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Lou Y.,BC Cancer Agency | McDonald P.C.,BC Cancer Agency | Oloumi A.,BC Cancer Agency | Chia S.,BC Cancer Agency | And 23 more authors.
Cancer Research | Year: 2011

Carbonic anhydrase IX (CAIX) is a hypoxia and HIF-1-inducible protein that regulates intra- and extracellular pH under hypoxic conditions and promotes tumor cell survival and invasion in hypoxic microenvironments. Interrogation of 3,630 human breast cancers provided definitive evidence of CAIX as an independent poor prognostic biomarker for distant metastases and survival. shRNA-mediated depletion of CAIX expression in 4T1 mouse metastatic breast cancer cells capable of inducing CAIX in hypoxia resulted in regression of orthotopic mammary tumors and inhibition of spontaneous lung metastasis formation. Stable depletion of CAIX in MDAMB-231 human breast cancer xenografts also resulted in attenuation of primary tumor growth. CAIX depletion in the 4T1 cells led to caspase-independent cell death and reversal of extracellular acidosis under hypoxic conditions in vitro. Treatment of mice harboring CAIX-positive 4T1 mammary tumors with novel CAIX-specific small molecule inhibitors that mimicked the effects of CAIX depletion in vitro resulted in significant inhibition of tumor growth and metastasis formation in both spontaneous and experimental models of metastasis, without inhibitory effects on CAIX-negative tumors. Similar inhibitory effects on primary tumor growth were observed in mice harboring orthotopic tumors comprised of lung metatstatic MDA-MB-231 LM2-4Luc+ cells. Our findings show that CAIX is vital for growth and metastasis of hypoxic breast tumors and is a specific, targetable biomarker for breast cancer metastasis. ©2011 AACR.


Anglesio M.S.,University of British Columbia | Kommoss S.,University of British Columbia | Clarke B.,University of Toronto | Galletta L.,Australian Ovarian Cancer Study Group | And 22 more authors.
Journal of Pathology | Year: 2013

Mucinous ovarian carcinomas (MCs) typically do not respond to current conventional therapy. We have previously demonstrated amplification of HER2 in 6 of 33 (18.2%) mucinous ovarian carcinomas (MCs) and presented anecdotal evidence of response with HER2-targeted treatment in a small series of women with recurrent HER2-amplified (HER2+) MC. Here, we explore HER2 amplification and KRAS mutation status in an independent cohort of 189 MCs and 199 mucinous borderline ovarian tumours (MBOTs) and their association to clinicopathological features. HER2 status was assessed by immunohistochemistry (IHC), FISH, and CISH, and interpreted per ASCO/CAP guidelines, with intratumoural heterogeneity assessment on full sections, where available. KRAS mutation testing was performed with Sanger sequencing. Stage and grade were associated with recurrence on both univariate and multivariate analysis (p < 0.001). Assessment of HER2 status revealed overexpression/amplification of HER2 in 29/154 (18.8%) MCs and 11/176 (6.2%) MBOTs. There was excellent agreement between IHC, FISH, and CISH assessment of HER2 status (perfect concordance of HER2 0 or 1+ IHC with non-amplified status, and 3+ IHC with amplified status). KRAS mutations were seen in 31/71 (43.6%) MCs and 26/33 (78.8%) MBOTs, and were near mutually exclusive of HER2 amplification. In the 189 MC cases, a total of 54 recurrences and 59 deaths (53 of progressive disease) were observed. Within MCs, either HER2 amplification/overexpression or KRAS mutation was associated with decreased likelihood of disease recurrence (p = 0.019) or death (p = 0.0041) when compared to cases with neither feature. Intratumoural heterogeneity was noted in 26% of HER2-overexpressing cases. These data support the stratification of MCs for the testing of new treatments, with HER2-targeted therapy as a viable option for HER2+ advanced or recurrent disease. Further research is required to delineate the molecular and clinical features of the ∼34% of MC cases with neither HER2 amplification nor KRAS mutations. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.


Lichner Z.,Li Ka Shing Knowledge Institute | Lichner Z.,University of Toronto | Scorilas A.,National and Kapodistrian University of Athens | White N.M.A.,Li Ka Shing Knowledge Institute | And 10 more authors.
American Journal of Pathology | Year: 2013

Clear cell renal cell carcinoma (ccRCC) is the most common tumor of the adult kidney, with an increasing rate of incidence. Recently, exome sequencing studies have revealed that the SWI/SNF (switch/sucrose nonfermentable) members PBRM1 and ARID1A are mutated in ccRCC, and it has also been suggested that aberrant chromatin regulation is a key step in kidney cancer pathogenesis. Herein, we show that down-regulation of another SW/SNF component, ARID1A, occurs frequently in ccRCC. We detected copy number loss of ARID1A in 16% of patients with ccRCC. Immunohistochemistry indicated that 67% of ccRCC (53 of 79) had significantly lower expression of BAF250a, the protein product of ARID1A, than did the matched normal kidney cortex. In parallel, we conducted in silico mRNA expression analysis on 404 ccRCC tumors and 167 normal kidney cortex samples using publicly available databases and confirmed significant down-regulation of ARID1A in 68.8% of patients. We also show that decreased BAF250a protein and ARID1A mRNA expression correlate with tumor stage and grade. Our results indicate that both the protein and mRNA levels of ARID1A are statistically significant prognostic markers for ccRCC. Even after controlling for other confounders in the multivariate analysis, BAF250 retained its prognostic significance. BAF250a IHC is easy to perform and represents a potential biomarker that could be incorporated in laboratory practice to enhance the accuracy of the existing prognostic models. Copyright © 2013 American Society for Investigative Pathology.


Wiegand K.C.,Applied Genomics | Wiegand K.C.,University of British Columbia | Lee A.F.,University of British Columbia | Lee A.F.,Vancouver General Hospital | And 18 more authors.
Journal of Pathology | Year: 2011

Mutation of the ARID1A gene and loss of the corresponding protein BAF250a has recently been described as a frequent event in clear cell and endometrioid carcinomas of the ovary. To determine whether BAF250a loss is common in other malignancies, immunohistochemistry (IHC) for BAF250a was performed on tissue microarrays (TMAs) in more than 3000 cancers, including carcinomas of breast, lung, thyroid, endometrium, kidney, stomach, oral cavity, cervix, pancreas, colon and rectum, as well as endometrial stromal sarcomas, gastrointestinal stromal tumours, sex cord-stromal tumours and four major types of lymphoma (diffuse large B cell lymphoma, primary mediastinal B cell lymphoma, mantle cell lymphoma and follicular lymphoma). We found that BAF250a loss is frequent in endometrial carcinomas but infrequent in other types of malignancies, with loss observed in 29% (29/101) of grade 1 or 2 and 39% (44/113) of grade 3 endometrioid carcinomas of the endometrium, 18% (17/95) of uterine serous carcinomas and 26% (6/23) of uterine clear cell carcinomas. Since endometrial cancers showed BAF250a loss, we stained whole tissue sections for BAF250a expression in nine cases of atypical hyperplasia and 10 cases of atypical endometriosis. Of the nine cases of complex atypical endometrial hyperplasia, all showed BAF250a expression; however, of 10 cases of atypical endometriosis (the putative precursor lesion for ovarian clear cell and endometrioid carcinoma), one case showed loss of staining for BAF250a in the atypical areas, with retention of staining in areas of non-atypical endometriosis. This was the sole case that recurred as an endometrioid carcinoma, indicating that BAF250a loss may be an early event in carcinogenesis. Since BAF250a loss is seen in endometrial carcinomas at a rate similar to that seen in ovarian carcinomas of clear cell and endometrioid type, and is uncommon in other malignancies, we conclude that loss of BAF250a is a particular feature of carcinomas arising from endometrial glandular epithelium. Copyright © 2011 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.


Wiegand K.C.,Applied Genomics | Wiegand K.C.,University of British Columbia | Shah S.P.,Applied Genomics | Al-Agha O.M.,Applied Genomics | And 51 more authors.
New England Journal of Medicine | Year: 2010

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. Copyright © 2010 Massachusetts Medical Society.


Alkushi A.,King Fahad National Guard Hospital | Kobel M.,University of Calgary | Kobel M.,Genetic Pathology Evaluation Center | Kalloger S.E.,University of British Columbia | Gilks C.B.,University of British Columbia
International Journal of Gynecological Pathology | Year: 2010

High-grade endometrial carcinomas are a heterogeneous group of tumors and include grade 3 endometrioid (EC-3), serous (SC), and clear cell carcinomas (CCC). There are conflicting data about the prognosis of these subtypes of high-grade endometrial carcinoma; this may be a result of lack of reproducibility in classifying tumor cell type. The purpose of this study was to examine differences in immunophenotype and outcome in a series of high-grade endometrial carcinomas, focusing on the comparison of EC-3 versus SC. We selected 180 endometrial carcinomas of SC, EC, or CCC type. No mixed carcinomas were included in the study. We chose the following immunohistochemical markers, estrogen receptor (ER), insulin-like growth factor 2 mRNA-binding protein 3 (IMP3), p16, p53, progesterone receptor (PR), and phosphatase and tensin homolog (PTEN) as being significantly differentially expressed in endometrial carcinoma subtypes. The tumors were stratified into 4 groups on the basis of their cell type and grade: EC grade 1 or 2, EC-3, SC, and CCC. Univariate survival analysis revealed significant differences in outcome between the 4 groups (P<0.0001), with significantly longer disease-specific survival for grade 1 or 2 EC versus EC-3 (P=0.0001), and EC-3 versus SC (P=0.0003). p16, PTEN, and IMP3 expression was observed more frequently in SC compared with EC-3 (P<0.0001, P=0.021, and P=0.031, respectively). These 3 markers showed the highest sensitivity and specificity in distinguishing between EC-3 and SC, with receiver operating characteristics area under the curve of 0.85, 0.69, and 0.71, respectively. ER and p53 approached but did not reach significance for differential expression in EC-3 versus SC (P=0.055 and P=0.068, respectively). A combination of p16 and PTEN predicts EC-3 versus SC with a sensitivity of 90.0% and specificity of 96.8%. p16 and PTEN can aid in distinguishing between EC-3 and SC of the endometrium, and are superior to ER, PR, and p53 for this purpose. EC-3 carcinomas have a significantly better prognosis than SC carcinomas of the endometrium. © 2010 International Society of Gynecological Pathologists.


Su L.,University of British Columbia | Sampaio A.V.,University of British Columbia | Jones K.B.,University of Utah | Jones K.B.,Howard Hughes Medical Institute | And 10 more authors.
Cancer Cell | Year: 2012

Synovial sarcoma is a translocation-associated sarcoma where the underlying chromosomal event generates SS18-SSX fusion transcripts. In vitro and in vivo studies have shown that the SS18-SSX fusion oncoprotein is both necessary and sufficient to support tumorigenesis; however, its mechanism of action remains poorly defined. We have purified a core SS18-SSX complex and discovered that SS18-SSX serves as a bridge between activating transcription factor 2 (ATF2) and transducin-like enhancer of split 1 (TLE1), resulting in repression of ATF2 target genes. Disruption of these components by siRNA knockdown or treatment with HDAC inhibitors rescues target gene expression, leading to growth suppression and apoptosis. Together, these studies define a fundamental role for aberrant ATF2 transcriptional dysregulation in the etiology of synovial sarcoma. © 2012 Elsevier Inc.


Al-Agha O.M.,Genetic Pathology Evaluation Center | Al-Agha O.M.,Applied Genomics | Huwait H.F.,Vancouver General Hospital | Chow C.,Genetic Pathology Evaluation Center | And 10 more authors.
American Journal of Surgical Pathology | Year: 2011

Sex cord-stromal tumors (SCSTs) of the ovary are relatively uncommon tumors. Diagnosis of SCST rests primarily on the histomorphology of these tumors, and tumors with an atypical or unconventional appearance can pose diagnostic challenges. Previously, we had identified FOXL2 (402C→G) mutation as being characteristic of adult granulosa cell tumors (aGCTs). However, molecular screening for this mutation is not always possible and adds time and cost to the diagnostic process. In this study, we investigated the potential diagnostic use of immunostaining for FOXL2 on formalin-fixed paraffin-embedded tissue sections. Using a commercially available polyclonal antiserum against FOXL2 protein, immunoexpression of FOXL2 was tested in 501 ovarian tumor samples, including 119 SCSTs, using whole tissue sections and tissue microarrays. Staining was correlated with FOXL2 mutation status. In addition, we compared FOXL2 immunoexpression with that of α-inhibin and calretinin, the 2 traditional immunomarkers of SCST, in a subset of 89 SCSTs. FOXL2 immunostaining was present in 95 of 119 (80%) SCSTs, including >95% of aGCTs, juvenile granulosa cell tumors, fibromas, and sclerosing stromal tumors. Only 50% of Sertoli-Leydig cell tumors (N=40) expressed FOXL2. One of 11 steroid cell tumors and 3 of 3 female adnexal tumors of probable Wolffian origin showed FOXL2 immunoreactivity, whereas all other non-SCSTs tested (N=368) were negative for FOXL2 expression. Thus, the sensitivity and specificity of FOXL2 immunoreactivity for SCST are 80% and 99%, respectively. The FOXL2 (402C→G) mutation was confirmed to be both a sensitive and relatively specific indicator of aGCT. Forty-five of 119 SCSTs were mutation positive. These cases were 39 of 42 (93%) aGCTs, 3 of 40 Sertoli-Leydig cell tumors, 2 of 5 thecomas, and 1 of 4 (25%) SCSTs of unclassified type. SCSTs harboring a FOXL2 mutation consistently immunoexpressed FOXL2 (44 of 45, 98%), but FOXL2 immunostaining was also seen in many SCSTs that lacked a mutation (49 of 73, 67%). FOXL2 immunostaining showed higher sensitivity for the diagnosis of SCST, compared with α-inhibin and calretinin, and FOXL2 staining was typically more intense in positive cases compared with either α-inhibin or calretinin. In the SCSTs that were negative for FOXL2 expression, α-inhibin and/or calretinin immunostaining yielded positive results. In conclusion, FOXL2 is a relatively sensitive and highly specific marker for SCST. FOXL2 staining is present in almost all SCSTs with a FOXL2 mutation, and also in a majority of SCSTs without a mutation. FOXL2, together with α-inhibin and calretinin, forms an immunomarker panel that will result in positive staining with 1 or more markers in essentially all cases of SCST. © 2011 Lippincott Williams & Wilkins.


Kalloger S.E.,Genetic Pathology Evaluation Center | Kalloger S.E.,Vancouver General Hospital | Kobel M.,University of Calgary | Leung S.,Genetic Pathology Evaluation Center | And 8 more authors.
Modern Pathology | Year: 2011

With the emerging evidence that the five major ovarian carcinoma subtypes (high-grade serous, clear cell, endometrioid, mucinous, and low-grade serous) are distinct disease entities, management of ovarian carcinoma will become subtype specific in the future. In an effort to improve diagnostic accuracy, we set out to determine if an immunohistochemical panel of molecular markers could reproduce consensus subtype assignment. Immunohistochemical expression of 22 biomarkers were examined on tissue microarrays constructed from 322 archival ovarian carcinoma samples from the British Columbia Cancer Agency archives, for the period between 1984 and 2000, and an independent set of 242 cases of ovarian carcinoma from the Gynaecologic Tissue Bank at Vancouver General Hospital from 2001 to 2008. Nominal logistic regression was used to produce a subtype prediction model for each of these sets of cases. These models were then cross-validated against the other cohort, and then both models were further validated in an independent cohort of 81 ovarian carcinoma samples from five different centers. Starting with data for 22 markers, full model fit, backwards, nominal logistic regression identified the same nine markers (CDKN2A, DKK1, HNF1B, MDM2, PGR, TFF3, TP53, VIM, WT1) as being most predictive of ovarian carcinoma subtype in both the archival and tumor bank cohorts. These models were able to predict subtype in the respective cohort in which they were developed with a high degree of sensitivity and specificity (κ statistics of 0.88±0.02 and 0.86±0.04, respectively). When the models were cross-validated (ie using the model developed in one case series to predict subtype in the other series), the prediction equation's performances were reduced (κ statistics of 0.70±0.04 and 0.61±0.04, respectively) due to differences in frequency of expression of some biomarkers in the two case series. Both models were then validated on the independent series of 81 cases, with very good to excellent ability to predict subtype (κ=0.85±0.06 and 0.78±0.07, respectively). A nine-marker immunohistochemical maker panel can be used to objectively support classification into one of the five major subtypes of ovarian carcinoma. © 2011 USCAP, Inc. All rights reserved.


PubMed | Genetic Pathology Evaluation Center, Cancer Research Center, Mount Sinai Hospital, University of Houston and University of Toronto
Type: Journal Article | Journal: The Journal of pathology | Year: 2016

We carried out whole genome and transcriptome sequencing on four tumour/normal pairs of epithelioid sarcoma. These index cases were supplemented with whole transcriptome sequencing of three additional tumours and three cell lines. Unlike rhabdoid tumour (the other major group of SMARCB1-negative cancers), epithelioid sarcoma shows a complex genome with a higher mutational rate, comparable to that of ovarian carcinoma. Despite this mutational burden, SMARCB1 mutations remain the most frequently recurring event and are probably critical drivers of tumour formation. Several cases show heterozygous SMARCB1 mutations without inactivation of the second allele, and we explore this further in vitro. Finding CDKN2A deletions in our discovery cohort, we evaluated CDKN2A protein expression in a tissue microarray. Six out of 16 cases had lost CDKN2A in greater than or equal to 90% of cells, while the remaining cases had retained the protein. Expression analysis of epithelioid sarcoma cell lines by transcriptome sequencing shows a unique profile that does not cluster with any particular tissue type or with other SWI/SNF-aberrant lines. Evaluation of the levels of members of the SWI/SNF complex other than SMARCB1 revealed that these proteins are expressed as part of a residual complex, similarly to previously studied rhabdoid tumour lines. This residual SWI/SNF is susceptible to synthetic lethality and may therefore indicate a therapeutic opportunity.

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