Molecular patterns of resistance to immune checkpoint blockade in melanoma.

Immune checkpoint blockade (ICB) has improved outcome for patients with metastatic melanoma but not all benefit from treatment. Several immune- and tumor intrinsic features are associated with clinical response at baseline. However, we need to further understand the molecular changes occurring during development of ICB resistance. Here, we collect biopsies from a cohort of 44 patients with melanoma after progression on anti-CTLA4 or anti-PD1 monotherapy. Genetic alterations of antigen presentation and interferon gamma signaling pathways are observed in approximately 25% of ICB resistant cases. Anti-CTLA4 resistant lesions have a sustained immune response, including immune-regulatory features, as suggested by multiplex spatial and T cell receptor (TCR) clonality analyses. One anti-PD1 resistant lesion harbors a distinct immune cell niche, however, anti-PD1 resistant tumors are generally immune poor with non-expanded TCR clones. Such immune poor microenvironments are associated with melanoma cells having a de-differentiated phenotype lacking expression of MHC-I molecules. In addition, anti-PD1 resistant tumors have reduced fractions of PD1+ CD8+ T cells as compared to ICB naïve metastases. Collectively, these data show the complexity of ICB resistance and highlight differences between anti-CTLA4 and anti-PD1 resistance that may underlie differential clinical outcomes of therapy sequence and combination.

Oncogenic translation directs spliceosome dynamics revealing an integral role for SF3A3 in breast cancer.

Splicing is a central RNA-based process commonly altered in human cancers; however, how spliceosomal components are co-opted during tumorigenesis remains poorly defined. Here we unravel the core splice factor SF3A3 at the nexus of a translation-based program that rewires splicing during malignant transformation. Upon MYC hyperactivation, SF3A3 levels are modulated translationally through an RNA stem-loop in an eIF3D-dependent manner. This ensures accurate splicing of mRNAs enriched for mitochondrial regulators. Altered SF3A3 translation leads to metabolic reprogramming and stem-like properties that fuel MYC tumorigenic potential in vivo. Our analysis reveals that SF3A3 protein levels predict molecular and phenotypic features of aggressive human breast cancers. These findings unveil a post-transcriptional interplay between splicing and translation that governs critical facets of MYC-driven oncogenesis.

Distinct mechanisms of resistance to fulvestrant treatment dictate level of ER independence and selective response to CDK inhibitors in metastatic breast cancer.

BACKGROUND: Resistance to endocrine treatment in metastatic breast cancer is a major clinical challenge. Clinical tools to predict both drug resistance and possible treatment combination approaches to overcome it are lacking. This unmet need is mainly due to the heterogeneity underlying both the mechanisms involved in resistance development and breast cancer itself. METHODS: To study the complexity of the mechanisms involved in the resistance to the selective estrogen receptor degrader (SERD) fulvestrant, we performed comprehensive biomarker analyses using several in vitro models that recapitulate the heterogeneity of developed resistance. We further corroborated our findings in tissue samples from patients treated with fulvestrant. RESULTS: We found that different in vitro models of fulvestrant resistance show variable stability in their phenotypes, which corresponded with distinct genomic alterations. Notably, the studied models presented adaptation at different cell cycle nodes to facilitate progression through the cell cycle and responded differently to CDK inhibitors. Cyclin E2 overexpression was identified as a biomarker of a persistent fulvestrant-resistant phenotype. Comparison of pre- and post-treatment paired tumor biopsies from patients treated with fulvestrant revealed an upregulation of cyclin E2 upon development of resistance. Moreover, overexpression of this cyclin was found to be a prognostic factor determining resistance to fulvestrant and shorter progression-free survival. CONCLUSIONS: These data highlight the complexity of estrogen receptor positive breast cancer and suggest that the development of diverse resistance mechanisms dictate levels of ER independence and potentially cross-resistance to CDK inhibitors.

SOX2 is a promising predictor of relapse and death in advanced stage high-grade serous ovarian cancer patients with residual disease after debulking surgery.

The transcription factor SOX2 is a well-established and important stem cell marker. Its role in cancer biology remains unclear, but it has been proposed to also be a marker of cancer stem cells. We investigated the role of SOX2 protein expression in women with high-grade serous ovarian cancer (HGSOC) to determine its potential prognostic and treatment predictive value. We constructed a tissue microarray of 130 advanced stage HGSOC tumors with an average of 6 cores each, stained for SOX2 protein expression and evaluated survival outcomes. We also treated two HGSOC cell lines with carboplatin and paclitaxel and measured SOX2 expression by RT-PCR and immunoblotting at different doses and time-points. Among patients with non-radical debulking surgery overall and progression-free survival were shorter for patients with SOX2 positive tumors (mean 26 vs. 39 months, log-rank test: p = .0076, and mean 14 vs. 19 months, p = .055, respectively). Knockdown of SOX2 in cell lines did not affect growth inhibition following chemotherapy treatment. Our results show that SOX2 has a strong prognostic potential among HGSOC patients with residual tumor tissue after debulking surgery and suggest that SOX2 expressing cells remaining after non-radical debulking surgery may constitute a subpopulation of cancer stem cells with greater tumor-initiating potential.

Extracellular lipid loading augments hypoxic paracrine signaling and promotes glioma angiogenesis and macrophage infiltration.

BACKGROUND: Primary brain tumors, in particular glioblastoma (GBM), remain among the most challenging cancers. Like most malignant tumors, GBM is characterized by hypoxic stress that triggers paracrine, adaptive responses, such as angiogenesis and macrophage recruitment, rescuing cancer cells from metabolic catastrophe and conventional oncological treatments. The unmet need of strategies to efficiently target tumor "stressness" represents a strong clinical motivation to better understand the underlying mechanisms of stress adaptation. Here, we have investigated how lipid loading may be involved in the paracrine crosstalk between cancer cells and the stromal compartment of the hypoxic tumor microenvironment. METHODS: Regions from patient GBM tumors with or without the lipid loaded phenotype were isolated by laser capture microdissection and subjected to comparative gene expression analysis in parallel with cultured GBM cells with or without lipid loading. The potential involvement of extracellular lipids in the paracrine crosstalk with stromal cells was studied by immunoprofiling of the secretome and functional studies in vitro as well as in various orthotopic GBM mouse models, including hyperlipidemic ApoE-/- mice. Statistical analyses of quantitative experimental methodologies were performed using unpaired Student's T test. For survival analyses of mouse experiments, log-rank test was used, whereas Kaplan-Meier was performed to analyze patient survival. RESULTS: We show that the lipid loaded niche of GBM patient tumors exhibits an amplified hypoxic response and that the acquisition of extracellular lipids by GBM cells can reinforce paracrine activation of stromal cells and immune cells. At the functional level, we show that lipid loading augments the secretion of e.g. VEGF and HGF, and may potentiate the cross-activation of endothelial cells and macrophages. In line with these data, in vivo studies suggest that combined local tumor lipid loading and systemic hyperlipidemia of ApoE-/- mice receiving a high fat diet induces tumor vascularization and macrophage recruitment, and was shown to significantly decrease animal survival. CONCLUSIONS: Together, these data identify extracellular lipid loading as a potentially targetable modulator of the paracrine adaptive response in the hypoxic tumor niche and suggest the contribution of the distinct lipid loaded phenotype in shaping the glioma microenvironment.

Claudin-4 Expression is Associated With Survival in Ovarian Cancer But Not With Chemotherapy Response.

The tight junction protein claudin-4 has been reported to be overexpressed in advanced ovarian cancer. We investigated the prognostic significance of claudin-4 overexpression and whether claudin-4 expression could predict platinum response in primary ovarian carcinoma (OC). Claudin-4 expression was evaluated by immunohistochemistry in a tissue microarray of 140 OCs. Multivariable Cox-regression models were used to assess the effect of claudin-4 overexpression on progression-free survival and overall survival (OS). Kaplan-Meier survival analyses and the logrank test were performed comparing claudin-4 high and low groups. The association between claudin-4 expression and platinum resistance was assessed using risk ratios and the Pearson χ test. A dataset of >1500 epithelial ovarian cancers was used to study the association between CLDN4 mRNA and survival. Of 140 evaluable cases, 71 (51%) displayed high claudin-4 expression. Claudin-4 overexpression predicted shorter 5-yr progression-free survival and OS in univariable analyses [hazard ratio (HR)=1.6 (1.1-2.5), P=0.020 and HR=1.6 (1.0-2.4), P=0.041, respectively]. Hazard of relapse was similar [HR=1.5 (1.0-2.4)] after adjustment for age, stage, type, and BRCA1/2 status in a multivariable analysis, but the evidence was slightly weaker (P=0.076). Validation in an external cohort confirmed the association between high expression of CLDN4 and poor 10-yr OS [HR=1.3 (1.1-1.5), P<0.001]. However, no confident association between claudin-4 and platinum sensitivity was found in our cohort [risk ratio=1.2 (0.7-2.0), P=0.3]. These findings suggest that high expression of claudin-4 may have a prognostic value in OC. The role of claudin-4 in the development of platinum resistance remains unclear.

Involvement of Chromatin Remodeling Genes and the Rho GTPases RhoB and CDC42 in Ovarian Clear Cell Carcinoma.

OBJECTIVE: Ovarian clear cell carcinomas (OCCCs) constitute a rare ovarian cancer subtype with distinct clinical features, but may nonetheless be difficult to distinguish morphologically from other subtypes. There is limited knowledge of genetic events driving OCCC tumorigenesis beyond ARID1A, which is reportedly mutated in 30-50% of OCCCs. We aimed to further characterize OCCCs by combined global transcriptional profiling and targeted deep sequencing of a panel of well-established cancer genes. Increased knowledge of OCCC-specific genetic aberrations may help in guiding development of targeted treatments and ultimately improve patient outcome. METHODS: Gene expression profiling of formalin-fixed, paraffin-embedded (FFPE) tissue from a cohort of the major ovarian cancer subtypes (cohort 1; n = 67) was performed using whole-genome cDNA-mediated Annealing, Selection, extension and Ligation (WG-DASL) bead arrays, followed by pathway, gene module score, and gene ontology analyses, respectively. A second FFPE cohort of 10 primary OCCCs was analyzed by targeted DNA sequencing of a panel of 60 cancer-related genes (cohort 2). Non-synonymous and non-sense variants affecting single-nucleotide variations and insertions or deletions were further analyzed. A tissue microarray of 43 OCCCs (cohort 3) was used for validation by immunohistochemistry and chromogenic in situ hybridization. RESULTS: Gene expression analyses revealed a distinct OCCC profile compared to other histological subtypes, with, e.g., ERBB2, TFAP2A, and genes related to cytoskeletal actin regulation being overexpressed in OCCC. ERBB2 was, however, not overexpressed on the protein level and ERBB2 amplification was rare in the validation cohort. Targeted deep sequencing revealed non-synonymous variants or insertions/deletions in 11/60 cancer-related genes. Genes involved in chromatin remodeling, including ARID1A, SPOP, and KMT2D were frequently mutated across OCCC tumors. CONCLUSION: OCCCs appear genetically heterogeneous, but harbor frequent alterations in chromatin remodeling genes. Overexpression of TFAP2A and ERBB2 was observed on the mRNA level in relation to other ovarian cancer subtypes. However, overexpression of ERBB2 was not reflected by HER2 amplification or protein overexpression in the OCCC validation cohort. In addition, Rho GTPase-dependent actin organization may also play a role in OCCC pathogenesis and warrants further investigation. The distinct biological features of OCCC discovered here may provide a basis for novel targeted treatment strategies.

Increased gene copy number of KIT and VEGFR2 at 4q12 in primary breast cancer is related to an aggressive phenotype and impaired prognosis.

Triple-negative breast cancer (TNBC) is associated with poor prognosis and no targeted treatments are available for TNBC. Drugs inhibiting tyrosine kinases, such as vascular endothelial growth factor receptor 2 (VEGFR2) and KIT, have shown some promising results for patients with TNBC. The aim of the study was to investigate whether gains and/or amplifications of VEGFR2 and KIT, located at 4q12, occur in TNBC. Fluorescence in situ hybridization (FISH) was used to quantify gene copy numbers of VEGFR2 and KIT in 83 primary human breast cancers including 31 TNBCs. Gains were defined as ≥ 4 gene copies in >40% of the cancer cells, whereas amplification was defined as CEP >2 in more than 10% of the cancer cells. A tumor was considered FISH positive for KIT and/or VEGFR2 if it displayed copy number gain and/or amplification. Ten (32%) of the TNBCs were VEGFR2 FISH positive and nine (29%) were KIT FISH positive, whereas non-TNBCs were FISH positive for VEGFR2 and KIT in nine (18%) cases for both genes, but no significant difference between TNBCs and non-TNBCs was found. FISH positivity for VEGFR2 and KIT was significantly correlated (χ(2) test, P < 0.001), and significantly related to ER negativity and high Nottingham histological grade (NHG). A significantly worse 5-year breast cancer specific survival (BCSS) was seen for FISH positive cases. Increased copy number of VEGFR2 and KIT thus has the potential of functioning as a novel predictive biomarker for selected targeted therapy particularly in the difficult-to-treat TNBC patient category.

Laser capture microdissection (LCM) and whole genome amplification (WGA) of DNA from normal breast tissue --- optimization for genome wide array analyses.

BACKGROUND: Laser capture microdissection (LCM) can be applied to tissues where cells of interest are distinguishable from surrounding cell populations. Here, we have optimized LCM for fresh frozen normal breast tissue where large amounts of fat can cause problems during microdissection. Since the amount of DNA needed for genome wide analyses, such as single nucleotide polymorphism (SNP) arrays, is often greater than what can be obtained from the dissected tissue, we have compared three different whole genome amplification (WGA) kits for amplification of DNA from LCM material. In addition, the genome wide profiling methods commonly used today require extremely high DNA quality compared to PCR based techniques and DNA quality is thus critical for successful downstream analyses. FINDINGS: We found that by using FrameSlides without glass backing for LCM and treating the slides with acetone after staining, the problems caused by excessive fat could be significantly decreased. The amount of DNA obtained after extraction from LCM tissue was not sufficient for direct SNP array analysis in our material. However, the two WGA kits based on Phi29 polymerase technology (Repli-g® (Qiagen) and GenomiPhi (GE Healthcare)) gave relatively long amplification products, and amplified DNA from Repli-g® gave call rates in the subsequent SNP analysis close to those from non-amplified DNA. Furthermore, the quality of the input DNA for WGA was found to be essential for successful SNP array results and initial DNA fragmentation problems could be reduced by switching from a regular halogen lamp to a VIS-LED lamp during LCM. CONCLUSIONS: LCM must be optimized to work satisfactorily in difficult tissues. We describe a work flow for fresh frozen normal breast tissue where fat is inclined to cause problems if sample treatment is not adapted to this tissue. We also show that the Phi29-based Repli-g® WGA kit (Qiagen) is a feasible approach to amplify DNA of high quality prior to genome wide analyses such as SNP profiling.