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Supplementary Data from Co-Targeting of MDM2 and CDK4/6 with Siremadlin and Ribociclib for the Treatment of Patients with Well-Differentiated or Dedifferentiated Liposarcoma: Results from a Proof-of-Concept, Phase Ib Study

Supplementary Data from Adjuvant Imatinib in Patients with GIST Harboring Exon 9 KIT Mutations: Results from a Multi-institutional European Retrospective Study

PDF file - 60K, Biological pathways overrepresented in "ARM" and internal trunk LMS

Complicated abdominal tissue response to pazopanib as fourth-line therapy in a woman with uterine PEComa. Panel A shows the baseline CT scan; panel B shows the complicated tissue response after 1 month of treatment.

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Supplementary Data from Lack of Prognostic Value of CTNNB1 Mutation Profile in Desmoid-Type Fibromatosis

Supplementary Figure 1F

Purpose: To analyze final long-term survival and clinical outcomes from the randomized phase III study of sunitinib in gastrointestinal stromal tumor patients after imatinib failure; to assess correlative angiogenesis biomarkers with patient outcomes.Experimental Design: Blinded sunitinib or placebo was given daily on a 4-week-on/2-week-off treatment schedule. Placebo-assigned patients could cross over to sunitinib at disease progression/study unblinding. Overall survival (OS) was analyzed using conventional statistical methods and the rank-preserving structural failure time (RPSFT) method to explore cross-over impact. Circulating levels of angiogenesis biomarkers were analyzed.Results: In total, 243 patients were randomized to receive sunitinib and 118 to placebo, 103 of whom crossed over to open-label sunitinib. Conventional statistical analysis showed that OS converged in the sunitinib and placebo arms (median 72.7 vs. 64.9 weeks; HR, 0.876; P = 0.306) as expected, given the cross-over design. RPSFT analysis estimated median OS for placebo of 39.0 weeks (HR, 0.505, 95% CI, 0.262–1.134; P = 0.306). No new safety concerns emerged with extended sunitinib treatment. No consistent associations were found between the pharmacodynamics of angiogenesis-related plasma proteins during sunitinib treatment and clinical outcome.Conclusions: The cross-over design provided evidence of sunitinib clinical benefit based on prolonged time to tumor progression during the double-blind phase of this trial. As expected, following cross-over, there was no statistical difference in OS. RPSFT analysis modeled the absence of cross-over, estimating a substantial sunitinib OS benefit relative to placebo. Long-term sunitinib treatment was tolerated without new adverse events. Clin Cancer Res; 18(11); 3170–9. ©2012 AACR.

Supplementary Figure 3

PDF file - 61K, Clinical characteristics of the molecular cohort (N=73)

PDF file - 60K, Biological pathways dysregulated in metastatic LMS

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Purpose:The European Organization for Research and Treatment of Cancer (EORTC) clinical phase II trial 90101 “CREATE” showed high antitumor activity of crizotinib, an inhibitor of anaplastic lymphoma kinase (ALK)/ROS1, in patients with advanced inflammatory myofibroblastic tumor (IMFT). However, recent findings suggested that other molecular targets in addition to ALK/ROS1 might also contribute to the sensitivity of this kinase inhibitor. We therefore performed an in-depth molecular characterization of archival IMFT tissue, collected from patients enrolled in this trial, with the aim to identify other molecular alterations that could play a role in the response to crizotinib.Experimental Design:Twenty-four archival IMFT samples were used for histopathological assessment and DNA/RNA evaluation to identify gene fusions, copy-number alterations (CNA), and mutations in the tumor tissue. Results were correlated with clinical parameters to assess a potential association between molecular findings and clinical outcomes.Results:We found 12 ALK fusions with 11 different partners in ALK-positive IMFT cases by Archer analysis whereas we did not identify any ROS1-rearranged tumor. One ALK-negative patient responding to crizotinib was found to have an ETV6–NTRK fusion in the tumor specimen. The CNA profile and mutational landscape of IMFT revealed extensive molecular heterogeneity. Loss of chromosome 19 (25% of cases) and PIK3CA mutations (9% of cases) were associated with shorter progression-free survival in patients receiving crizotinib.Conclusions:We identified multiple genetic alterations in archival IMFT material and provide further insight into the molecular profile of this ultra-rare, heterogeneous malignancy, which may potentially translate into novel treatment approaches for this orphan disease.

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Supplementary Figure from Lack of Prognostic Value of CTNNB1 Mutation Profile in Desmoid-Type Fibromatosis

PDF file, 60KB, Progression-free (panel A) and overall (panel B) survival according to the mitosis count. HPF: high power field.

PDF file - 74k, Complete Longitudinal Analyses of the Randomized, Placebo-controlled, Phase III Trial of Sunitinib in Patients with Gastrointestinal Stromal Tumor Following Imatinib Failure

Purpose: Altered expression of cell cycle/apoptosis key regulators may promote tumor progression, reflect secondary genetic/epigenetic events, and impair the effectiveness of therapy. Their expression pattern might then identify gastrointestinal stromal tumor (GIST) patient subgroups with different response to imatinib and elucidate novel therapeutic targets.Experimental Design: Immunohistochemical evaluation of expression of p53, p16, p21, CHK2, CCND1, BCL2, CDK4, and MDM2 was done on 353 histologically validated GIST patients enrolled into a European/Australasian phase III trial. TP53 was screened for mutations in cases with presumptive nonfunctional protein; that is, high p53 and low expression of the two downstream molecules p21 and MDM2. Results were correlated with clinicopathologic data, KIT/PDGFRA mutation status, and imatinib dosage.Results: Frequent impaired expression was found for BCL2 (78%), CHK2 (53%), p53 (50%), and p16 (47%). Stomach-originating GISTs showed significantly lower expression of p21, p16, and BCL2. KIT/PDGFRA wild-type GISTs had significant lower expression of CDK4. Eighty-eight percent of the high p53 expressers show low downstream target activation, indicating a nonfunctional p53 route. Of these high p53 expressers, 16.4% harbor a detectable TP53 mutation. Multivariate analysis, including previously identified markers, showed an independent effect of p53 and p16 on progression-free survival (PFS). Patients with high level of CHK2 and p21 showed significantly better PFS upon a high-dose regimen.Conclusions: Impaired p53, p16, BCL2, and CHK2 expression is common in advanced GISTs. Distinct patterns of expression correlate with tumor site, genotype, and PFS. Cell cycle/apoptosis maintenance is instrumental for optimal response to imatinib.

Purpose:Perivascular epitheliod cell tumors (PEComas) are rare mesenchymal neoplasms for which the role of systemic treatments is not established as there are no published prospective clinical trials or sufficiently large retrospective case series. The aim of this study is to clarify the activity of conventional chemotherapy and biological agents in advanced/metastatic PEComas.Experimental Design:This was an observational, retrospective, international study that included patients with advanced/metastatic PEComa treated with systemic therapy at 5 European sarcoma reference centers and within the Italian Rare Cancer Network. Survival analyses were performed using the Kaplan–Meier method and the Cox hazards regression models.Results:A total of 53 patients were included. Cytotoxic chemotherapy regimens were active only in a small proportion of PEComas. Gemcitabine-based regimens [objective response rate (ORR): 20%, median progression-free survival (PFS): 3.4 months] seemed to have the same activity of anthracycline-based regimens (ORR: 13%, median PFS: 3.2 months). Antiangiogenic agents resulted in disease stabilization in some patients, with a number having density changes/tissue response on imaging, with an ORR of 8.3% and a median PFS of 5.4 months. mTOR inhibitors were the most active agents, with an ORR of 41% and a median PFS of 9 months.Conclusions:Our study provides data for the selection of systemic therapy in patients with advanced/metastatic PEComa: mTOR inhibitors are the most active agents. Antiangiogenics and chemotherapy with gemcitabine-based regimens or anthracycline-based regimens are options in further line, but with a lower response rate and PFS.

Figure S1. Disease free survival grouped according to the presence of KIT ex. 11 mutations: KITdel-inc557/558 vs. other deletions vs. duplications vs. substitutions overall (p

Supplementary Data from Trabectedin plus Durvalumab in Patients with Advanced Pretreated Soft Tissue Sarcoma and Ovarian Carcinoma (TRAMUNE): An Open-Label, Multicenter Phase Ib Study

Supplementary Figure from Co-Targeting of MDM2 and CDK4/6 with Siremadlin and Ribociclib for the Treatment of Patients with Well-Differentiated or Dedifferentiated Liposarcoma: Results from a Proof-of-Concept, Phase Ib Study

PDF file - 60K, Biological pathways overrepresented in "REARRANGED" LMS and LMS of the extremities

Purpose:CDKN2A loss is frequent in gastrointestinal stromal tumors (GISTs) and associated with aggressive outcome. Palbociclib is a CDK4 inhibitor with preclinical antitumor efficacy in tumors with P16/CDKN2A loss.Patients and Methods:This is a multicenter single-arm phase II clinical trial assessing safety and efficacy of palbociclib in patients with advanced GIST bearing CDKN2A gene loss. Adults with unresectable locally advanced or metastatic, refractory to at least imatinib and sunitinib, measurable and documented progressive disease (PD) as per RECIST 1.1, and CDKN2A deletion centrally assessed were eligible. Patients received palbociclib 125 mg orally daily on a 21 days on/7 days off dosing schedule, until PD or unacceptable toxicity. The primary endpoint was 4-month non-PD rate according to RECIST 1.1.Results:As of May 2017, 71 patients had been included in the study, and 29 patients (40.3%) met the molecular eligibility requirement. Twenty-five patients (86.2%) had grade 1–2 adverse events (AEs) and 12 patients (41.4%) grade 3–4 AEs possibly related to the drug. The planned interim statistical analysis performed after central histologic and radiological review showed that 19 (86.4%) out of the first 22 evaluable patients had PD at 4 months. CDKN2A status had no impact either on overall survival or outcome on previous standard lines of treatment. Translational analysis suggested upregulation of CCNE1 or downregulation of CDKN1A/P21 or LRRC3B as potential mechanisms of resistance.Conclusions:Palbociclib has no significant clinical activity as a single agent in P16/CDKN2A–deleted GIST refractory to imatinib and sunitinib.

Purpose:The role of surgery for first relapse locally recurrent retroperitoneal sarcoma (RPS-LR1) is uncertain. We report outcomes of the largest RPS-LR1 series and propose a new prognostic nomogram.Experimental Design:Patients with consecutive RPS-LR1 without distant metastases who underwent resection at 22 centers (2002–2011) were included. Endpoints were disease-free and overall survival (DFS, OS) and crude-cumulative-incidence (CCI) of local/distant recurrence from second surgery. Nomograms predicting DFS and OS from second surgery were developed and validated (calibration plots); discrimination was assessed (Harrell C index).Results:Of 684 patients identified, full prognostic variable data were available for 602. Initial surgery for primary RPS was performed at our institutions in 188 patients (31%) and elsewhere in 414 (69%). At a median follow-up of 119 months [Interquartile range (IQR), 80–169] from initial surgery and 75 months (IQR 50–105) from second surgery, 6-year DFS and OS were 19.2% [95% confidence interval (CI), 16.0–23.0%] and 54.1% (95% CI, 49.8–58.8%), respectively. Recurrence patterns and survival probability were histology-specific, with liposarcoma subtypes having the highest 6-year CCI of second local recurrence (LR, 60.2%–70.9%) and leiomyosarcoma (LMS) having higher 6-year CCI of distant metastasis (DM, 36.3%). Nomograms included age at second surgery, multifocality, grade, completeness of second surgery, histology, chemotherapy/radiotherapy at first surgery, and number of organs resected at first surgery. OS and DFS nomograms showed good calibration and discriminative ability (C index 0.70 and 0.67, respectively).Conclusions:We developed nomograms to predict DFS and OS for patients undergoing RPS-LR1 resection. Nomograms provide individualized, disease-relevant estimations of survival for RPS-LR1 patients and assist in clinical decisions.

Purpose: Platelet-derived growth factor receptor-alpha (PDGFRA) mutations are found in approximately 5% to 7% of advanced gastrointestinal stromal tumors (GIST). We sought to extensively assess the activity of imatinib in this subgroup.Experimental Design: We conducted an international survey among GIST referral centers to collect clinical data on patients with advanced PDGFRA-mutant GISTs treated with imatinib for advanced disease.Results: Fifty-eight patients were included, 34 were male (59%), and median age at treatment initiation was 61 (range, 19–83) years. The primary tumor was gastric in 40 cases (69%). Thirty-two patients (55%) had PDGFRA-D842V substitutions whereas 17 (29%) had mutations affecting other codons of exon 18, and nine patients (16%) had mutation in other exons. Fifty-seven patients were evaluable for response, two (4%) had a complete response, eight (14%) had a partial response, and 23 (40%) had stable disease. None of 31 evaluable patients with D842V substitution had a response, whereas 21 of 31 (68%) had progression as their best response. Median progression-free survival was 2.8 [95% confidence interval (CI), 2.6–3.2] months for patients with D842V substitution and 28.5 months (95% CI, 5.4–51.6) for patients with other PDGFRA mutations. With 46 months of follow-up, median overall survival was 14.7 months for patients with D842V substitutions and was not reached for patients with non-D842V mutations.Conclusions: This study is the largest reported to date on patients with advanced PDGFRA-mutant GISTs treated with imatinib. Our data confirm that imatinib has little efficacy in the subgroup of patients with D842V substitution in exon 18, whereas other mutations appear to be sensitive to imatinib. Clin Cancer Res; 18(16); 4458–64. ©2012 AACR.

Purpose:Trabectedin has shown preclinical synergy with immune checkpoint inhibitors in preclinical models.Patients and Methods:TRAMUNE is a phase Ib study investigating the combination of trabectedin with durvalumab through a dose escalation phase and two expansion cohorts, soft tissue sarcoma (STS) and ovarian carcinoma. Trabectedin was given at three dose levels (1 mg/m2, 1.2 mg/m2, and 1.5 mg/m2) on day 1, in combination with durvalumab, 1,120 mg on day 2, every 3 weeks. The primary endpoints were the recommended phase II dose (RP2D) of trabectedin combined with durvalumab and the objective response rate (ORR) as per RECIST 1.1. The secondary endpoints included safety, 6-month progression-free rate (PFR), progression-free survival (PFS), overall survival, and biomarker analyses.Results:A total of 40 patients were included (dose escalation, n = 9; STS cohort, n = 16; ovarian carcinoma cohort, n = 15, 80% platinum resistant/refractory). The most frequent toxicities were grade 1–2 fatigue, nausea, neutropenia, and alanine/aspartate aminotransferase increase. One patient experienced a dose-limiting toxicity at dose level 2. Trabectedin at 1.2 mg/m2 was selected as the RP2D. In the STS cohort, 43% of patients experienced tumor shrinkage, the ORR was 7% [95% confidence interval (CI), 0.2–33.9], and the 6-month PFR was 28.6% (95% CI, 8.4–58.1). In the ovarian carcinoma cohort, 43% of patients experienced tumor shrinkage, the ORR was 21.4% (95% CI, 4.7–50.8), and the 6-month PFR was 42.9% (95% CI, 17.7–71.1). Baseline levels of programmed death-ligand 1 expression and CD8-positive T-cell infiltrates were associated with PFS in patients with ovarian carcinoma.Conclusions:Combining trabectedin and durvalumab is manageable. Promising activity is observed in patients with platinum-refractory ovarian carcinoma.See related commentary by Digklia et al., p. 1745

Specific agents used

PDF file - 52K, Kaplan-Meier curves of overall survival according to age (A), tumor size (B), tumor location (C) and tumor grade (D)

Immunohistochemical analysis of FOXP3 in primary breast tumors showed that a high number of tumor-infiltrating regulatory T cells (Ti-Treg) within lymphoid infiltrates surrounding the tumor was predictive of relapse and death, in contrast to those present within the tumor bed. Ex vivo analysis showed that these tumor-infiltrating FOXP3+ T cells are typical Treg based on their CD4+CD25highCD127lowFOXP3+ phenotype, their anergic state on in vitro stimulation, and their suppressive functions. These Ti-Treg could be selectively recruited through CCR4 as illustrated by (a) selective blood Treg CCR4 expression and migration to CCR4 ligands, (b) CCR4 down-regulation on Ti-Treg, and (c) correlation between Ti-Treg in lymphoid infiltrates and intratumoral CCL22 expression. Importantly, in contrast to other T cells, Ti-Treg are selectively activated locally and proliferate in situ, showing T-cell receptor engagement and suggesting specific recognition of tumor-associated antigens (TAA). Immunohistochemical stainings for ICOS, Ki67, and DC-LAMP show that Ti-Treg were close to mature DC-LAMP+ dendritic cells (DC) in lymphoid infiltrates but not in tumor bed and were activated and proliferating. Furthermore, proximity between Ti-Treg, CD3+, and CD8+ T cells was documented within lymphoid infiltrates. Altogether, these results show that Treg are selectively recruited within lymphoid infiltrates and activated by mature DC likely through TAA presentation, resulting in the prevention of effector T-cell activation, immune escape, and ultimately tumor progression. This study sheds new light on Treg physiology and validates CCR4/CCL22 and ICOS as therapeutic targets in breast tumors, which represent a major health problem. [Cancer Res 2009;69(5):2000–9]

Supplementary Text - PDF file 132K, Supplementary Material and methods, and Supplementary figure legends

Supplementary Figure Legend. Legends for Supplementary Figures S1-S6.

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PDF file, 67K, Identification of pDC and mDC in blood and tissues.

PDF file, 64K, Breast TUMSN, but not BCCSN, inhibit IFN-α production by CpGA- or Flu-activated pDC in a dose-dependent manner.

Supplementary Data from Regulatory T Cells Recruited through CCL22/CCR4 Are Selectively Activated in Lymphoid Infiltrates Surrounding Primary Breast Tumors and Lead to an Adverse Clinical Outcome

PDF file, 46K, Purified healthy pDC were cultured with IL-3 either alone or in combination with 25% of TUMSN for 40 h and then incubated for 5 days with allogeneic naive CD4+ T cells.

Supplementary Figures 1-4 from Quantitative and Functional Alterations of Plasmacytoid Dendritic Cells Contribute to Immune Tolerance in Ovarian Cancer

PDF file - 197K, Supplemental Table 1. Characteristics of GIST patients for the immunohistochemistry study. Supplemental Table 2. Characteristics of GIST and STS patients studied in flow cytometry. Supplemental Table 3: Antibodies for flow cytometry and IHC. Supplemental Figure 1. Correlation between T and NK cell infiltrates and the 3-digit Miettinen score. Supplemental Figure 2. GIST TIls are mainly composed of CD4+ T cells.

PDF file, 114K, Overall survival and relapse-free survival of patients according to the presence of CD123+ pDC in primary breast carcinoma.

Supplementary Figures S1-S6. S1: Sensitivity to IGF�1R inhibitor AZ12253801 is influenced by levels of DVL3 protein but not IGF�1R or INSR S2: Effects of HUNK depletion on IGF signalling S3: Sensitivity to IGF�1R inhibition is enhanced by proximal but not distal WNT pathway inhibition S4: Investigating effects of DVL3 on IGF�induced ERK activation in prostate and breast cancer cells S5: ERK activation induced by DVL inhibition is independent of EGFR and IRS�1, and involves complex formation with adaptor proteins Grb2, DAB2 and SOS S6: DVL3 immunohistochemistry

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Supplementary Tables 1-2 from Quantitative and Functional Alterations of Plasmacytoid Dendritic Cells Contribute to Immune Tolerance in Ovarian Cancer

Supplementary Methods and References. Description of additional methods and procedures used in the study. Also includes supplementary references.

Supplementary Tables S1-S8. S1: Antibodies used for western blotting, immunoprecipitation and immunofluorescence S2: siRNA libraries and individual siRNAs used in this study S3: Sequences of qPCR primers S4: Z' values in primary siRNA screens S5: Candidate resistance mediators tested in second round screens S6: Validation of screen hits S7: DVL3 expression and clinical parameters in breast and prostate cancer S8:DVL3 IPS on tumor tissue from 22 patients recruited to trials of IGF-1R antibodies

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PDF file, 768K, IHC analysis on frozen BT sections was performed using anti-BDCA2 (brown) together with anti-CD3 (green, a) or anti- cytokeratine (green, b) antibodies (x10)

Supplementary Figures - PDF file 322K, S1. Spontaneous MMTVneu display high Her2/neu expression. S2. FoxP3 expression among CD4+ CD25+ T cells correlates with CD45RB negativity. S3. NEU15WT TApDC display unaltered uptake ability. S4. NEU15WT TApDC display unaltered TLR7 and TLR9 expression. S5. Fold changes in gene expression upon intratumoral TLR7L injection. S6. Intratumoral TLR7L or CpG injection induced distinct immune cell recruitment. S7. List of flurochrome-conjugated antibodies used for multi-parametric flow cytometry analysis

PDF file - 296K, Figure S1: ICOS is mainly expressed by TA-Treg. Figure S2: pDC expanded Treg sustain suppressive activity. Figure S3: ICOS-L is highly expressed on R848-activated HD-pDC. Figure S4: ICOS blockade reduces Treg but not Tconv proliferation induced by pDC without impacting mDC/T cell activation. Figure S5: Strong IL-10 secretion in culture of BT single cell suspension. Figure S6: Correlation between TA-pDC and TA-Treg percentages in fresh primary BT and in retrospective IHC cohort

PDF file - 103K, Table S1: Clinical characteristic of the cohort of BT patients enrolled for TMA analysis (n=120) Table S2: clones of mAbs used for the multi-parametric flow cytometry analyses