in situ hybridization analysis was performed to evaluate for EGFR amplification and PTEN deletion. Histopathological features and MIB-1 LI were similar to adult GBMs. p53 protein expression was observed in 63%. Although EGFR protein overexpression was noted in 23% of cases, corresponding amplification of the EGFR gene was rare (5.5%). Deletion of the PTEN gene was also equally rare (5.5%). One case showed polysomy (chromosomal gains) of chromosomes 7 and 10. Loss of p16 and p27 immunoexpression was observed in 68% and 54% of cases, respectively. In pediatric de novo/primary GBMs, deletion of PTEN and EGFR amplification are rare, while p53 alterations are more frequent compared to primary adult GBMs. Frequency of loss of p16 and p27 immunoexpression is similar to their adult counterparts. This suggests that pediatric malignant gliomas are distinctly different from adult GBMs, highlighting the need for identification of molecular targets that may be adopted for future novel therapeutic strategies.

The human RNA polymerase II-associated factor (hPAF) complex is comprised of five subunits that include hPaf1, parafibromin, hLeo1, hCtr9 and hSki8. This multifaceted complex was first identified in yeast (yPAF) and subsequently in Drosophila and humans. Recent advances in the study on hPAF have revealed various functions of the complex in humans that are similar to yPAF, including efficient transcription elongation, mRNA quality control and cell cycle regulation. A major component of the hPAF complex, hPaf1, is amplified and overexpressed in pancreatic cancer. The parafibromin subunit of the hPAF complex is a product of the hereditary hyperparathyroidism type 2 (HRPT-2) tumor-suppressor gene, which is mutated in the germ line of hyperparathyroidism-jaw tumor patients. This review evaluates the role of the hPAF complex and its individual subunits in endocrine and pancreatic cancers. It focuses on the functions of the hPAF complex and its individual subunits and dysregulation of the complex, thus providing an insight into its potential involvement in the development of endocrine cancers and other tumor types.

Increased MYCN gene copy number is a characteristic property of neurogenic tumors. Fluorescence in situ hybridization (FISH) and array-based comparative genomic hybridization (array-CGH) are traditionally used to determine MYCN amplification for tumor stratification. A unique ability of real-time quantitative polymerase chain reaction (qPCR) to determine gene copy number, even within a small percent of observed tumor cells, and can be more appropriate. MYCN genomic copy number from 44 human brain tumors (22 medulloblastomas and 22 neurocytomas) was determined by means of FISH, array-CGH, and qPCR. By qPCR, with the original set of oligonucleotides, 17 out of 44 (38.6%) tumors were found to contain a 1.3- to 2.9-fold increase of MYCN defined as low-level gain. An absolute qPCR method was used to get high accuracy of results. Strong correlation was observed between the three methods: for medulloblastomas, r=1 (P<0.01) between FISH and array-CGH and r=0.92 (P<0.01) between qPCR and FISH/array-CGH. For neurocytomas, r=0.9 (P<0.01) between FISH and array-CGH and r=0.34/0.43 (P<0.01) between qPCR and FISH/array-CGH. Absolute qPCR assays possess high precision compared to other conventional methods and can be used for accurate and quickness detection of MYCN status (low-level gene gain and amplification).

Pathology archives provide unique and abundant opportunities to investigate human carcinogenesis and identify potential targets for cancer therapy. Microwaving was introduced into various procedures used in histopathology two decades ago, although the precise mechanisms underlying its effectiveness in any of the procedures, including antigen retrieval, acceleration of fixation and nucleic acid hybridization, are not known. Since microwaving was first applied to fluorescence in situ hybridization (FISH), many pathologists and researchers have enjoyed the benefits of excellent preservation of histological structures as well as good retrieval of FISH signals by this method. Microwave-assisted fluorescence in situ hybridization (MW-FISH) has proved to be especially useful in retrospective investigations of tissues fixed and preserved for long periods of time, and the success rates in the randomly selected pathology archives have been greater (70-95%) than by the conventional protocol ( Collapse

Patients with glioblastoma (astrocytoma, World Health Organization grade IV) exhibit 2-year survival rates of less than 20% despite significant advances in therapeutic options available to patients. Epidermal growth factor receptor (EGFR) hyperexpression is one of the most commonly encountered abnormalities in this tumor. However, EGFR expression, amplification, and mutations are poorly predictive of patient survival. Investigators have taken to exploiting the sensitivities of activated downstream targets in the EGFR second messenger pathways to certain inhibitory drugs to downregulate their neoplastic messages promoting cell growth and inhibiting cell death.

It is important to both gain some understanding of the functional significance of these pathways and to understand the role the pathologist might play in characterizing the activation status of certain downstream messenger proteins that are targeted in these brain tumor therapies. We have reviewed the literature regarding histologic assays that have been incorporated into trials of these new drugs and report on the methods used to study these proteins and the conclusions of these studies.

Literature review and primary material from Duke University (Durham, NC) Department of Pathology archives.

To date, drug trial reports indicate that identification of the presence of the EGFR variant, EGFRvIII, and measurement of the activated downstream targets, phospho-Akt, phospho-S6, and phospho-MAPK, may be useful in predicting sensitivity to some of the EGFR kinase inhibitors. No studies to date have identified prognostic significance related to immunoreactivity status among any of these markers that is independent of histologic grade.

Genetic instabilities are believed to be one of the major causes of developing a cancer phenotype in humans. During the progression of cancer, aberrant expression of proteins, either owing to genetic (amplification, mutation and deletion) or epigenetic modifications (DNA methylation and histone deacetylation), contributes in different ways to the development of cancer. By differential screening analysis, an amplification of the 19q13 locus containing a novel pancreatic differentiation 2 (PD2) gene was identified. PD2 is the human homolog of the yeast RNA polymerase II-associated factor 1 (yPaf1) and is part of the human RNA polymerase II-associated factor (hPAF) complex. hPAF is comprised of five subunits that include PD2/hPaf1, parafibromin, hLeo1, hCtr9 and hSki8. This multifaceted complex was first identified in yeast (yPAF) and subsequently in Drosophila and human. Recent advances in the study on PAF have revealed various functions of the complex in human, which are similar to yPAF, including efficient transcription elongation, mRNA quality control and cell-cycle regulation. Although the precise function of this complex in cancer is not clearly known, some of its subunits have been linked to a malignant phenotype. Its core subunit, PD2/hPaf1, is amplified and overexpressed in many cancers. Further, an overexpression of PD2/hPaf1 results in the induction of a transformed phenotype, suggesting its possible involvement in tumorigenesis. The parafibromin subunit of the hPAF complex is a product of the HRPT-2 (hereditary hyperparathyroidism type 2) tumor suppressor gene, which is mutated in the germ line of hyperparathyroidism-jaw tumor patients. This review focuses on the functions of the PAF complex and its individual subunits, the interaction of the subunits with each other and/or with other molecules, and dysregulation of the complex, providing an insight into its potential involvement in the development of cancer.

To analyze the clinical and molecular characteristics of malignant transformation (MT) of low-grade glioma (LGG) in children.

The clinical, radiologic, and histologic characteristics of children treated at our institution who experienced MT of LGG were reviewed. Molecular alterations in these tumors were analyzed by fluorescent in situ hybridization, immunohistochemistry, and TP53 sequencing. Cumulative incidence estimate and risk factors for MT were determined for 65 patients with grade 2 astrocytoma treated at our institution during the study interval.

Eleven patients who experienced MT were identified (median age at diagnosis of LGG, 13.3 years). Initial diagnoses were grade 2 astrocytoma (n = 6) and other grade 1/2 gliomas (n = 5). The median latency of MT was 5.1 years. Histologic diagnoses after MT were glioblastoma (n = 7) and other high-grade gliomas (n = 4). The 15-year cumulative incidence estimate of MT among 65 patients with grade 2 astrocytoma was 6.7% +/- 3.9%; no risk factor analyzed, including radiotherapy, was associated with MT. Tissue was available for molecular analysis in all patients, including nine with samples obtained before and after MT. TP53 overexpression was more common after MT. Deletions of RB1 and/or CDKN2A were observed in 71% of LGGs and in 90% of tumors after MT. PTEN pathway abnormalities occurred in 76% of patients. One of five oncogenes analyzed (PDGFRA) was amplified in one patient.

The molecular abnormalities that occur during MT of LGG in children are similar to those observed in primary and secondary glioblastoma in adults.

Most adult glioblastoma multiformes (GBMs) present in patients 45-70 years old; tumors occurring at the extremes of the adult age spectrum are uncommon, and seldom studied. We hypothesized that young-adult GBMs would differ from elderly-adult and from pediatric GBMs. Cases were identified from years 1997 to 2005. Demographic and histological features, MIB-1 and TP53 immunohistochemical findings and epidermal growth factor receptor (EGFR) amplification status by fluorescence in situ hybridization were compiled and correlated with survival. Twenty-eight (74%) of our 38 young-adult GBM patients had primary de novo tumors, two of which occurred in patients with cancer syndromes. Two additional GBMs were radiation-induced and eight (21%) were secondary GBMs. Seven patients were identified as long-term (>3 years) survivors. Six of 38 cases manifested unusual morphological features, including three epithelioid GBMs, one rhabdoid GBM, one gliosarcoma and one small cell GBM containing abundant, refractile, eosinophilic inclusions. MIB-1 index emerged as the most important prognosticator of survival (P < 0.005). Although there was a trend between extent of necrosis, TP53 immunohistochemical expression, and EGFR amplification status and survival, none reached statistical significance. GBMs in young adults are a more inhomogeneous tumor group than GBMs occurring in older adult patients and show features that overlap with both pediatric and adult GBMs.

Prostate cancer (CaP) is characterized by the accumulation of both genetic and epigenetic alterations that transform premalignant lesions to invasive carcinoma. However, the molecular events underlying this critical transition are poorly understood. One of the important genes that might play a role in CaP development is the PTEN gene. At the present time, there has been no systematic analysis of the incidence of genomic PTEN deletion by fluorescence in situ hybridization (FISH) in CaP and associated preneoplastic histologic lesions. This study assesses the frequency of PTEN deletion by interphase FISH analysis in CaP and prostatic intra-epithelial neoplasia (PIN). Dual-color FISH was performed using DNA probes for bands 10q23.3 (PTEN locus) and chromosome 10 centromere using 35 radical prostatectomy specimens. PTEN deletions were not found in 3/3 of stroma, 6/6 samples of benign glandular epithelium, and 12/12 samples of low-grade PIN. However, PTEN deletions were found in 3/13 (23%) of high-grade PIN and 24/35 (68%) of CaP. Concordance was observed between PTEN deletion status and the overall cellular PTEN protein expression levels, as assessed by immunohistochemistry. The high frequency of PTEN deletion observed in CaP versus precursor lesions implicates a pivotal role for PTEN haploinsufficiency in the transition from preneoplastic PIN to CaP. Moreover, this observation is an important consideration for novel therapeutic trials in CaP in which biologic efficacy is influenced by the activity level of PTEN. These findings draw attention to the usefulness of this relatively simple FISH assay for future applications in clinical laboratories.