Stephen Yip

ARID1A Mutations in Endometriosis-Associated Ovarian Carcinomas

BACKGROUND Ovarian clear-cell and endometrioid carcinomas may arise from endometriosis, but the molecular events involved in this transformation have not been described. METHODS We sequenced the whole transcriptomes of 18 ovarian clear-cell carcinomas and 1 ovarian clear-cell carcinoma cell line and found somatic mutations in ARID1A (the AT-rich interactive domain 1A [SWI-like] gene) in 6 of the samples. ARID1A encodes BAF250a, a key component of the SWI–SNF chromatin remodeling complex. We sequenced ARID1A in an additional 210 ovarian carcinomas and a second ovarian clear-cell carcinoma cell line and measured BAF250a expression by means of immunohistochemical analysis in an additional 455 ovarian carcinomas. RESULTS ARID1A mutations were seen in 55 of 119 ovarian clear-cell carcinomas (46%), 10 of 33 endometrioid carcinomas (30%), and none of the 76 high-grade serous ovarian carcinomas. Seventeen carcinomas had two somatic mutations each. Loss of the BAF250a protein correlated strongly with the ovarian clear-cell carcinoma and endometrioid carcinoma subtypes and the presence of ARID1A mutations. In two patients, ARID1A mutations and loss of BAF250a expression were evident in the tumor and contiguous atypical endometriosis but not in distant endometriotic lesions. CONCLUSIONS These data implicate ARID1A as a tumor-suppressor gene frequently disrupted in ovarian clear-cell and endometrioid carcinomas. Since ARID1A mutation and loss of BAF250a can be seen in the preneoplastic lesions, we speculate that this is an early event in the transformation of endometriosis into cancer. (Funded by the British Columbia Cancer Foundation and the Vancouver General Hospital–University of British Columbia Hospital Foundation.).

Recurrent activating ACVR1 mutations in diffuse intrinsic pontine glioma.

Diffuse intrinsic pontine gliomas (DIPGs) are highly infiltrative malignant glial neoplasms of the ventral pons that, due to their location within the brain, are unsuitable for surgical resection and consequently have a universally dismal clinical outcome. The median survival time is 9–12 months, with neither chemotherapeutic nor targeted agents showing substantial survival benefit in clinical trials in children with these tumors. We report the identification of recurrent activating mutations in the ACVR1 gene, which encodes a type I activin receptor serine/threonine kinase, in 21% of DIPG samples. Strikingly, these somatic mutations (encoding p.Arg206His, p.Arg258Gly, p.Gly328Glu, p.Gly328Val, p.Gly328Trp and p.Gly356Asp substitutions) have not been reported previously in cancer but are identical to mutations found in the germ line of individuals with the congenital childhood developmental disorder fibrodysplasia ossificans progressiva (FOP) and have been shown to constitutively activate the BMP–TGF-β signaling pathway. These mutations represent new targets for therapeutic intervention in this otherwise incurable disease.

MSH6 Mutations Arise in Glioblastomas during Temozolomide Therapy and Mediate Temozolomide Resistance

Purpose: Over the past few years, the alkylating agent temozolomide has become the standard-of-care therapy for patients with glioblastoma, the most common brain tumor. Recently, large-scale cancer genome sequencing efforts have identified a hypermutation phenotype and inactivating MSH6 mismatch repair gene mutations in recurrent, post-temozolomide glioblastomas, particularly those growing more rapidly during temozolomide treatment. This study aimed to clarify the timing and role of MSH6 mutations in mediating glioblastoma temozolomide resistance. Experimental Design:MSH6 sequence and microsatellite instability (MSI) status were determined in matched prechemotherapy and postchemotherapy glioblastomas identified by The Cancer Genome Atlas (TCGA) as having posttreatment MSH6 mutations. Temozolomide-resistant lines were derived in vitro through selective growth under temozolomide, and the MSH6 gene was sequenced in resistant clones. The role of MSH6 inactivation in mediating resistance was explored using lentiviral short hairpin RNA knockdown and MSH6 reconstitution. Results:MSH6 mutations were confirmed in posttreatment TCGA glioblastomas but absent in matched pretreatment tumors. The posttreatment hypermutation phenotype displayed a signature bias toward CpC transitions and was not associated with MSI. In vitro modeling through exposure of an MSH6 wild-type glioblastoma line to temozolomide resulted in resistant clones; one clone showed an MSH6 mutation, Thr1219Ile, that had been independently noted in two treated TCGA glioblastomas. Knockdown of MSH6 in the glioblastoma line U251 increased resistance to temozolomide cytotoxicity and reconstitution restored cytotoxicity in MSH6-null glioma cells. Conclusions:MSH6 mutations are selected in glioblastomas during temozolomide therapy both in vitro and in vivo and are causally associated with temozolomide resistance.

Distinct evolutionary trajectories of primary high-grade serous ovarian cancers revealed through spatial mutational profiling

High‐grade serous ovarian cancer (HGSC) is characterized by poor outcome, often attributed to the emergence of treatment‐resistant subclones. We sought to measure the degree of genomic diversity within primary, untreated HGSCs to examine the natural state of tumour evolution prior to therapy. We performed exome sequencing, copy number analysis, targeted amplicon deep sequencing and gene expression profiling on 31 spatially and temporally separated HGSC tumour specimens (six patients), including ovarian masses, distant metastases and fallopian tube lesions. We found widespread intratumoural variation in mutation, copy number and gene expression profiles, with key driver alterations in genes present in only a subset of samples (eg PIK3CA, CTNNB1, NF1). On average, only 51.5% of mutations were present in every sample of a given case (range 10.2–91.4%), with TP53 as the only somatic mutation consistently present in all samples. Complex segmental aneuploidies, such as whole‐genome doubling, were present in a subset of samples from the same individual, with divergent copy number changes segregating independently of point mutation acquisition. Reconstruction of evolutionary histories showed one patient with mixed HGSC and endometrioid histology, with common aetiologic origin in the fallopian tube and subsequent selection of different driver mutations in the histologically distinct samples. In this patient, we observed mixed cell populations in the early fallopian tube lesion, indicating that diversity arises at early stages of tumourigenesis. Our results revealed that HGSCs exhibit highly individual evolutionary trajectories and diverse genomic tapestries prior to therapy, exposing an essential biological characteristic to inform future design of personalized therapeutic solutions and investigation of drug‐resistance mechanisms.

Concurrent CIC mutations, IDH mutations, and 1p/19q loss distinguish oligodendrogliomas from other cancers

Oligodendroglioma is characterized by unique clinical, pathological, and genetic features. Recurrent losses of chromosomes 1p and 19q are strongly associated with this brain cancer but knowledge of the identity and function of the genes affected by these alterations is limited. We performed exome sequencing on a discovery set of 16 oligodendrogliomas with 1p/19q co‐deletion to identify new molecular features at base‐pair resolution. As anticipated, there was a high rate of IDH mutations: all cases had mutations in either IDH1 (14/16) or IDH2 (2/16). In addition, we discovered somatic mutations and insertions/deletions in the CIC gene on chromosome 19q13.2 in 13/16 tumours. These discovery set mutations were validated by deep sequencing of 13 additional tumours, which revealed seven others with CIC mutations, thus bringing the overall mutation rate in oligodendrogliomas in this study to 20/29 (69%). In contrast, deep sequencing of astrocytomas and oligoastrocytomas without 1p/19q loss revealed that CIC alterations were otherwise rare (1/60; 2%). Of the 21 non‐synonymous somatic mutations in 20 CIC‐mutant oligodendrogliomas, nine were in exon 5 within an annotated DNA‐interacting domain and three were in exon 20 within an annotated protein‐interacting domain. The remaining nine were found in other exons and frequently included truncations. CIC mutations were highly associated with oligodendroglioma histology, 1p/19q co‐deletion, and IDH1/2 mutation (p < 0.001). Although we observed no differences in the clinical outcomes of CIC mutant versus wild‐type tumours, in a background of 1p/19q co‐deletion, hemizygous CIC mutations are likely important. We hypothesize that the mutant CIC on the single retained 19q allele is linked to the pathogenesis of oligodendrogliomas with IDH mutation. Our detailed study of genetic aberrations in oligodendroglioma suggests a functional interaction between CIC mutation, IDH1/2 mutation, and 1p/19q co‐deletion. Copyright

Molecular pathology in adult gliomas: diagnostic, prognostic, and predictive markers

Over the past 10 years, there has been an increasing use of molecular markers in the assessment and management of adult malignant gliomas. Some molecular signatures are used diagnostically to help pathologists classify tumours, whereas others are used to estimate prognosis for patients. Most crucial, however, are those markers that are used to predict response to certain therapies, thereby directing clinicians to a particular treatment while avoiding other potentially deleterious therapies. Recently, large-scale genome-wide surveys have been used to identify new biomarkers that have been rapidly developed as diagnostic and prognostic tools. Given these developments, the pace of discovery of new molecular assays will quicken to facilitate personalised medicine in the setting of malignant glioma.

Targeting placental growth factor/neuropilin 1 pathway inhibits growth and spread of medulloblastoma

Medulloblastoma is the most common pediatric malignant brain tumor. Although current therapies improve survival, these regimens are highly toxic and are associated with significant morbidity. Here, we report that placental growth factor (PlGF) is expressed in the majority of medulloblastomas, independent of their subtype. Moreover, high expression of PlGF receptor neuropilin 1 (Nrp1) correlates with poor overall survival in patients. We demonstrate that PlGF and Nrp1 are required for the growth and spread of medulloblastoma: PlGF/Nrp1 blockade results in direct antitumor effects in vivo, resulting in medulloblastoma regression, decreased metastasis, and increased mouse survival. We reveal that PlGF is produced in the cerebellar stroma via tumor-derived Sonic hedgehog (Shh) and show that PlGF acts through Nrp1-and not vascular endothelial growth factor receptor 1-to promote tumor cell survival. This critical tumor-stroma interaction-mediated by Shh, PlGF, and Nrp1 across medulloblastoma subtypes-supports the development of therapies targeting PlGF/Nrp1 pathway.

Maintenance of primary tumor phenotype and genotype in glioblastoma stem cells

The clinicopathological heterogeneity of glioblastoma (GBM) and the various genetic and phenotypic subtypes in GBM stem cells (GSCs) are well described. However, the relationship between GSCs and the corresponding primary tumor from which they were isolated is poorly understood. We have established GSC-enriched neurosphere cultures from 15 newly diagnosed GBM specimens and examined the relationship between the histopathological and genomic features of GSC-derived orthotopic xenografts and those of the respective patient tumors. GSC-initiated xenografts recapitulate the distinctive cytological hallmarks and diverse histological variants associated with the corresponding patient GBM, including giant cell and gemistocytic GBM, and primitive neuroectodermal tumor (PNET)-like components. This indicates that GSCs generate tumors that preserve patient-specific disease phenotypes. The majority of GSC-derived intracerebral xenografts (11 of 15) demonstrated a highly invasive behavior crossing the midline, whereas the remainder formed discrete nodular and vascular masses. In some cases, GSC invasiveness correlated with preoperative MRI, but not with the status of PI3-kinase/Akt pathways or O(6)-methylguanine methyltransferase expression. Genome-wide screening by array comparative genomic hybridization and fluorescence in situ hybridization revealed that GSCs harbor unique genetic copy number aberrations. GSCs acquiring amplifications of the myc family genes represent only a minority of tumor cells within the original patient tumors. Thus, GSCs are a genetically distinct subpopulation of neoplastic cells within a GBM. These studies highlight the value of GSCs for preclinical modeling of clinically relevant, patient-specific GBM and, thus, pave the way for testing novel anti-GSC/GBM agents for personalized therapy.

Applications and limitations of radiomics.

Radiomics is an emerging field in quantitative imaging that uses advanced imaging features to objectively and quantitatively describe tumour phenotypes. Radiomic features have recently drawn considerable interest due to its potential predictive power for treatment outcomes and cancer genetics, which may have important applications in personalized medicine. In this technical review, we describe applications and challenges of the radiomic field. We will review radiomic application areas and technical issues, as well as proper practices for the designs of radiomic studies.

Molecular Diagnostic Testing in Malignant Gliomas: A Practical Update on Predictive Markers

Advances in understanding the molecular underpinnings of cancer and in molecular diagnostic technologies have changed the clinical practice of oncologic pathology. The development of targeted therapies against specific molecular alterations in cancer further portends changes in the role of the pathology laboratory to guide such custom therapies. To reconcile the flood of scientific discoveries in this area, the promises of highly touted novel therapeutics, and the practicality of applying this knowledge to the day-to-day practice of clinical neuropathology, the present review highlights the operative differences between diagnostic, predictive, and prognostic markers, and discusses issues surrounding the transition of prospective biomarkers to routine laboratory implementation. This review focuses on 3 predictive molecular markers that are either in clinical use or are contemplated for use in the evaluation of malignant gliomas: assessment of 1p/19q loss in oligodendroglial tumors, examination of O6-methylguanine DNA methyltransferase promoter methylation status in glioblastomas, and molecular dissection of the epidermal growth factor receptor-phosphatidylinositol 3-kinase pathway in glioblastomas. Implementation of such predictive markers is not straightforward and requires critical review of the available literature and attention to practical laboratory, compliance, financial, and clinical management issues.