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1.  Diagnostic Value of Next-Generation Sequencing in an Unusual Sphenoid Tumor 
The Oncologist  2014;19(6):623-630.
The authors report the case of a patient for whom whole genome sequencing was instrumental in assisting in the correct diagnosis of the tumor based on the finding of homozygous SMARCB1 loss. This resulted in a switch in chemotherapy protocol, leading to diminution of the distant metastases. They conclude that cancer diagnostics is an area that can greatly benefit from the comprehensiveness of a whole genome analysis.
Extraordinary advancements in sequencing technology have made what was once a decade-long multi-institutional endeavor into a methodology with the potential for practical use in a clinical setting. We therefore set out to examine the clinical value of next-generation sequencing by enrolling patients with incurable or ambiguous tumors into the Personalized OncoGenomics initiative at the British Columbia Cancer Agency whereby whole genome and transcriptome analyses of tumor/normal tissue pairs are completed with the ultimate goal of directing therapeutics. First, we established that the sequencing, analysis, and communication with oncologists could be completed in less than 5 weeks. Second, we found that cancer diagnostics is an area that can greatly benefit from the comprehensiveness of a whole genome analysis. Here, we present a scenario in which a metastasized sphenoid mass, which was initially thought of as an undifferentiated squamous cell carcinoma, was rediagnosed as an SMARCB1-negative rhabdoid tumor based on the newly acquired finding of homozygous SMARCB1 deletion. The new diagnosis led to a change in chemotherapy and a complete nodal response in the patient. This study also provides additional insight into the mutational landscape of an adult SMARCB1-negative tumor that has not been explored at a whole genome and transcriptome level.
doi:10.1634/theoncologist.2013-0390
PMCID: PMC4041668  PMID: 24807916
Sphenoid; Rhabdoid; Atypical teratoid/rhabdoid tumor; Next-generation sequencing; SMARCB1
2.  Identification of cytotoxic agents disrupting synovial sarcoma oncoprotein interactions by proximity ligation assay 
Oncotarget  2016;7(23):34384-34394.
Conventional cytotoxic therapies for synovial sarcoma provide limited benefit. Drugs specifically targeting the product of its driver translocation are currently unavailable, in part because the SS18-SSX oncoprotein functions via aberrant interactions within multiprotein complexes. Proximity ligation assay is a recently-developed method that assesses protein-protein interactions in situ. Here we report use of the proximity ligation assay to confirm the oncogenic association of SS18-SSX with its co-factor TLE1 in multiple human synovial sarcoma cell lines and in surgically-excised human tumor tissue. SS18-SSX/TLE1 interactions are disrupted by class I HDAC inhibitors and novel small molecule inhibitors. This assay can be applied in a high-throughput format for drug discovery in fusion-oncoprotein associated cancers where key effector partners are known.
doi:10.18632/oncotarget.8882
PMCID: PMC5085163  PMID: 27120803
synovial sarcoma; proximity ligation assay; drug screening; HDAC inhibitors; protein-protein association
3.  Prognostic Significance of Progesterone Receptor–Positive Tumor Cells Within Immunohistochemically Defined Luminal A Breast Cancer 
Journal of Clinical Oncology  2012;31(2):203-209.
Purpose
Current immunohistochemical (IHC)-based definitions of luminal A and B breast cancers are imperfect when compared with multigene expression-based assays. In this study, we sought to improve the IHC subtyping by examining the pathologic and gene expression characteristics of genomically defined luminal A and B subtypes.
Patients and Methods
Gene expression and pathologic features were collected from primary tumors across five independent cohorts: British Columbia Cancer Agency (BCCA) tamoxifen-treated only, Grupo Español de Investigación en Cáncer de Mama 9906 trial, BCCA no systemic treatment cohort, PAM50 microarray training data set, and a combined publicly available microarray data set. Optimal cutoffs of percentage of progesterone receptor (PR) –positive tumor cells to predict survival were derived and independently tested. Multivariable Cox models were used to test the prognostic significance.
Results
Clinicopathologic comparisons among luminal A and B subtypes consistently identified higher rates of PR positivity, human epidermal growth factor receptor 2 (HER2) negativity, and histologic grade 1 in luminal A tumors. Quantitative PR gene and protein expression were also found to be significantly higher in luminal A tumors. An empiric cutoff of more than 20% of PR-positive tumor cells was statistically chosen and proved significant for predicting survival differences within IHC-defined luminal A tumors independently of endocrine therapy administration. Finally, no additional prognostic value within hormonal receptor (HR) –positive/HER2-negative disease was observed with the use of the IHC4 score when intrinsic IHC-based subtypes were used that included the more than 20% PR-positive tumor cells and vice versa.
Conclusion
Semiquantitative IHC expression of PR adds prognostic value within the current IHC-based luminal A definition by improving the identification of good outcome breast cancers. The new proposed IHC-based definition of luminal A tumors is HR positive/HER2 negative/Ki-67 less than 14%, and PR more than 20%.
doi:10.1200/JCO.2012.43.4134
PMCID: PMC3532392  PMID: 23233704
4.  Tissue Microarrays in Clinical Oncology 
Seminars in radiation oncology  2008;18(2):89-97.
The tissue microarray is a recently-implemented, high-throughput technology for the analysis of molecular markers in oncology. This research tool permits the rapid assessment of a biomarker in thousands of tumor samples, using commonly available laboratory assays such as immunohistochemistry and in-situ hybridization. Although introduced less than a decade ago, the TMA has proven to be invaluable in the study of tumor biology, the development of diagnostic tests, and the investigation of oncological biomarkers. This review describes the impact of TMA-based research in clinical oncology and its potential future applications. Technical aspects of TMA construction, and the advantages and disadvantages inherent to this technology are also discussed.
doi:10.1016/j.semradonc.2007.10.006
PMCID: PMC2292098  PMID: 18314063
5.  Establishment and characterization of a new human myxoid liposarcoma cell line (DL-221) with the FUS-DDIT3 translocation 
Myxoid liposarcoma has the pathognomonic fusion oncogene FUS-DDIT3 encoding a chimeric transcription factor. Metastatic risk is higher with an increased round cell component and has been linked to aberrations involving the IGFR/PI3K/AKT pathway. These molecular insights have yet to translate to targeted therapies and the lack of experimental models is a major hindrance. We describe the initial in-depth characterization of a new cell line (DL-221) and establishment of a mouse xenograft model.
The cell line DL-221 was derived from a metastatic pleural lesion showing myxoid and round cell histology. This newly established cell line was characterized for phenotypic properties and molecular cytogenetic profile, using PCR, COBRA-FISH and western blot. Next-generation whole exome sequencing was performed to further characterize the cell line and the parent tumor. NOD-SCID-IL2R gamma knockout mice were xenograft hosts.
DL-221 cells grew an adhering monolayer and COBRA-FISH showed an aneuploid karyotype with t(12;16)(q13;p11) and several other rearrangements; RT-PCR demonstrated a FUS-DDIT3 fusion transcript type 1. Both the cell line and the original tumor harbored a TP53 compound heterozygous mutation in exon 4 and 7 and were wild type for PIK3CA. Moreover, among the 1254 variants called by whole exome sequencing, there was 77% concordance between the cell line and parent tumor. The recently described hotspot mutation in the TERT promoter region in myxoid liposarcomas was also found at C228T in DL-221. Xenografts suitable for additional pre-clinical studies were successfully established in mice after subcutaneous injection.
The established DL-221 cell line is the only published available myxoid liposarcoma cell line that underwent spontaneous immortalization, without requiring SV40 transformation. The cell line and its xenograft model are unique and helpful tools to study the biology and novel potential targeted treatment approaches for myxoid liposarcoma.
doi:10.1038/labinvest.2016.64
PMCID: PMC4965313  PMID: 27270875
Myxoid liposarcoma; round cell liposarcoma; translocation; FUS-DDIT3; TP53; PIK3CA; HDAC; TERT; CTAG1B; exome sequencing; targeted therapy
6.  Targeting Human Cancer by a Glycosaminoglycan Binding Malaria Protein 
Cancer cell  2015;28(4):500-514.
SUMMARY
Plasmodium falciparum engineer infected erythrocytes to present the malarial protein, VAR2CSA, which binds a distinct type chondroitin sulfate (CS) exclusively expressed in the placenta. Here, we show that the same CS modification is present on a high proportion of malignant cells and that it can be specifically targeted by recombinant VAR2CSA (rVAR2). In tumors, placental-like CS chains are linked to a limited repertoire of cancer-associated proteoglycans including CD44 and CSPG4. The rVAR2 protein localizes to tumors in vivo and rVAR2 fused to diphtheria toxin or conjugated to hemiasterlin compounds strongly inhibits in vivo tumor cell growth and metastasis. Our data demonstrate how an evolutionarily refined parasite-derived protein can be exploited to target a common, but complex, malignancy-associated glycosaminoglycan modification.
Graphical Abstract
doi:10.1016/j.ccell.2015.09.003
PMCID: PMC4790448  PMID: 26461094
7.  αB-crystallin Expression in Breast Cancer is Associated with Brain Metastasis 
NPJ breast cancer  2015;1:15014.
Background/Objectives
The molecular chaperone αB-crystallin is expressed in estrogen receptor, progesterone receptor and human epidermal growth factor receptor-2 “triple-negative” breast carcinomas and promotes brain and lung metastasis. We examined αB-crystallin expression in primary breast carcinomas with metastatic data to evaluate its association with prognosis and site-specific metastases.
Methods
αB-crystallin gene (CRYAB) expression was examined using publically available global-gene expression data (n=855 breast tumors) with first site of distant metastasis information (“855Met”). αB-crystallin protein expression was determined by immunohistochemistry using the clinically annotated tissue microarray (n=3987 breast tumors) from British Columbia Cancer Agency (BCCA). Kaplan-Meier and multivariable Cox regression analyses were used to evaluate the prognostic value of αB-crystallin. Multivariable logistic regression analysis was used to evaluate risks of αB-crystallin and other markers for site of metastasis.
Results
In the 855Met dataset, αB-crystallin gene (CRYAB) expression was an independent predictor of brain as the first distant site of relapse (HR = 1.2, (95% CI 1.0-1.4), P = 0.021). In the BCCA series, αB-crystallin protein expression was an independent prognostic marker of poor breast cancer specific survival (HR = 1.3, (95% CI 1.1-1.6), P = 0.014). Among patients with metastases, αB-crystallin was the strongest independent predictor of brain metastasis (OR = 2.99 (95% CI 1.83-4.89), P < 0.0001) and the only independent predictor of brain as the first site of distant metastasis (OR = 3.15 (95% CI1.43-6.95), P = 0.005). αB-crystallin was also associated with worse survival (3.0 versus 4.7 months, P = 0.007).
Conclusions
αB-crystallin is a promising biomarker to identify breast cancer patients at high risk for early relapse in the brain, independent of ER and HER2 status.
doi:10.1038/npjbcancer.2015.14
PMCID: PMC5027912  PMID: 27656679
αB-crystallin; metastasis; triple-negative breast cancer; tissue microarray; biomarker; brain
8.  Responsiveness of Intrinsic Subtypes to Adjuvant Anthracycline Substitution in the NCIC.CTG MA.5 Randomized Trial 
Purpose
Recent studies suggest that intrinsic breast cancer subtypes may differ in their responsiveness to specific chemotherapy regimens. We examined this hypothesis on NCIC.CTG MA.5, a clinical trial randomizing premenopausal women with node-positive breast cancer to adjuvant CMF (cyclophosphamide-methotrexate-fluorouracil) versus CEF (cyclophosphamide-epirubicin-fluorouracil) chemotherapy.
Experimental Design
Intrinsic subtype was determined for 476 tumors using the quantitative reverse transcriptase PCR PAM50 gene expression test. Luminal A, luminal B, HER2-enriched (HER2-E), and basal-like subtypes were correlated with relapse-free survival (RFS) and overall survival (OS), estimated using Kaplan-Meier plots and log-rank testing. Multivariable Cox regression analyses determined significance of interaction between treatment and intrinsic subtypes.
Results
Intrinsic subtypes were associated with RFS (P = 0005) and OS (P < 0.0001) on the combined cohort. The HER2-E showed the greatest benefit from CEF versus CMF, with absolute 5-year RFS and OS differences exceeding 20%, whereas there was a less than 2% difference for non-HER2-E tumors (interaction test P = 0.03 for RFS and 0.03 for OS). Within clinically defined Her2+ tumors, 79% (72 of 91) were classified as the HER2-E subtype by gene expression and this subset was strongly associated with better response to CEF versus CMF (62% vs. 22%, P = 0.0006). There was no significant difference in benefit between CEF and CMF in basal-like tumors [n = 94; HR, 1.1; 95% confidence interval (CI), 0.6−.1 for RFS and HR, 1.3; 95% CI, 0.7−2.5 for OS].
Conclusion
HER2-E strongly predicted anthracycline sensitivity. The chemotherapy-sensitive basal- like tumors showed no added benefit for CEF over CMF, suggesting that nonanthracycline regimens may be adequate in this subtype although further investigation is required.
doi:10.1158/1078-0432.CCR-11-2956
PMCID: PMC3743660  PMID: 22351696
9.  A comparison of PAM50 intrinsic subtyping with immunohistochemistry and clinical prognostic factors in tamoxifen-treated estrogen receptor positive breast cancer 
Purpose
To compare clinical, immunohistochemical and gene expression models of prognosis applicable to formalin-fixed, paraffin-embedded blocks in a large series of estrogen receptor positive breast cancers, from patients uniformly treated with adjuvant tamoxifen.
Methods
qRT-PCR assays for 50 genes identifying intrinsic breast cancer subtypes were completed on 786 specimens linked to clinical (median followup 11.7 years) and immunohistochemical (ER, PR, HER2, Ki67) data. Performance of predefined intrinsic subtype and Risk-Of-Relapse scores was assessed using multivariable Cox models and Kaplan-Meier analysis. Harrell’s C index was used to compare fixed models trained in independent data sets, including proliferation signatures.
Results
Despite clinical ER positivity, 10% of cases were assigned to non-Luminal subtypes. qRT-PCR signatures for proliferation genes gave more prognostic information than clinical assays for hormone receptors or Ki67. In Cox models incorporating standard prognostic variables, hazard ratios for breast cancer disease specific survival over the first 5 years of followup, relative to the most common Luminal A subtype, are 1.99 (95% CI: 1.09–3.64) for Luminal B, 3.65 (1.64–8.16) for HER2-enriched and 17.71 (1.71–183.33) for the basal like subtype. For node-negative disease, PAM50 qRT-PCR based risk assignment weighted for tumor size and proliferation identifies a group with >95% 10 yr survival without chemotherapy. In node positive disease, PAM50-based prognostic models were also superior.
Conclusion
The PAM50 gene expression test for intrinsic biological subtype can be applied to large series of formalin-fixed paraffin-embedded breast cancers, and gives more prognostic information than clinical factors and immunohistochemistry using standard cutpoints.
doi:10.1158/1078-0432.CCR-10-1282
PMCID: PMC2970720  PMID: 20837693
10.  Kaposi Sarcoma of the Adrenal Gland Resembling Epithelioid Angiosarcoma: A Case Report 
Sarcoma  2011;2011:898257.
Patients with human immunodeficiency virus infection are known to have increased risk of various neoplasms, including Kaposi sarcoma, which classically involves the skin and mucosal locations. The anaplastic variant of Kaposi sarcoma is rare and poorly documented in the literature. It is characterised clinically by a more aggressive behaviour and increased metastatic potential, and histologically by increased cellularity, mitotic rate, and rarely by epithelioid angiosarcoma-like morphology. We report herein a 64-year-old man with a long-standing history of human immunodeficiency virus infection who developed a right adrenal tumor with a high-grade anaplastic angiosarcoma-like morphology. Immunohistochemistry for human herpes virus-8 was strongly positive in the tumor cells. To the best of our knowledge, this is the first report of an anaplastic Kaposi sarcoma in the adrenal gland.
doi:10.1155/2011/898257
PMCID: PMC3153921  PMID: 21845069
11.  Dual loss of the SWI/SNF complex ATPases SMARCA4/BRG1 and SMARCA2/BRM is highly sensitive and specific for small cell carcinoma of the ovary, hypercalcaemic type 
The Journal of Pathology  2015;238(3):389-400.
Abstract
Small cell carcinoma of the ovary, hypercalcaemic type (SCCOHT) is a lethal and sometimes familial ovarian tumour of young women and children. We and others recently discovered that over 90% of SCCOHTs harbour inactivating mutations in the chromatin remodelling gene SMARCA4 with concomitant loss of its encoded protein SMARCA4 (BRG1), one of two mutually exclusive ATPases of the SWI/SNF chromatin remodelling complex. To determine the specificity of SMARCA4 loss for SCCOHT, we examined the expression of SMARCA4 by immunohistochemistry in more than 3000 primary gynaecological tumours. Among ovarian tumours, it was only absent in clear cell carcinoma (15 of 360, 4%). In the uterus, it was absent in endometrial stromal sarcomas (4 of 52, 8%) and high‐grade endometrioid carcinomas (2 of 338, 1%). Recent studies have shown that SMARCA2 (BRM), the other mutually exclusive ATPase of the SWI/SNF complex, is necessary for survival of tumour cells lacking SMARCA4. Therefore, we examined SMARCA2 expression and discovered that all SMARCA4‐negative SCCOHTs also lacked SMARCA2 protein by IHC, including the SCCOHT cell lines BIN67 and SCCOHT1. Among ovarian tumours, the SMARCA4/SMARCA2 dual loss phenotype appears completely specific for SCCOHT. SMARCA2 loss was not due to mutation but rather from an absence of mRNA expression, which was restored by treatment with the histone deacetylase inhibitor trichostatin A. Re‐expression of SMARCA4 or SMARCA2 inhibited the growth of BIN67 and SCCOHT1 cell lines. Our results indicate that SMARCA4 loss, either alone or with SMARCA2, is highly sensitive and specific for SCCOHT and that restoration of either SWI/SNF ATPase can inhibit the growth of SCCOHT cell lines. © 2015 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.
doi:10.1002/path.4633
PMCID: PMC4832362  PMID: 26356327
small cell carcinoma; hypercalcaemic type; rhabdoid tumour; SMARCA4/BRG1; SMARCA2/BRM; SMARCB1/INI1; SWI/SNF; HDAC inhibitor; trichostatin A; epigenetic silencing
12.  Therapeutic Antibodies Targeting CSF1 Impede Macrophage Recruitment in a Xenograft Model of Tenosynovial Giant Cell Tumor 
Sarcoma  2010;2010:174528.
Tenosynovial giant cell tumor is a neoplastic disease of joints that can cause severe morbidity. Recurrences are common following local therapy, and no effective medical therapy currently exists. Recent work has demonstrated that all cases overexpress macrophage colony-stimulating factor (CSF1), usually as a consequence of an activating gene translocation, resulting in an influx of macrophages that form the bulk of the tumor. New anti-CSF1 drugs have been developed; however there are no preclinical models suitable for evaluation of drug benefits in this disease. In this paper, we describe a novel renal subcapsular xenograft model of tenosynovial giant cell tumor. Using this model, we demonstrate that an anti-CSF1 monoclonal antibody significantly inhibits host macrophage infiltration into this tumor. The results from this model support clinical trials of equivalent humanized agents and anti-CSF1R small molecule drugs in cases of tenosynovial giant cell tumor refractory to conventional local therapy.
doi:10.1155/2010/174528
PMCID: PMC2957133  PMID: 20981142
13.  Ki67 Index, HER2 Status, and Prognosis of Patients With Luminal B Breast Cancer 
Background
Gene expression profiling of breast cancer has identified two biologically distinct estrogen receptor (ER)-positive subtypes of breast cancer: luminal A and luminal B. Luminal B tumors have higher proliferation and poorer prognosis than luminal A tumors. In this study, we developed a clinically practical immunohistochemistry assay to distinguish luminal B from luminal A tumors and investigated its ability to separate tumors according to breast cancer recurrence-free and disease-specific survival.
Methods
Tumors from a cohort of 357 patients with invasive breast carcinomas were subtyped by gene expression profile. Hormone receptor status, HER2 status, and the Ki67 index (percentage of Ki67-positive cancer nuclei) were determined immunohistochemically. Receiver operating characteristic curves were used to determine the Ki67 cut point to distinguish luminal B from luminal A tumors. The prognostic value of the immunohistochemical assignment for breast cancer recurrence-free and disease-specific survival was investigated with an independent tissue microarray series of 4046 breast cancers by use of Kaplan–Meier curves and multivariable Cox regression.
Results
Gene expression profiling classified 101 (28%) of the 357 tumors as luminal A and 69 (19%) as luminal B. The best Ki67 index cut point to distinguish luminal B from luminal A tumors was 13.25%. In an independent cohort of 4046 patients with breast cancer, 2847 had hormone receptor–positive tumors. When HER2 immunohistochemistry and the Ki67 index were used to subtype these 2847 tumors, we classified 1530 (59%, 95% confidence interval [CI] = 57% to 61%) as luminal A, 846 (33%, 95% CI = 31% to 34%) as luminal B, and 222 (9%, 95% CI = 7% to 10%) as luminal–HER2 positive. Luminal B and luminal–HER2-positive breast cancers were statistically significantly associated with poor breast cancer recurrence-free and disease-specific survival in all adjuvant systemic treatment categories. Of particular relevance are women who received tamoxifen as their sole adjuvant systemic therapy, among whom the 10-year breast cancer–specific survival was 79% (95% CI = 76% to 83%) for luminal A, 64% (95% CI = 59% to 70%) for luminal B, and 57% (95% CI = 47% to 69%) for luminal–HER2 subtypes.
Conclusion
Expression of ER, progesterone receptor, and HER2 proteins and the Ki67 index appear to distinguish luminal A from luminal B breast cancer subtypes.
doi:10.1093/jnci/djp082
PMCID: PMC2684553  PMID: 19436038
14.  Synergism of Heat Shock Protein 90 and Histone Deacetylase Inhibitors in Synovial Sarcoma 
Sarcoma  2009;2009:794901.
Current systemic therapies have little curative benefit for synovial sarcoma. Histone deacetylase (HDAC) inhibitors and the heat shock protein 90 (Hsp90) inhibitor 17-AAG have recently been shown to inhibit synovial sarcoma in preclinical models. We tested combinations of 17-AAG with the HDAC inhibitor MS-275 for synergism by proliferation and apoptosis assays. The combination was found to be synergistic at multiple time points in two synovial sarcoma cell lines. Previous studies have shown that HDAC inhibitors not only induce cell death but also activate the survival pathway NF-κB, potentially limiting therapeutic benefit. As 17-AAG inhibits activators of NF-κB, we tested if 17-AAG synergizes with MS-275 through abrogating NF-κB activation. In our assays, adding 17-AAG blocks NF-κB activation by MS-275 and siRNA directed against histone deacetylase 3 (HDAC3) recapitulates the effects of MS-275. Additionally, we find that the NF-κB inhibitor BAY 11-7085 synergizes with MS-275. We conclude that agents inhibiting NF-κB synergize with HDAC inhibitors against synovial sarcoma.
doi:10.1155/2009/794901
PMCID: PMC2659882  PMID: 19325926
15.  Defining Breast Cancer Intrinsic Subtypes by Quantitative Receptor Expression 
The Oncologist  2015;20(5):474-482.
This study compared centrally performed clinical assays of quantitative ER, PR, and HER2 expression with molecular intrinsic subtypes identified by an open-source, centroid-based, subtype predictor in tumors collected across three phase III randomized clinical trials and determined the molecular populations within strongly hormone receptor-positive tumors, borderline tumors, and triple-negative tumors.
Purpose.
To determine intrinsic breast cancer subtypes represented within categories defined by quantitative hormone receptor (HR) and HER2 expression.
Methods.
We merged 1,557 cases from three randomized phase III trials into a single data set. These breast tumors were centrally reviewed in each trial for quantitative ER, PR, and HER2 expression by immunohistochemistry (IHC) stain and by reverse transcription-quantitative polymerase chain reaction (RT-qPCR), with intrinsic subtyping by research-based PAM50 RT-qPCR assay.
Results.
Among 283 HER2-negative tumors with <1% HR expression by IHC, 207 (73%) were basal-like; other subtypes, particularly HER2-enriched (48, 17%), were present. Among the 1,298 HER2-negative tumors, borderline HR (1%–9% staining) was uncommon (n = 39), and these tumors were heterogeneous: 17 (44%) luminal A/B, 12 (31%) HER2-enriched, and only 7 (18%) basal-like. Including them in the definition of triple-negative breast cancer significantly diminished enrichment for basal-like cancer (p < .05). Among 106 HER2-positive tumors with <1% HR expression by IHC, the HER2-enriched subtype was the most frequent (87, 82%), whereas among 127 HER2-positive tumors with strong HR (>10%) expression, only 69 (54%) were HER2-enriched and 55 (43%) were luminal (39 luminal B, 16 luminal A). Quantitative HR expression by RT-qPCR gave similar results. Regardless of methodology, basal-like cases seldom expressed ER/ESR1 or PR/PGR and were associated with the lowest expression level of HER2/ERBB2 relative to other subtypes.
Conclusion.
Significant discordance remains between clinical assay-defined subsets and intrinsic subtype. For identifying basal-like breast cancer, the optimal HR IHC cut point was <1%, matching the American Society of Clinical Oncology and College of American Pathologists guidelines. Tumors with borderline HR staining are molecularly diverse and may require additional assays to clarify underlying biology.
doi:10.1634/theoncologist.2014-0372
PMCID: PMC4425383  PMID: 25908555
Breast cancer; Intrinsic subtypes; Receptor expression
16.  Synovial sarcoma: recent discoveries as a roadmap to new avenues for therapy 
Cancer discovery  2015;5(2):124-134.
Oncogenesis in synovial sarcoma is driven by the chromosomal translocation t(X,18; p11,q11), which generates an in-frame fusion of the SWI/SNF subunit SS18 to the C-terminal repression domains of SSX1 or SSX2. Proteomic studies have identified an integral role of SS18-SSX in the SWI/SNF complex, and provide new evidence for mistargeting of polycomb-repression in synovial sarcoma. Two recent in vivo studies are highlighted, providing additional support for the importance of Wnt signaling in synovial sarcoma: one uses a conditional mouse model where knockout of beta-catenin prevents tumor formation, and another uses a small molecule inhibitor of beta-catenin in xenograft models.
doi:10.1158/2159-8290.CD-14-1246
PMCID: PMC4320664  PMID: 25614489
synovial sarcoma; Wnt; beta-catenin; Akt; epigenetic; translocation; SWI/SNF
17.  Cancer genomics: why rare is valuable 
Rare conditions are sometimes ignored in biomedical research because of difficulties in obtaining specimens and limited interest from fund raisers. However, the study of rare diseases such as unusual cancers has again and again led to breakthroughs in our understanding of more common diseases. It is therefore unsurprising that with the development and accessibility of next-generation sequencing, much has been learnt from studying cancers that are rare and in particular those with uniform biological and clinical behavior. Herein, we describe how shotgun sequencing of cancers such as granulosa cell tumor, endometrial stromal sarcoma, epithelioid hemangioendothelioma, ameloblastoma, small-cell carcinoma of the ovary, clear-cell carcinoma of the ovary, nonepithelial ovarian tumors, chondroblastoma, and giant cell tumor of the bone has led to rapidly translatable discoveries in diagnostics and tumor taxonomies, as well as providing insights into cancer biology.
doi:10.1007/s00109-015-1260-8
PMCID: PMC4366545  PMID: 25676695
Next-generation sequencing; Formefruste; Rare tumors; Genomics
18.  An International Ki67 Reproducibility Study 
Background
In breast cancer, immunohistochemical assessment of proliferation using the marker Ki67 has potential use in both research and clinical management. However, lack of consistency across laboratories has limited Ki67’s value. A working group was assembled to devise a strategy to harmonize Ki67 analysis and increase scoring concordance. Toward that goal, we conducted a Ki67 reproducibility study.
Methods
Eight laboratories received 100 breast cancer cases arranged into 1-mm core tissue microarrays—one set stained by the participating laboratory and one set stained by the central laboratory, both using antibody MIB-1. Each laboratory scored Ki67 as percentage of positively stained invasive tumor cells using its own method. Six laboratories repeated scoring of 50 locally stained cases on 3 different days. Sources of variation were analyzed using random effects models with log2-transformed measurements. Reproducibility was quantified by intraclass correlation coefficient (ICC), and the approximate two-sided 95% confidence intervals (CIs) for the true intraclass correlation coefficients in these experiments were provided.
Results
Intralaboratory reproducibility was high (ICC = 0.94; 95% CI = 0.93 to 0.97). Interlaboratory reproducibility was only moderate (central staining: ICC = 0.71, 95% CI = 0.47 to 0.78; local staining: ICC = 0.59, 95% CI = 0.37 to 0.68). Geometric mean of Ki67 values for each laboratory across the 100 cases ranged 7.1% to 23.9% with central staining and 6.1% to 30.1% with local staining. Factors contributing to interlaboratory discordance included tumor region selection, counting method, and subjective assessment of staining positivity. Formal counting methods gave more consistent results than visual estimation.
Conclusions
Substantial variability in Ki67 scoring was observed among some of the world’s most experienced laboratories. Ki67 values and cutoffs for clinical decision-making cannot be transferred between laboratories without standardizing scoring methodology because analytical validity is limited.
doi:10.1093/jnci/djt306
PMCID: PMC3888090  PMID: 24203987
19.  Prognostic significance of FOXP3+ tumor-infiltrating lymphocytes in breast cancer depends on estrogen receptor and human epidermal growth factor receptor-2 expression status and concurrent cytotoxic T-cell infiltration 
Introduction
The infiltration of FOXP3+ regulatory T cells into invasive tumors has been reported to be associated with survival in a variety of cancers. The prognostic significance of FOXP3+ tumor-infiltrating lymphocytes (TILs) in breast cancer, however, remains controversial.
Methods
FOXP3+ TILs were assessed by immunohistochemistry on tissue microarrays constructed from a well-defined cohort of 3,992 breast cancer patients linked to detailed demographic, biomarker, treatment and outcome data. Survival analyses were performed using the Kaplan-Meier function and Cox proportional hazards regression models to evaluate the association of FOXP3+ TILs with breast cancer-specific survival, stratified by intrinsic subtype and cytotoxic T-cell infiltration status (as defined by CD8 immunohistochemistry).
Results
The presence of high numbers of FOXP3+ TILs was significantly associated with young age, high grade, estrogen receptor (ER) negativity, concurrent CD8+ cytotoxic T-cell infiltration, and human epidermal growth factor receptor-2 positive (HER2+)/ER− and core basal subtypes. On multivariate survival analysis, a high level of FOXP3+ TILs was significantly associated with poor survival in ER+ breast cancers that lacked CD8+ T-cell infiltrates (hazard ratio (HR) = 1.30, 95% confidence interval (CI) = 1.02 to 1.66). However, in ER− breast cancers, FOXP3+ TILs were strongly associated with improved survival in the HER2+/ER− subgroup, particularly in those with co-existent CD8+ T-cell infiltrates (HR = 0.48, 95% CI = 0.23 to 0.98), for which the presence of high levels of FOXP3+ TILs was independent of standard clinical prognostic factors.
Conclusions
FOXP3+ regulatory TILs are a poor prognostic indicator in ER+ breast cancer, but a favorable prognostic factor in the HER2+/ER− subtype. The prognostic value of FOXP3+ TILs in breast cancer differs depending on ER and HER2 expression status and CD8+ T-cell infiltration.
Electronic supplementary material
The online version of this article (doi:10.1186/s13058-014-0432-8) contains supplementary material, which is available to authorized users.
doi:10.1186/s13058-014-0432-8
PMCID: PMC4303113  PMID: 25193543
20.  Developing a new generation of breast cancer clinical gene expression tests 
When treatment decisions are based purely on clinicopathological factors, many women with estrogen receptor-positive/human epidermal growth factor receptor 2-negative cancers are overtreated. Gene expression profiles are valuable clinical tools that stratify the recurrence risk to identify patients most likely to benefit from adjuvant systemic therapies. Building upon greater understanding of tumor biology and more rigorous approaches to validation (including independent studies with a high level of evidence), several second-generation multigene tests have been developed. In the previous issue, Martin and colleagues report the third clinical validation study for EndoPredict, a distributed assay to assess risk of distant recurrences in estrogen receptor-positive/human epidermal growth factor receptor 2-negative women. The authors confirm the assay’s independent prognostic value in premenopausal and postmenopausal, node-positive women treated with contemporary chemotherapy followed by endocrine therapy. EndoPredict did not, however, predict benefit from adding paclitaxel. Predictive signatures for selecting among chemotherapy regimens remain an area needing further development.
doi:10.1186/bcr3688
PMCID: PMC4100317  PMID: 25928431
21.  Genome-wide transcriptome analyses reveals p53 inactivation mediated loss of miR-34a expression in malignant peripheral nerve sheath tumors 
The Journal of pathology  2010;220(1):58-70.
Malignant peripheral nerve sheath tumors (MPNSTs) are aggressive soft tissue tumors that occur either sporadically or in patients with Neurofibromatosis Type 1. The malignant transformation of the benign neurofibroma to MPNST is incompletely understood at the molecular level. We have determined the gene expression signature for benign and malignant PNSTs and found that the major trend in malignant transformation from neurofibroma to MPNST consists of the loss of expression of a large number of genes, rather than widespread increase in gene expression. Relatively few genes are expressed at higher levels in MPNSTs and these include genes involved in cell proliferation and genes implicated in tumor metastasis. In addition, a gene expression signature indicating p53 inactivation is seen in the majority of MPNSTs. Subsequent microRNA profiling of benign and malignant PNSTs indicated a relative downregulation of miR-34a in most MPNSTs compared to neurofibromas. In vitro studies using the cell lines MPNST-14 (NF1 mutant) and MPNST-724 (from a non-NF1 individual) show that exogenous expression of p53 or miR-34a promotes apoptotic cell death. In addition, exogenous expression of p53 in MPNST cells induces miR-34a and other miRNAs. Our data shows that p53 inactivation and subsequent loss of expression of miR-34a may significantly contribute to the MPNST development. Collectively, our findings suggests that deregulation of miRNAs have a potential role in the malignant transformation process in peripheral nerve sheath tumors.
doi:10.1002/path.2633
PMCID: PMC4058327  PMID: 19890883
peripheral nerve sheath tumors; malignant transformation; expression profiling; microRNA
22.  Myxoid liposarcoma in a 91-year-old patient 
Background
Myxoid liposarcoma is a mesenchymal malignancy most commonly presenting in young adults. This tumor is known for its characteristic chromosomal rearrangement at the DDIT3 locus.
Results
We report a case of myxoid liposarcoma in a 91-year-old, the oldest known patient with this disease-entity. FISH analysis of the DDIT3 and FUS loci demonstrate the pathognomonic chromosomal alteration in the setting of predominantly round cell histology on biopsy, confirmed by RT-PCR.
Conclusion
Myxoid liposarcoma affects mostly young adults but can be seen in the elderly population. Molecular and cytogenetic assays are helpful auxiliaries to histology in the setting of unusual histology and clinical presentation.
doi:10.1186/1755-8166-6-50
PMCID: PMC3843574  PMID: 24252207
Liposarcoma; Myxoid liposarcoma; Round cell liposarcoma; Advanced age; DDIT3; FUS; FISH
23.  Cyclin D1 as a diagnostic immunomarker for endometrial stromal sarcoma with YWHAE-FAM22 rearrangement 
Endometrial stromal sarcoma (ESS) characterized by YWHAE-FAM22 genetic fusion is histologically higher-grade and clinically more aggressive than ESS with JAZF1-SUZ12 or equivalent genetic rearrangements, hence it is clinically important to recognize this subset of ESS. To identify diagnostic immunomarkers for this biologically-defined ESS subset, we compared gene expression profiles from YWHAE-FAM22 ESS, JAZF1-rearranged ESS and uterine leiomyosarcomas. These studies showed consistent upregulation of cyclin D1 in YWHAE-FAM22 ESS compared to JAZF1-SUZ12 ESS. Immunohistochemically, the high-grade round cell component of all 12 YWHAE-FAM22 ESS demonstrated diffuse (≥70%) moderate-to-strong nuclear cyclin D1 staining and this diffuse positivity was not seen in 34 ESS with JAZF1 and equivalent genetic rearrangements or in 21 low-grade ESS with no demonstrable genetic rearrangements. In a series of 243 non-ESS pure uterine mesenchymal and mixed epithelial-mesenchymal tumors, only 2 of 8 undifferentiated endometrial sarcomas with nuclear uniformity and 1 of 80 uterine leiomyosarcomas demonstrate diffuse cyclin D1 immunoreactivity. Both cyclin D1-positive undifferentiated endometrial sarcomas showed diffuse strong CD10 staining, which is consistently absent in the high-grade round cell component of YWHAE-FAM22 ESS. The low-grade spindle cell component of YWHAE-FAM22 ESS showed a spatially heterogeneous cyclin D1 staining pattern that was weaker and less diffuse overall. Our findings indicate that cyclin D1 is a sensitive and specific diagnostic immunomarker for YWHAE-FAM22 ESS. When evaluating high-grade uterine sarcomas, cyclin D1 can be included in the immunohistochemical panel as an indicator of YWHAE-FAM22 ESS.
doi:10.1097/PAS.0b013e31825fa931
PMCID: PMC3444748  PMID: 22982899
Endometrial stromal sarcoma; round cell; YWHAE-FAM22; cyclin D1; JAZF1-SUZ12
24.  SS18-SSX2 and the mitochondrial apoptosis pathway in mouse and human synovial sarcomas 
Oncogene  2012;32(18):2365-2375.e5.
Synovial sarcoma is a deadly malignancy with limited sensitivity to traditional cytotoxic chemotherapy. SS18-SSX fusion oncogene expression characterizes human synovial sarcomas and drives oncogenesis in a mouse model. Elevated expression of BCL2 is considered a consistent feature of the synovial sarcoma expression profile. Our objective was to evaluate the expression of apoptotic pathway members in synovial sarcomas and interrogate the impact of modulating SS18-SSX expression on this pathway. We show in human and murine synovial sarcoma cells that SS18-SSX increases BCL2 expression, but represses other anti-apoptotic genes, including MCL1 and BCL2A1. This repression is achieved by directly suppressing expression via binding through ATF2 to the cyclic AMP response element in the promoters of these genes and recruiting TLE1/Groucho. The suppression of these two anti-apoptotic pathways silences the typical routes by which other tumors evade BH3-domain peptidomimetic pharmacotherapy. We show that mouse and human synovial sarcoma cells are sensitive in vitro to ABT-263, a BH3-peptidomimetic, much more so than are other tested cancer cell lines. ABT-263 also enhances the sensitivity of these cells to doxorubicin, a traditional cytotoxic chemotherapy used for synovial sarcoma. We also demonstrate the capacity of ABT-263 to stunt synovial sarcomagenesis in vivo in a genetic mouse model. These data recommend pursuit of BH3-peptidomimetic pharmacotherapy in human synovial sarcomas.
doi:10.1038/onc.2012.247
PMCID: PMC3756901  PMID: 22797074
synovial sarcoma; apoptosis; chemotherapy; targeted therapy; mouse model
25.  Effect of continuous statistically standardized measures of estrogen and progesterone receptors on disease-free survival in NCIC CTG MA.12 Trial and BC Cohort 
Introduction
We hypothesized improved inter-laboratory comparability of estrogen receptor (ER) and progesterone receptor (PgR) across different assay methodologies with adjunctive statistical standardization, akin to bone mineral density (BMD) z-scores. We examined statistical standardization in MA.12, a placebo-controlled pre-menopausal trial of adjuvant tamoxifen with locally assessed hormone receptor +/- tumours, and in a cohort of post-menopausal British Columbia (BC) tamoxifen-treated patients.
Methods
ER and PgR were centrally assessed for both patient groups with real time quantitative reverse transcription polymerase chain reaction (qPCR) and immunohistochemistry (IHC). Effects on disease-free survival (DFS) were investigated separately for 345 MA.12 and 673 BC patients who had both qPCR and IHC assessments. Comparisons utilized continuous laboratory units and statistically standardized z-scores. Univariate categorization of ER/PgR was by number of standard deviations (SD) above or below the mean (z-score ≥1.0 SD below mean; z-score <1.0 SD below mean; z-score ≤1.0 SD above mean; z-score >1.0 SD above mean). Exploratory multivariate examinations utilized step-wise Cox regression.
Results
Median follow-up for MA.12 was 9.7 years; for BC patients, 11.8 years. For MA.12, 101 of 345 (29%) patients were IHC ER-PgR-. ER was not univariately associated with DFS (qPCR, P = 0.19; IHC, P = 0.08), while PgR was (qPCR, P = 0.09; IHC, P = 0.04). For BC patients, neither receptor was univariately associated with DFS: for ER, PCR, P = 0.36, IHC, P = 0.24; while for PgR, qPCR, P = 0.17, IHC, P = 0.31. Multivariately, MA.12 patients randomized to tamoxifen had significantly better DFS (P = 0.002 to 0.005) than placebo. Meanwhile, jointly ER and PgR were not associated with DFS whether assessed by qPCR or by IHC in all patients, or in the subgroup of patients with IHC positive stain, for pooled or separate treatment arms. Different results by type of continuous unit supported the concept of ER level being relevant for medical decision-making. For postmenopausal BC tamoxifen patients, higher qPCR PgR was weakly associated with better DFS (P = 0.06).
Conclusions
MA.12 pre-menopausal patients in a placebo-controlled tamoxifen trial had similar multivariate prognostic effects with statistically standardized hormone receptors when tumours were assayed by qPCR or IHC, for hormone receptor +/- and + tumours. The BC post-menopausal tamoxifen cohort did not exhibit a significant prognostic association of ER or PgR with DFS. Adjunctive statistical standardization is currently under investigation in other NCIC CTG endocrine trials.
doi:10.1186/bcr3465
PMCID: PMC3978444  PMID: 23972025

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