Torsten Nielsen

The genomic subtyping of breast cancers into luminal A, luminal B, human epidermal growth factor receptor-2 (Her2)-Enriched and basal-like has remarkably advanced breast cancer diagnosis, treatment, and outcome. However, comparatively few advancements have been made in identifying biomarkers that can tailor treatments for the aggressive basal-like subtype. This group is clinically approximated as triple negative breast cancer (TNBC), characterized by immunohistochemical negativity for estrogen receptor, progesterone receptor, and Her2. However, this definition identifies a biologically heterogenous group, highlighting the complexity of guiding therapeutic choices for TNBC including those with basal-like molecular biology.My research goals have included identifying improved diagnostic biomarkers that can guide better therapeutic options for these aggressive cancers in a clinically-applicable manner using immunohistochemistry, RNA expression, and proteomic profiling. First, applying an optimized immunohistochemical panel, defined by nestin positivity or INPP4B negativity, on 239 specimens from the phase III SBG0102 clinical trial, I was able to identify those with a basal-like gene expression subtype and to predict which metastatic breast cancer patients benefit from gemcitabine chemotherapy. Then, using a 770-gene RNA panel targeting multiple biological mechanisms and additional 30-custom genes related to capecitabine metabolism on 111 TNBC specimens from the phase III FinXX adjuvant capecitabine trial, I found that genes and metagenes related to immune response (cytotoxic cells, PDL2), endothelial, mast cells and 38 individual genes are the most significantly associated with capecitabine benefit. Finally, to characterize the heterogeneity of basal-like and triple negative breast cancers beyond current genomic classifications, I used a method called SP3-Clinical Tissue Proteomics, compatible with routine clinical specimens, for comprehensive protein profiling which revealed distinct subgroups within basal-like and triple negative breast cancers. Specifically, within 88 TNBC samples, four proteomic clusters were evident, which had features of “basal-immune hot”, “basal-immune cold”, “mesenchymal”, and “luminal”, each with distinct clinical outcomes. This work sets a foundation for developing clinically-applicable tests that can tackle the diversity of basal-like and triple negative breast cancers using standard formalin-fixed pathology clinical trial materials. The findings presented may guide the selection of breast cancer patients for clinical trial evaluation of existing chemotherapy or emerging therapies.

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Sarcomas are aggressive cancers of the connective tissues, such as bone, muscle, cartilage, and fat. Despite their diverse origins, sarcomas are predominantly treated by surgery and radiation, as conventional chemotherapy has limited benefit for most subtypes. When sarcomas recur or metastasize, there are few options for systemic therapy, and prognosis is very poor. Despite advancements in our understanding of the molecular drivers of sarcomas, almost no new treatments have proven benefit for metastatic sarcomas. Immunotherapy has shown value for other cancers, such as melanoma and lung cancer; however, sarcomas lag behind the common cancers in our understanding of their immune microenvironment and potential for treatment with immunotherapeutics. Early trials using single-agent immune checkpoint inhibitors in sarcomas delivered mixed results, but these studies somewhat indiscriminately lumped together different sarcoma subtypes that might have critical immunological differences. My study employs tissue microarrays incorporating 1360 sarcoma specimens (spanning 23 subtypes) to characterize immune infiltrates and expression of targetable immune biomarkers, using immunohistochemistry. Genomically-complex sarcoma types – driven by mutations and/or copy-number alterations – are found to have much higher levels of lymphocytic and phagocytic immune infiltrates than translocation-associated sarcomas. Across nearly all subtypes, tumor-associated macrophages outnumber tumor-infiltrating lymphocytes, predominately M2 (anti-inflammatory) macrophages. Expression of the target of first-generation immune checkpoint PD-(L)1 is uniformly low, but expression of LAG-3 and TIM-3 – emerging immune checkpoints – is significantly more common. Expression of anti-phagocytic immune checkpoint CD47 is yet more predominant, displaying all-or-nothing expression with 100% positivity seen in over half of positive cases. To further characterize the lymphocytic response, T-cell receptor (TCR) sequencing was performed on specimens from 25 sarcoma patients on a clinical trial of tremelimumab (anti-CTLA-4) with durvalumab (anti-PD-L1). We found that the TCR repertoire is richer and more diverse among the genomically-complex sarcomas relative to the translocation-associated sarcomas, and following immune checkpoint blockade, we observed an overall increase in the clonality of the peripheral TCR repertoire. My study demonstrates a tangible positive relationship between genomic complexity and immunogenicity, and highlights novel immune checkpoints of relevance to sarcomas. As such, this work provides the essential translational background to direct the use of immunotherapy in sarcoma management.

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Immunotherapy is dramatically changing the landscape of cancer treatment and is becoming incorporated into the standard of care for some tumor types. Until recently, breast cancer has not been generally considered particularly immunogenic. However, breast cancer is a heterogeneous disease and increasing evidence suggests that patients with basal-like breast cancer, an aggressive subtype lacking targeted therapy options, may be amenable to immunotherapy. My research goals have included the investigation and clinical characterization of two emerging targetable immune checkpoint biomarkers: lymphocyte-activation gene 3 (LAG-3) and the T-cell Immunoglobulin and Mucin domain-containing molecule 3 (TIM-3), by applying immunohistochemistry to a well-annotated tissue microarray cohort of 3,992 breast cancers. As an additional research goal, I evaluated a novel in situ multiplex biomarker assessment method (Nanostring-based digital spatial profiling-DSP) for its compatibility with breast cancer tissue microarrays, to generate immune profiles from patient surgical specimens. I report that the expression of LAG-3 or TIM-3 on intra-epithelial tumor-infiltrating lymphocytes (iTILs) was observed in a minority of cases (11%) in the whole cohort, but was significantly enriched in basal-like breast cancers (33% and 28% of basal-like breast cancers being infiltrated with LAG-3+ and TIM-3+iTILs, respectively). Furthermore, I found that LAG-3+iTILs and TIM-3+iTILs were present in breast cancers co-infiltrated with established immunotherapy targets (program cell death-1/PD-1 and its ligand, PD-L1). In multivariate analyses, LAG-3+iTILs or TIM-3+iTILs were independent favorable prognostic factors in breast cancer patients. In the last part of the thesis, I profiled the tumor immune microenvironment of two basal-like-enriched breast cancer cohorts, quantifying the expression of 31 immuno-oncology biomarkers using DSP. I then validated the digital counts for CD8 and PD1 by comparing with immunohistochemistry, and CD45 digital counts by comparing with hematoxylin & eosin-stained stromal TILs counts. Lastly, I identified a 4-biomarker signature indicative of a pre-existing immunity in breast cancer patients. The body of work presented here may help guide the selection of breast cancer patients for clinical trial evaluation of emerging immunotherapy agents. Furthermore, I show that digital spatial profiling technology can efficiently and quantitatively profile immune expression on breast cancer patient specimens using only a tiny fraction from precious tumor samples.

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Myxoid liposarcoma is a malignant fatty tumour that develops in the deep soft tissue. While local control rates are good, current chemotherapy options remain ineffective against metastatic disease. Myxoid liposarcoma is characterized by a balanced translocation involving FUS (12q13) and DDIT3 (16p11). The resulting FUS-DDIT3 oncoprotein is proposed to function as an aberrant transcription factor but its exact mechanism of action has remained unclear, rendering it difficult to formulate rational strategies for targeted therapy. A current gap in knowledge behind the oncogenic functions of FUS-DDIT3 is the lack of comprehensive data on its interactome, which could identify the oncoprotein's key partners. This study utilized immunoprecipitation-mass spectrometry to identify the FUS-DDIT3 interactome in whole cell lysates of myxoid liposarcoma cells, and results showed an enrichment of RNA processing proteins. RNA-seq analysis was performed on myxoid liposarcoma cells after FUS-DDIT3 knockdown to look for changes in the alternative splicing profile, but no evidence of such changes was seen. Further TMT-labeled immunoprecipitation-mass spectrometry analyses in nuclear lysates of myxoid liposarcoma cells showed that members of several chromatin regulatory complexes were present in the FUS-DDIT3 interactome. These complexes included the NuRD, SWI/SNF, PRC1, PRC2 and MLL1 COMPASS-like complexes. Co-immunoprecipitation experiments validated the association of FUS-DDIT3 with BRG1/SMARCA4, BAF155/SMARCC1, BAF57/SMARCE1, HDAC2, KDM1A, and MTA1. Knockdown of FUS-DDIT3 also reduced H3K27ac levels at the promoter of a gene target, PTX3. Other sarcoma fusion oncoproteins have been reported to interact with chromatin regulators and affect histone modifications or chromatin remodeling as one of their oncogenic mechanisms. Data from this study suggests that FUS-DDIT3 may utilize a similar epigenetic mechanism of action, providing potential candidates for targeted therapy as epigenetic aberrations are potentially reversible by existing and emerging epigenetic drugs.

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Epithelioid sarcoma is a soft tissue tumor with an unusual predilection for the distal extremities in young adults. Despite wide-margin resections the 10-year survival is in the range of 50%. The biology of epithelioid sarcoma remains incompletely understood, but one key feature is the loss of SMARCB1. We use whole genome sequencing of five cases of epithelioid sarcoma matched to normal germline DNA, looking for mutations other than SMARCB1. These index cases are supplemented with three additional tumors and three cell lines that undergo whole transcriptome sequencing and are analyzed for somatic point mutations, copy number changes, translocations, and expression patterns. Unlike the situation in other SMARCB1 inactivated tumors, we find a complex genome with a relatively high mutational burden. However, aberrations of SMARCB1 remain the only consistent mutation. Some cases do not show biallelic DNA-level inactivation of this gene which leads us to examine other possible second-hit silencing mechanisms. With the significance attributed to SMARCB1 loss in the genomic landscape of epithelioid sarcoma, we explore two approaches, namely EZH2 and SMARCA4 inhibitions, to specifically exploit this abnormality and suggest novel therapeutic options.

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Synovial sarcoma is an aggressive soft tissue cancer affecting primarily adolescents and young adults and characterized by the known chimeric fusion oncoprotein SS18-SSX resulting from a t(X;18) translocation. Oncoprotein-mediated interactions with chromatin-modification proteins are known to influence transformation; however targeted therapies are not currently available. Patients remain at high-risk for local recurrence and metastasis and see a 10-year survival rate of approximately 50%. Accordingly, it was hypothesized that therapeutic compounds able to disrupt the SS18-SSX driving complex would be clinically effective against synovial sarcoma. The proximity ligation assay was employed to study the drug-mediated disruption of the key SS18-SSX-mediated protein interactions that drive sarcomagenesis, and was developed for use to demonstrate targeted drug efficacy. Results identified HDAC inhibitors as potent in disrupting the oncogenic protein complex. The combination of HDAC and proteasome inhibitors was further found to be synergistic against synovial sarcoma. This pre-clinical study was used as support for a novel clinical trial proposal for translocation-associated soft tissue sarcomas. The effects of HDAC inhibition on genome-wide transcription patterns was further assessed by RNA-seq analysis. Uniquely to synovial sarcoma, it was revealed that HDAC inhibition elicits loss of the SS18-SSX oncoprotein and provokes significant pro-apoptotic BIK expression correlating to CDKN2A reactivation. From this, the contributing role of CDKN2A reactivation and reactive oxygen species accumulation in HDAC-inhibitor mediated apoptosis in synovial sarcoma was demonstrated.

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Gene expression profiling of breast cancer delineates a particularly aggressive subtype referred to as “basal-like”, which comprises ~15% of all cases, afflicts younger women and is refractory to endocrine and anti-HER2 therapies. Immunohistochemical surrogate definitions for basal-like breast cancer, such as the ER/PR/HER2 triple negative phenotype and models incorporating positive expression of cytokeratin 5 (CK5/6) and/or epidermal growth factor receptor are more amenable to implementation in a clinical setting. Despite this and the fact that basal-like breast carcinomas are being increasingly recognized as a distinct clinical entity, there is no diagnostic method used and reported in routine practice. Without a reproducible test to identify this aggressive subtype in the clinic there will be no ability to establish clearly defined intake criteria for subtype-specific clinical trials, translating to no progress in the management of this form of the disease and little change in breast cancer survival rates for the foreseeable future.A first evaluation of performance of the triple negative definition and various surrogate immunopanels for basal-like breast cancer in clinical laboratories is described in the initial chapters of this dissertation. Considerable staining variability of individual biomarkers included in immunopanels typically led to only moderate concordance with a gene expression gold standard for identification of basal-like breast carcinomas. Lack of standardization was the underlying reason for all of the observed variability, supporting the notion that further standardization efforts through continual participation in external quality assurance programs are needed before routine diagnosis of basal-like breast carcinomas could be made in a clinical setting.In light of this, we sought to identify more easy-to-interpret and robust biomarkers for this disproportionately deadly type of breast cancer. A parallel comparison of 46 proposed immunohistochemical biomarkers of basal-like breast cancer was performed against a gene expression profile gold standard. Results from that survey determined that loss of expression of INPP4B and positive expression of nestin had the strongest associations with this aggressive subtype. Paving the way for further studies, this comprehensive immunohistochemical biomarker survey is a necessary step to determine an optimized surrogate immunopanel that best defines basal-like breast cancer in a practical and clinically-accessible way.

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Gene expression profiles have identified five major molecular breast cancer subtypes (Luminal A, Luminal B, Basal-like, HER2+/estrogen receptor− , and Normal Breast-like) that show significant differences in survival. The cost and complexity of gene expression technology has impeded its clinical implementation. By comparison, immunohistochemistry is an economical technique applicable to the standard formalin-fixed, paraffin-embedded material commonly used in hospital labs, and has the advantage of simultaneously interpretation with histomorphology. In this thesis, I hypothesize that a surrogate panel of immunohistochemical biomarkers can be developed to discriminate the breast cancer biological subtypes. The main study cohort consists of over 4000 primary invasive breast tumors, assembled into tissue microarrays. These patients were referred to the British Columbia Cancer Agency between 1986-1992 and have staging, pathology, treatment and follow-up information. In summary, our results demonstrate that (1) the rabbit monoclonal antibody, SP1, is an improved standard for immunohistochemiscal estrogen receptor assessment in breast cancer; (2) the transcription factor, GATA-3, is almost exclusively expressed among estrogen receptor positive tumors but does not seem to predict for tamoxifen response among estrogen receptor positive patients; (3) the proliferation marker, Ki-67, together with HER2 can segregate Luminal A from Luminal B subtypes, which carry distinct risks for breast cancer relapse and death; and (4) the inclusion of the basal markers EGFR and ck5/6 to “triple negative” breast cancers provides a more specific definition of basal-like breast cancer that better predicts patient survival. These results consistently demonstrate that an immunopanel of six biomarkers (estrogen receptor, progesterone receptor, HER2, Ki-67, epidermal growth factor receptor and cytokeratin 5/6) can be readily applied to standard pathology specimens to subtype breast cancer samples based on their underlying molecular biology. These findings have been considered sufficient to justify application of this panel onto NCIC (MA5, MA12) and CALGB (9341 and 9741) clinical trials specimens. This followup work which is underway and will determine if the six marker immunopanel can guide decisions about which patients need aggressive systemic drug treatment, and thereby ensure patients get the most effective, individualized adjuvant systemic therapy for their breast tumor.

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Synovial sarcoma is a soft tissue tumor defined by the presence of t(X;18)(p11.2;q11.2), fusing the SYT (SS18) gene on chromosome 18 and one of three SSX genes on chromosome X. T(X;18) results in production of a fusion protein (SYT-SSX) that is thought to underlie synovial sarcoma pathogenesis through aberrant targeting of both activating (trithorax, SWI/SNF) and repressing (Polycomb) transcription factors when expressed in a stem or progenitor-like cellular background.Clinically, synovial sarcomas present considerable diagnostic and therapeutic challenges. Whereas the classical biphasic histology is distinctive, the more common monophasic histology can be difficult to differentiate from other spindle cell tumors. In these situations, detection of t(X;18) is the gold standard for diagnosis, but it is a specialized and time-consuming process. Immunohistochemistry can be helpful, but no marker that is both highly sensitive and specific is available. Here I describe a fluorescence in situ hybridization based method employing an SYT break-apart probe set that can expedite detection of t(X;18). I also report that TLE1, which was identified in gene expression studies as a good discriminator of synovial sarcoma from other mesenchymal tumors, is a highly sensitive and specific immunohistochemical marker for synovial sarcoma. Both of these novel diagnostic techniques are applicable to small tissue samples such as core needle biopsies and are now being used clinically.The diagnosis of synovial sarcoma carries a poor prognosis and the 10-year overall survival rate is approximately 50%, most of whom are young adults. The addition of chemotherapy to surgical resection (the mainstay of treatment) does not appear to improve overall survival. Thus, there is a strong need for development of a clinically effective systemic therapy to improve patient outcome. I describe preclinical studies that demonstrate the Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) inhibits proliferation of synovial sarcoma by inducing apoptosis and that this is associated with degradation of multiple receptor tyrosine kinases and disruption of the SYT-SSX-β-catenin interaction. I also identify a subset of synovial sarcoma cells, typified by expression of CD133, which exhibit stem-like properties and are relatively resistant to doxorubicin but susceptible to 17-AAG at clinically relevant concentrations.

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