Meta-analysis of whole-genome gene expression datasets assessing the effects of IDH1 and IDH2 mutations in isogenic disease models.

Mutations in isocitrate dehydrogenase 1 (IDH1) and IDH2 are oncogenic drivers to a variable extent in several tumors, including gliomas, acute myeloid leukemia (AML), cholangiocarcinoma, melanoma, and thyroid carcinoma. The pathobiological effects of these mutations vary considerably, impeding the identification of common expression profiles. We performed an expression meta-analysis between IDH-mutant (IDHmut) and IDH-wild-type (IDHwt) conditions in six human and mouse isogenic disease models. The datasets included colon cancer cells, glioma cells, heart tissue, hepatoblasts, and neural stem cells. Among differentially expressed genes (DEGs), serine protease 23 (PRSS23) was upregulated in four datasets, i.e., in human colon carcinoma cells, mouse heart tissue, mouse neural stem cells, and human glioma cells. Carbonic anhydrase 2 (CA2) and prolyl 3-hydroxylase 2 (P3H2) were upregulated in three datasets, and SOX2 overlapping transcript (SOX2-OT) was downregulated in three datasets. The most significantly overrepresented protein class was termed intercellular signal molecules. An additional DEG set contained genes that were both up- and downregulated in different datasets and included oxidases and extracellular matrix structural proteins as the most significantly overrepresented protein classes. In conclusion, this meta-analysis provides a comprehensive overview of the expression effects of IDH mutations shared between different isogenic disease models. The generated dataset includes biomarkers, e.g., PRSS23 that may gain relevance for further research or clinical applications in IDHmut tumors.

Genetic relationship between Hashimoto`s thyroiditis and papillary thyroid carcinoma with coexisting Hashimoto`s thyroiditis.

Hashimoto's thyroiditis (HT) is present in the background of around 30% of papillary thyroid carcinomas (PTCs). The genetic predisposition effect of this autoimmune condition is not thoroughly understood. We analyzed the microarray expression profiles of 13 HT, eight PTCs with (w/) coexisting HT, six PTCs without (w/o) coexisting HT, six micro PTCs (mPTCs), and three normal thyroid (TN) samples. Based on a false discovery rate (FDR)-adjusted p-value ≤ 0.05 and a fold change (FC) > 2, four comparison groups were defined, which were HT vs. TN; PTC w/ HT vs. TN; PTC w/o HT vs. TN; and mPTC vs. TN. A Venn diagram displayed 15 different intersecting and non-intersecting differentially expressed gene (DEG) sets, of which a set of 71 DEGs, shared between the two comparison groups HT vs. TN ∩ PTC w/ HT vs. TN, harbored the relatively largest number of genes related to immune and inflammatory functions; oxidative stress and reactive oxygen species (ROS); DNA damage and DNA repair; cell cycle; and apoptosis. The majority of the 71 DEGs were upregulated and the most upregulated DEGs included a number of immunoglobulin kappa variable genes, and other immune-related genes, e.g., CD86 molecule (CD86), interleukin 2 receptor gamma (IL2RG), and interferon, alpha-inducible protein 6 (IFI6). Upregulated genes preferentially associated with other gene ontologies (GO) were, e.g., STAT1, MMP9, TOP2A, and BRCA2. Biofunctional analysis revealed pathways related to immunogenic functions. Further data analysis focused on the set of non-intersecting 358 DEGs derived from the comparison group of HT vs. TN, and on the set of 950 DEGs from the intersection of all four comparison groups. In conclusion, this study indicates that, besides immune/inflammation-related genes, also genes associated with oxidative stress, ROS, DNA damage, DNA repair, cell cycle, and apoptosis are comparably more deregulated in a data set shared between HT and PTC w/ HT. These findings are compatible with the conception of a genetic sequence where chronic inflammatory response is accompanied by deregulation of genes and biofunctions associated with oncogenic transformation. The generated data set may serve as a source for identifying candidate genes and biomarkers that are practical for clinical application.

Comprehensive molecular biomarker identification in breast cancer brain metastases.

BACKGROUND: Breast cancer brain metastases (BCBM) develop in about 20-30% of breast cancer (BC) patients. BCBM are associated with dismal prognosis not at least due to lack of valuable molecular therapeutic targets. The aim of the study was to identify new molecular biomarkers and targets in BCBM by using complementary state-of-the-art techniques. METHODS: We compared array expression profiles of three BCBM with 16 non-brain metastatic BC and 16 primary brain tumors (prBT) using a false discovery rate (FDR) p < 0.05 and fold change (FC) > 2. Biofunctional analysis was conducted on the differentially expressed probe sets. High-density arrays were employed to detect copy number variations (CNVs) and whole exome sequencing (WES) with paired-end reads of 150 bp was utilized to detect gene mutations in the three BCBM. RESULTS: The top 370 probe sets that were differentially expressed between BCBM and both BC and prBT were in the majority comparably overexpressed in BCBM and included, e.g. the coding genes BCL3, BNIP3, BNIP3P1, BRIP1, CASP14, CDC25A, DMBT1, IDH2, E2F1, MYCN, RAD51, RAD54L, and VDR. A number of small nucleolar RNAs (snoRNAs) were comparably overexpressed in BCBM and included SNORA1, SNORA2A, SNORA9, SNORA10, SNORA22, SNORA24, SNORA30, SNORA37, SNORA38, SNORA52, SNORA71A, SNORA71B, SNORA71C, SNORD13P2, SNORD15A, SNORD34, SNORD35A, SNORD41, SNORD53, and SCARNA22. The top canonical pathway was entitled, role of BRCA1 in DNA damage response. Network analysis revealed key nodes as Akt, ERK1/2, NFkB, and Ras in a predicted activation stage. Downregulated genes in a data set that was shared between BCBM and prBT comprised, e.g. BC cell line invasion markers JUN, MMP3, TFF1, and HAS2. Important cancer genes affected by CNVs included TP53, BRCA1, BRCA2, ERBB2, IDH1, and IDH2. WES detected numerous mutations, some of which affecting BC associated genes as CDH1, HEPACAM, and LOXHD1. CONCLUSIONS: Using complementary molecular genetic techniques, this study identified shared and unshared molecular events in three highly aberrant BCBM emphasizing the challenge to detect new molecular biomarkers and targets with translational implications. Among new findings with the capacity to gain clinical relevance is the detection of overexpressed snoRNAs known to regulate some critical cellular functions as ribosome biogenesis.

Pleomorphism and drug resistant cancer stem cells are characteristic of aggressive primary meningioma cell lines.

BACKGROUND: Meningioma tumors arise in arachnoid membranes, and are the most reported central nervous system (CNS) tumors worldwide. Up to 20% of grade I meningioma tumors reoccur and currently predictive cancer stem cells (CSCs) markers for aggressive and drug resistant meningiomas are scarce. METHODS: Meningioma tissues and primary cell lines were investigated using whole transcriptome microarray analysis, immunofluorescence staining of CSCs markers (including CD133, Sox2, Nestin, and Frizzled 9), and drug treatment with cisplatin or etoposide. RESULTS: Unsupervised hierarchical clustering of six meningioma samples separated tissues into two groups. Analysis identified stem cells related pathways to be differential between the two groups and indicated the de-regulation of the stem cell associated genes Reelin (RELN), Calbindin 1 (CALB1) and Anterior Gradient 2 Homolog (AGR2). Immunofluorescence staining for four tissues confirmed stemness variation in situ. Biological characterization of fifteen meningioma primary cell lines concordantly separated cells into two functionally distinct sub-groups. Pleomorphic cell lines (NG type) grew significantly faster than monomorphic cell lines (G type), had a higher number of cells that express Ki67, and were able to migrate aggressively in vitro. In addition, NG type cell lines had a lower expression of nuclear Caspase-3, and had a significantly higher number of CSCs co-positive for CD133+ Sox2+ or AGR2+ BMI1+. Importantly, these cells were more tolerant to cisplatin and etoposide treatment, showed a lower level of nuclear Caspase-3 in treated cells and harbored drug resistant CSCs. CONCLUSION: Collectively, analyses of tissues and primary cell lines revealed stem cell associated genes as potential targets for aggressive and drug resistant meningiomas.

Microarray expression profiling identifies genes, including cytokines, and biofunctions, as diapedesis, associated with a brain metastasis from a papillary thyroid carcinoma.

Brain metastatic papillary thyroid carcinomas (PTCs) are afflicted with unfavorable prognosis; however, the underlying molecular genetics of these rare metastases are virtually unknown. In this study, we compared whole transcript microarray expression profiles of a BRAF mutant, brain metastasis from a PTC, including its technical replicate (TR), with eight non-brain metastatic PTCs and eight primary brain tumors. The top 95 probe sets (false discovery rate (FDR) p-value < 0.05 and fold change (FC) > 2) that were differentially expressed between the brain metastatic PTC, including the TR, and both, non-brain metastatic PTCs and primary brain tumors were in the vast majority upregulated and comprise, e.g. ROS1, MYBPH, SLC18A3, HP, SAA2-SAA4, CP, CCL20, GFAP, RNU1-120P, DMBT1, XDH, CXCL1, PI3, and NAPSA. Cytokines were represented by 10 members in the top 95 probe sets. Pathway and network analysis (p-value < 0.05 and FC > 2) identified granulocytes adhesion and diapedesis as top canonical pathway. Most significant upstream regulators were lipopolysaccharide, TNF, NKkB (complex), IL1A, and CSF2. Top networks categorized under diseases & functions were entitled migration of cells, cell movement, cell survival, apoptosis, and proliferation of cells. Probe sets that were significantly shared between the brain metastatic PTC, the TR, and primary brain tumors include CASP1, CASP4, C1R, CC2D2B, RNY1P16, WDR72, LRRC2, ZHX2, CITED1, and the noncoding transcript AK128523. Taken together, this study identified a set of candidate genes and biofunctions implicated in, so far nearly uncharacterized, molecular processes of a brain metastasis from a PTC.

Microarray Expression Data Identify DCC as a Candidate Gene for Early Meningioma Progression.

Meningiomas are the most common primary brain tumors bearing in a minority of cases an aggressive phenotype. Although meningiomas are stratified according to their histology and clinical behavior, the underlying molecular genetics predicting aggressiveness are not thoroughly understood. We performed whole transcript expression profiling in 10 grade I and four grade II meningiomas, three of which invaded the brain. Microarray expression analysis identified deleted in colorectal cancer (DCC) as a differentially expressed gene (DEG) enabling us to cluster meningiomas into DCC low expression (3 grade I and 3 grade II tumors), DCC medium expression (2 grade I and 1 grade II tumors), and DCC high expression (5 grade I tumors) groups. Comparison between the DCC low expression and DCC high expression groups resulted in 416 DEGs (p-value<0.05; fold change>2). The most significantly downregulated genes in the DCC low expression group comprised DCC, phosphodiesterase 1C (PDE1C), calmodulin-dependent 70kDa olfactomedin 2 (OLFM2), glutathione S-transferase mu 5 (GSTM5), phosphotyrosine interaction domain containing 1 (PID1), sema domain, transmembrane domain (TM) and cytoplasmic domain, (semaphorin) 6D (SEMA6D), and indolethylamine N-methyltransferase (INMT). The most significantly upregulated genes comprised chromosome 5 open reading frame 63 (C5orf63), homeodomain interacting protein kinase 2 (HIPK2), and basic helix-loop-helix family, member e40 (BHLHE40). Biofunctional analysis identified as predicted top upstream regulators beta-estradiol, TGFB1, Tgf beta complex, LY294002, and dexamethasone and as predicted top regulator effectors NFkB, PIK3R1, and CREBBP. The microarray expression data served also for a comparison between meningiomas from female and male patients and for a comparison between brain invasive and non-invasive meningiomas resulting in a number of significant DEGs and related biofunctions. In conclusion, based on its expression levels, DCC may constitute a valid biomarker to identify those benign meningiomas at risk for progression.

Epidemiological trends of histopathologically WHO classified CNS tumors in developing countries: systematic review.

BACKGROUND: Many developing countries are lagging behind in reporting epidemiological data for individual central nervous system (CNS) tumors. This paper aimed to elicit patterns for the epidemiology of individual World Health Organization (WHO) classified CNS tumors in countries registered by WHO as "developing". MATERIALS AND METHODS: Cyber search was carried out through 66 cancer networks/registries and 181 PubMed published papers that reported counts of CNS tumors for the period of 2009-2012. The relationship between the natural log of incidence Age Standardized Rate (ASR) reported by Globocan and Latitude/ Longitude was investigated. RESULTS: Registries for 21 countries displayed information related to CNS tumors. In contrast tends for classified CNS tumor cases were identified for 38 countries via 181 PubMed publications. Extracted data showed a majority of unclassified reported cases [PubMed (38 countries, 45.7%), registries (21 countries, 96.1%)]. For classified tumors, astrocytic tumors were the most frequently reported type [PubMed (38 countries, 1,245 cases, 15.7%), registries (21 countries, 627 cases, 1.99%]. A significant linear regression relationship emerged between latitudes and reported cases of CNS tumors. CONCLUSIONS: Previously unreported trends of frequencies for individually classified CNS tumors were elucidated and a possible link of CNS tumors occurrence with geographical location emerged.

The innovative safe fixative for histology, histopathology, and immunohistochemistry techniques: "pilot study using shellac alcoholic solution fixative".

The concerns over health and workplace hazards of formalin fixative, joined to its cross-linking of molecular groups that results in suboptimal immunohistochemistry, led us to search for an innovative safe fixative. Shellac is a natural material which is used as a preservative in foods and pharmaceutical industries. This study was undertaken to evaluate the fixation adequacy and staining quality of histopathological specimens fixed in the "shellac alcoholic solution" (SAS), and also to determine the validity of immunohistochemical staining of SAS-fixed material in comparison to those fixed in formalin. Fresh samples from 26 cases from various human tissues were collected at the frozen section room of King Abdulaziz University Hospital, and fixed in SAS fixative or in neutral buffered formaldehyde (NBF) for 12, 18, 24, and 48 h, and processed for paraffin sectioning. Deparaffinized sections were stained with hematoxylin and eosin (H&E) and immunostained for different antigens. The tissues fixed in SAS for >18 h showed best staining quality of H&E comparable to NBF-fixed tissues. Comparison of the immunohistochemical staining of different tissues yielded nearly equivalent readings with good positive nuclear staining quality in both fixatives. These findings support the fixation and preservation adequacy of SAS. Furthermore, it was concluded that the good staining quality obtained with SAS-fixed tissues, which was more or less comparable with the quality obtained with the formalin fixed tissues, supports the validity of this new solution as a good innovative fixative.

Primary intracranial germ cell tumors.

BACKGROUND: Primary intracranial germ cell tumors are rare (ICGCTs) and usually localized in the pineal and suprasellar regions of the brain. They are divided into histologic types: Germinoma, teratoma choriocarcinoma, embryonal carcinoma, yolk sac tumor, and malignant mixed germ cell tumors (MMGCTs). Neuroimaging evaluation is useful to distinguish between the types of ICGCTs. Germinoma is highly sensitive to radiotherapy or/and chemotherapy, and is potentially curable without surgery. MMGCTs are treated with the combination of chemotherapy and radiation, with a poorer prognosis. ICGCTs warrant correct pre-operative diagnosis in order to decide on appropriate management. AIM: To report the clinicopathological and immunohistochemical findings in three cases of primary intracranial germ-cell tumor. MATERIALS AND METHODS: Three cases of intracranial germ cell tumors inclusive of both genders and all brain regions were retrieved from the archives of the Anatomical Pathology Department at King Abdul Aziz University Hospital, Jeddah between the years, 1995-2011, through a computerized search. RESULTS: Based on histological examination, they were respectively diagnosed as MMGCTs (two cases) and germinoma. Clinical, radiological, pathological characteristics and immunohistochemical profile of the three ICGCTs are presented along with the review of recent literature. CONCLUSION: ICGCTs are rare tumors affecting males more than females, and at the end of three years follow-up in the present study, treatment morbidity appears to be low with no recurrence observed among surviving patients, indicating that suprasellar and basal ganglia ICGCTs may have a favorable prognosis regardless of histological type. Pineal MMGCTs may have an aggressive course.

Childhood primary glomerular diseases in the western region of Saudi Arabia.

We report our institute experience on primary glomerular disease in children in the western region of Saudi Arabia over the last 18 years (1988 to 2006). A total of 169 cases were identified as primary glomerular diseases in children and adolescent with age range from first year of life till 18 years. Minimal change disease and focal segmental glomerulosclerosis were the commonly encountered primary glomerular diseases (20.1%and 19.5% respectively), mesangioproliferative glomerulonephritis IgM nephropathy (14.8%), IgA nephropathy (10.7%), postinfectious glomerulonephritis (9.5%), membranous glomerulonephritis (7.1%), membranoproliferative glomerulonephritis (5.9%) and mesangioproliferative glomerulonephritis with negative immunofluorescence (5.9%). The less frequently encountered primary glomerular diseases were congenital nephritic syndrome Finnish type (2.4%), Alport syndrome (2.4%), dense deposit disease (1.2%), and mesangio-proliferative glomerulonephritis with IgG positive (0.6%). We concluded that minimal change disease and focal segmental glomerulosclerosis are the most common primary glomerular disorder en-countered in children in our series and with similar age distribution.

Frequency of benign and preinvasive breast diseases.

OBJECTIVE: The importance of benign proliferative and non-invasive breast lesions as a risk factor preceding the development of invasive mammary carcinoma is well established in the literature. The objective of this study is to estimate the magnitude of benign proliferative diseases as well as mammary intra-epithelial neoplasia in the Western region of the Kingdom of Saudi Arabia (KSA), in order to encourage nationwide breast cancer screening programs for early detection of the high risk proliferative and pre-invasive breast lesions. METHODS: We reviewed histopathology records (reports and slides of selected cases) of 2129 breast cases including mastectomies and breast biopsies from January 1985 to December 2002 in King Abdul-Aziz University Hospital and King Khalid National Guard Hospital, Jeddah, KSA. All the cases and diagnosis are listed and reclassified using systematized nomenclature of medicine (SNOMED) coding system and then regrouped based on the associated risk factors of developing breast carcinoma. RESULTS: Two thousand one hundred and twenty-nine reports were reviewed and 2343 diagnosis were identified as some cases had more than one diagnosis. The total of benign diagnosis were 1504 after exclusion of malignant diagnosis (558), normal breast tissue, gynecomastia, and non-mammary tissue (281). All diagnosis (1504) were reclassified based on anatomical prognostic indicators into non-proliferative (1283/1504), proliferative (140/1504), atypical hyperplasia (AH) (8/1504), and carcinoma in situ (CIS) (73/1504). We compare our findings with the literature and we found that the percentage of benign non-proliferative diagnosis was 85.3% that is higher than the literature 69.7%. Proliferative diseases were 9.3% and atypical hyperplasia was 0.5%, which was lower than the literature 26.2% and 3.6%. On the other hand, CIS diagnosis was 4.9%, which is much higher than the reported literature 1.7%. The study findings could be explained on the basis of higher prevalence of benign breast lesions in our population, or it is related to the number of cases studied, or to the diagnostic criteria followed initially. CONCLUSION: These findings should encourage us to refine our diagnostic criteria of proliferative diseases, AH and CIS (mammary intraepithelial neoplasia [MIN]). In addition, we strongly encourage a breast cancer screening program, nationwide.

Profile of Pap smear cytology in the Western region of Saudi Arabia.

OBJECTIVE: Cervical carcinoma is an important women's health problem in the Western countries. There are only few published data on this disease in the Kingdom of Saudi Arabia (KSA). The aim of this study is to evaluate the abnormal cytological entities detected by cervical Pap smear in Western region of KSA and to discuss the importance of Pap smears screening programs. METHODS: Retrospective review of all the cervical smears present at the King Abdul-Aziz University Hospital, Jeddah, KSA from 1984 to 2000. The reports of 22089 smears were retrieved from the file of the pathology department. RESULTS: There were 368 (1.66%) abnormal Pap smears out of 22089 smears. Out of these 368 abnormal Pap smears there were 62 (16.8%, mean age 37 year) cervical intraepithelial neoplasia grade 1, 45 (12.2%, mean age 38.5) cervical intraepithelial neoplasia grade 2, 27 (7.3%, mean age 40.5) cervical intraepithelial neoplasia grade 3, 22 (6%, mean age 38.5) positive for malignant cells, 36 (9.8%, mean age 40.5) atypical endocervical cells, 88 (23.9%, mean age 39) atypical squamous cells, 9 (2.4%, mean age 40.5) squamous metaplasia with atypia, 26 (7.1%, mean age 45) squamous cell carcinoma, 6 (1.6%, mean age 36.5) reparative atypia, 2 (5.4% mean age 35) herpes virus changes, 19 (5.1%, mean age 37.5) human papilloma virus changes, 5 (1.4%, mean age 55) adenocarcinoma of endometrium, and 7 (1.9%, men age 43.5) adenocarcinoma of endocervix. CONCLUSION: Cervical intraepithelial neoplasia and invasive cervical carcinoma are less common in KSA compared to the Western countries, however, cervical screening programs are necessary nationwide to estimate the actual magnitude of cervical carcinoma and its precursor lesions.

Role of fine needle aspiration in diagnosing breast lesions.

OBJECTIVE: To determine the diagnostic efficacy of breast fine needle aspiration (FNA), using 72 cases that were having both FNA cytology and follow-up histology diagnosis. The study results were compared with results of 27 other studies in the literature. A review of literature regarding the additional benefits of performing "Triple Test" in increasing the cumulative diagnostic accuracy of FNA is also included. METHODS: Our study group consisted of 72 FNA cytologies of female breasts performed at our institution and followed by a histological diagnosis. The following parameters: Sensitivity, specificity, accuracy, predictive values, false positive and negative fraction rates were determined to establish the diagnostic efficacy of the breast FNA. RESULTS: The sensitivity of FNA procedure was 98.4% and specificity 60%, with the predictive value for positive diagnosis 93.9% and for negative diagnosis 85.7%. The overall diagnostic accuracy was 93%. The false positive fraction was 6% and the false negative fraction was 14.2%. The false positive and false negative cases were recorded as having a minimal effect on patient management, as all the false positive and negative diagnosis' were picked up at intra operative frozen sections, and hence no over or under treatment was given to the patients due to these FNA results. CONCLUSION: Fine needle aspiration breast biopsy is an efficient tool and yields a definitive diagnosis, and its use for routine diagnosis must be encouraged since it has high positive (93.9%) and negative (85.7%) predictive values.

Profile of breast pathology at Asir Central hospital - Review of 312 breast cases.

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