XLS file 30K, Accumulation of chromosomal aberrations in stepwise generated cell lines.

Supplementary Table S1 from WDR19 Expression is Increased in Prostate Cancer Compared with Normal Cells, but Low-Intensity Expression in Cancers is Associated with Shorter Time to Biochemical Failures and Local Recurrence

Links to supplementary video clips (VCS1+VCS2) and description of supplementary optical datasets (ODS1+ODS2)

Correlation between copy number status determined by FISH and microarrays.

PDF file - 59 KB, Overview of patient characteristics for the three cohorts collected and treated at Haukeland University Hospital, Bergen, Norway

Supplementary Tables 1-4 from Increased Expression of SIM2-s Protein Is a Novel Marker of Aggressive Prostate Cancer

PDF file - 68 KB, Subset of endometrioid cases, retrospective population based series. Cox' proportional hazard regression model used to estimate the prognostic value of ER in relation to traditional prognostic markers in endometrial carcinoma.

PDF file 659K, Supplementary Materials and Methods, Figures,and Tables.

Metastatic castration-resistant prostate cancer (mCRPC) is an immunologically cold disease with dismal outcomes. Cryoablation destroys cancer tissue, releases tumor-associated antigens and creates a pro-inflammatory microenvironment, while dendritic cells (DCs) activate immune responses through processing of antigens. Immunotherapy combinations could enhance the anti-tumor efficacy. This open-label, single-arm, single-center phase I trial determined the safety and tolerability of combining cryoablation and autologous immature DC, without and with checkpoint inhibitors. Immune responses and clinical outcomes were evaluated. Patients with mCRPC, confirmed metastases and intact prostate gland were included. The first participants underwent prostate cryoablation with intratumoral injection of autologous DCs in a 3 + 3 design. In the second part, patients received cryoablation, the highest acceptable DC dose, and checkpoint inhibition with either ipilimumab or pembrolizumab. Sequentially collected information on adverse events, quality of life, blood values and images were analyzed by standard descriptive statistics. Neither dose-limiting toxicities nor adverse events > grade 3 were observed in the 18 participants. Results indicate antitumor activity through altered T cell receptor repertoires, and 33% durable (> 46 weeks) clinical benefit with median 40.7 months overall survival. Post-treatment pain and fatigue were associated with circulating tumor cell (CTC) presence at inclusion, while CTC responses correlated with clinical outcomes. This trial demonstrates that cryoimmunotherapy in mCRPC is safe and well tolerated, also for the highest DC dose (2.0 × 10 8 ) combined with checkpoint inhibitors. Further studies focusing on the biologic indications of antitumor activity and immune system activation could be considered through a phase II trial focusing on treatment responses and immunologic biomarkers.

PDF file - 84K, Gene Set Enrichment Analysis (www.broadinstitute.org/gsea/msigdb). Gene sets (GO categories, computational gene sets and curated gene sets) within FDR<10% related to cell cycle/cell proliferation and being enriched in tumors with high level of pStathmin(S38).

PDF - 254K, Supplemental Figure 1: In vivo proliferation of angiogenic and non-angiogenic cell clones. Supplemental Figure 2: Chromosomal locations of genes with relative overexpression in either angiogenic or revertant clones. Supplemental Figure 3: Canonical pathways overrepresented in angiogenic and revertant clones.

PDF file - 43 KB, Clustering of transcriptional data in relation to ER and ESR1 expression

Conventional type 1 dendritic cells (cDC1) and conventional type 2 dendritic cells (cDC2) have attracted increasing attention as alternatives to monocyte-derived dendritic cells (moDCs) in cancer immunotherapy. Use of cDCs for therapy has been hindered by their low numbers in peripheral blood. In the present study, we found that extensive spontaneous apoptosis and cDC death in culture within 24hrs represent an additional challenge. Different media conditions that maintain cDC viability and function were investigated. CD141+ cDC1 and CD1c+ cDC2 were isolated from healthy blood donor buffy coats. Low viabilities were found with CellGenix DC, RPMI-1640, and X-VIVO 15 standard culture media and with several supplements at 24hrs and 48hrs. Among multiple factors it was found that GM-CSF improved both cDC1 and cDC2 viability, whereas Flt3-L and IL-4 only increased viability of cDC1 and cDC2, respectively. Combinations of these three cytokines improved viability of both cDCs further, both at 24hrs and 48hrs time points. Although these cytokines have been extensively investigated for their role in myeloid cell differentiation, and are also used clinically, their effects on mature cDCs remain incompletely known, in particular effects on pro-inflammatory or tolerogenic cDC features. HLA-DR, CD80, CD83, CD86, PD-L1 and PD-L2 cDC membrane expressions were relatively little affected by GM-CSF, IL-4 and Flt3-L cytokine supplements compared to the strong induction following Toll-like receptor (TLR) stimulation for 24hrs. With minor exceptions the three cytokines appeared to be permissive to the TLR-induced marker expression. Allogeneic mixed leukocyte reaction showed that the cytokines promoted T-cell proliferation and revealed a potential to boost both Th1 and Th2 polarizing cytokines. GM-CSF and Flt3-L and their combination improved the capability of cDC1 for dextran uptake, while in cDC2, dextran capture was improved by GM-CSF. The data suggest that GM-CSF, IL-4 and Flt3-L and combinations might be beneficial for DC viability and function in vitro. Limited viability of cDCs could be a confounding variable experimentally and in immunotherapy.

PDF file - 40K, Unsupervised hierarchical clustering of gene expression data from endometrial carcinoma. The pStathmin(S38) high cases are significantly overrepresented in two of the clusters with aggressive tumors.

PDF file - 69 KB, Comparing clinic-pathologic data in the different cohorts; the primary investigation series, prospective validation series and the external microarray data set are compared to the retrospective population based series.

XLS file 2928K, Changed gene expression in D5HS comparing to EPT2-D5 cells

e14708 Background: Despite improvements, metastatic castration-resistant prostate cancer (mCRPC) still has dismal overall survival (OS). We treated 18 mCRPC patients with dendritic cell (DC)-based cryoimmunotherapy (CryoIT) as monotherapy or combined with checkpoint inhibitors to determine safety and immune response. Methods: mCRPC patients were biopsied before cryoablation, and subsequently injected intratumorally with autologous immature DCs. DC doses were escalated in a 3+3 design (n=9) followed by dose expansion combined with either ipilimumab (n=6) or pembrolizumab (n=3). The disease burden was assessed by PSA values, iRECISTv1.1 (PET/CT, MRI and bone scintigraphy at baseline, 14, 22 and 46 weeks) and circulating tumor cells (CTC; CellSearch). Immune response was monitored by ultradeep T-cell receptor sequencing. Results: The patients had aggressive cancer, with ISUP grade group 4 and 5 in 72% (13/18) at diagnosis and in 100% at inclusion. At baseline, higher ALP correlated with higher regulatory T cells (p=0.047), FoxP3+/CD3+ratios (p=0.014) and FoxP3+/CD8+ratios (p=0.004) in tissues. Median OS and progression-free survival (PFS) were 40.7 and 10.5 months, respectively. Of the 8 patients alive 43 to 85 months post-CryoIT, 7 had progressed and received other treatment. Early decreases in PSA (p=0.002) and LDH (p=0.01) levels 6 weeks after CryoIT indicated better outcomes at 22 weeks. Longer OS correlated with lower tissue ratios of CD4+/CD3+cells (p=0.002) and CD4+/CD8+cells (p=0.007). All patients with increased numbers of new T-cell clonotypes after CryoIT and CTC at the pretreatment level of 1-4 cells/ 7.5 mL blood changed to negative during follow-up up to 72 weeks. Conclusions: CryoIT demonstrates non-inferior OS and PFS compared to other mCRPC trials, and therapy response was suggested by decreased CTCs and induction of new T-cell clonotypes post-CryoIT. Lower CD4 + cell fractions of lymphocytes in the TME indicate better OS after CryoIT. CryoIT appears not to decrease effects of post-treatment recurrence therapies. Clinical trial information: NCT02423928 . [Table: see text]

PDF file - 132 KB, Selection of single genes (Tab A) and transcription factor gene sets (Tab B) differentially expressed between ER negative and ER positive endometrial carcinomas (primary investigation set).

PDF file - 50K, Validations series. Cox's proportional hazard regression model used to estimate the prognostic value of pStathmin(S38) in endometrial carcinomas, in relation to histopathologic variables and Stathmin.

Supplementary Figure 2 Legend from Increased Expression of SIM2-s Protein Is a Novel Marker of Aggressive Prostate Cancer