Supplemental Figure 3. Selected BTM geneset enrichments (FDR < 0,25) of SD (patient 002) and PD patients (004 and 008) at tumor site. Gene names of enriched genesets are publicly available online in supplemental data for original paper23.

Supplemental Table 1. Quantitative Immunohistochemistry (IHC). In the patient 002 with clinical benefit (stable disease, right two columns), IHC analysis of tumor showed increased CD4 (60x), CD8 (10x), PD1 (20x) and PDL1 (3x). In patients with disease (001, 004, 005, 008), quantitative IHC showed unchanged or decreased levels of CD4, CD8, PD-1, and PDL-1 over treatment periods. Individual antigens were quantified using the CRI Inform software (Perkin Elmer) which applies user-directed antigen thresholds to generate percentages normalized to the total tumor area.

Supplemental Figure 2. Hierarchical clustering of total gene expression of PBMC and tumor RNA samples. Only genes commonly expressed in PBMC and tumor samples are shown. Of note, up-regulation of specific genes at tumor site may come from tumor rather immune cells. On x-axis, each column represents patient at specific treatment time point, as numbered below. For example, 002- C1W1 is patient 002 at time point Cycle 1 week1. 1 - 002_Screen 12 - 004_Screen 2 - 002_C1W1 13 - 004_C1W2 3 - 002_C1W3 14 - 008_Screen 4 - 002_C1W7 15 - 008_C1W1 5 - 002_C1W10 16 - 008_C1W3 6 - 002_C2W11 17 - 008_C1W7 7 - 002_C2W12 18 - 008_C1W10 8 - 002_C2W13 9 - 002_C2W17 10 - 002_C2W20 11 - 002_C2W26

Supplemental Figure 4. Selected Broad Institute C7 immune geneset enrichments (FDR < 0,25). Gene names of enriched genesets are publicly available online at MSigDB: http://software.broadinstitute.org/gsea/msigdb/index.jsp.

Supplemental Table 2. List of genes, forming gene signature sets shown at Figure 6.

Supplemental Figure 1. Enrichments of Broad collection of immunological gene signatures in PBMC of SD (patient 002) and PD patients (patients 004 and 008). (A) non-supervised hierarchical clustering of selected gene signatures (FDR < 0,25). Gene names of enriched genesets are publicly available online at MSigDB: http://software.broadinstitute.org/gsea/msigdb/index.jsp. (B) Comparison of genesets enrichment scores between SD and PD patients. Positive values indicate enrichment in SD patient, while negative values - enrichment in PD patients. Enrichments were calculated based on outputs of GSEA analysis. X-axis: [Signal(SD) - Signal(PD)] / Signal(SD), where Signal is enrichment signal strength of geneset in patient 002 with SD response. Y-axis: [NES(SD) - NES(PD)] / NES(SD), where NES(SD) is normalized enrichment score of geneset in patient 002.

Abstract Purpose: Polyinosinic-polycytidylic acid-poly-l-lysine carboxymethylcellulose (poly-ICLC), a synthetic double-stranded RNA complex, is a ligand for toll-like receptor-3 and MDA-5 that can activate immune cells, such as dendritic cells, and trigger natural killer cells to kill tumor cells. Patients and Methods: In this pilot study, eligible patients included those with recurrent metastatic disease in whom prior systemic therapy (head and neck squamous cell cancer and melanoma) failed. Patients received 2 treatment cycles, each cycle consisting of 1 mg poly-ICLC 3× weekly intratumorally (IT) for 2 weeks followed by intramuscular (IM) boosters biweekly for 7 weeks, with a 1-week rest period. Immune response was evaluated by immunohistochemistry (IHC) and RNA sequencing (RNA-seq) in tumor and blood. Results: Two patients completed 2 cycles of IT treatments, and 1 achieved clinical benefit (stable disease, progression-free survival 6 months), whereas the remainder had progressive disease. Poly-ICLC was well tolerated, with principal side effects of fatigue and inflammation at injection site ( Conclusions: Poly-ICLC was well tolerated in patients with solid cancer and generated local and systemic immune responses, as evident in the patient achieving clinical benefit. These results warrant further investigation and are currently being explored in a multicenter phase II clinical trial (NCT02423863). Clin Cancer Res; 24(20); 4937–48. ©2018 AACR.

e14010 Background: Polyinosinic-polycytidylic acid (poly-ICLC) is stabilized double stranded RNA comprising carboxymethylcellulose, polyinosinic-polycytidylic acid, and poly-L-lysine RNA. Poly-ICLC is a ligand for toll like receptor 3 (TLR3) expressed on dendritic cells and is under clinical development for the treatment of viral infections and tumors. Pilot studies in pediatric brain cancer have shown efficacy.1 Poly-ICLC can be given intra-muscularly (IM) to induce systemic inflammation and/or intratumorally (IT) to induce immune infiltration of tumor. Methods: Three patients are currently enrolled on this study. The planned sample size is 15. Eligible patients include those ≥18 years old with unresectable melanoma, squamous cell cancer of the head and neck, breast cancer, squamous cell cancer of the skin or basal cell cancer. Patients with sarcoma are also eligible and must be ≥14 years old. Patients must have an ECOG performance status of 2 or better, a life expectancy of 6 months and must have progressed on prior systemic therapy or despite local irradiation. Brain metastases are allowable. 1 mg Poly-ICLC is administered IT three times a week for two weeks followed by biweekly IM injection for 7 weeks and 1 week of rest. If stable disease or better is observed, this 10 week cycle will be repeated and if there is continuous response than a maintenance cycle of IM poly-ICLC will be offered. The primary objectives of the study are to determine safety and evaluate therapeutic effect as assessed by progression free survival at 26 weeks. The secondary objectives are to determine whether injected and/or non-injected lesions will regress and to assess immunogenicity of this poly-ICLC regimen. Results: Not yet available. Conclusions: Not yet available. Clinical Trial Information: NCT01984892 1. Hartman LL, Crawford JR, Makale MT, et al. Pediatric Phase II Trials of Poly-ICLC in the Management of Newly Diagnosed and Recurrent Brain Tumors. Journal of pediatric hematology/oncology 2013. Clinical trial information: NCT01984892.

Rigosertib (ON 01910.Na), a synthetic novel benzyl styryl sulfone, was administered to 28 patients with advanced cancer in a Phase I trial in order to characterize its pharmacokinetic profile, determine the dose-limiting toxicities (DLT), define the recommended phase II dose (RPTD) and to document any antitumor activity. Patients with advanced malignant neoplasms refractory to standard therapy were given escalating doses of rigosertib (50, 100, 150, 250, 325, 400, 650, 850, 1,050, 1,375, 1,700 mg/m2/24h) as a 3-day continuous infusion (CI) every 2 weeks. An accelerated Fibonacci titration schedule with specified decreases for toxicities was used for escalation until grade ≥2 toxicity occurred. Intrapatient dose escalation was allowed if toxicity was grade ≤2 and the disease remained stable. Plasma pharmacokinetics (PK) and urinary PK assessments were studied in the 1st and 4th cycles. Twenty-nine patients (12 men and 17 women; age 36-87 y with a median of 63 y) were registered, but one died before study drug was given. Twenty-eight patients received a median of 3 cycles of therapy. Most common grade ≥2 toxicities attributable to rigosertib included fatigue, anorexia, vomiting and constipation. DLTs included muscular weakness, hyponatremia, neutropenia, delirium and confusional state. Risk factors for severe toxicities include preexisting neurological dysfunction or advanced gynecologic cancer after pelvic surgery. Rigosertib pharmacokinetics showed rapid plasma distribution phases and urinary excretion. Elevations in plasma Cmax and AUC due to decreases in plasma clearance were associated with acute grade ≥3 toxicities. Of 22 evaluable patients, 9 (41%) achieved a best overall response of stable disease; all other patients (n=13; 59%) progressed. The median progression-free survival time was 50 days (95% confidence interval [CI]: 37-80 days). Nine (41%) patients survived for over 1 y. In summary, prolonged IV infusions of rigosertib were generally well tolerated. Nine (41%) patients achieved stable disease and 9 (41%) patients survived for over 1 year. The RPTD appears to be 850 mg/m2/24hr CI x 3 days. (ClinicalTrials.gov identifier: NCT01538537).

Rigosertib (ON 01910.Na), a synthetic novel benzyl styryl sulfone, was administered to 28 patients with advanced cancer in a Phase I trial in order to characterize its pharmacokinetic profile, determine the dose-limiting toxicities (DLT), define the recommended phase II dose (RPTD) and to document any antitumor activity. Patients with advanced malignant neoplasms refractory to standard therapy were given escalating doses of rigosertib (50, 100, 150, 250, 325, 400, 650, 850, 1,050, 1,375, 1,700 mg/m(2)/24h) as a 3-day continuous infusion (CI) every 2 weeks. An accelerated Fibonacci titration schedule with specified decreases for toxicities was used for escalation until grade ≥2 toxicity occurred. Intrapatient dose escalation was allowed if toxicity was grade ≤2 and the disease remained stable. Plasma pharmacokinetics (PK) and urinary PK assessments were studied in the 1st and 4th cycles. Twenty-nine patients (12 men and 17 women; age 36-87 y with a median of 63 y) were registered, but one died before study drug was given. Twenty-eight patients received a median of 3 cycles of therapy. Most common grade ≥2 toxicities attributable to rigosertib included fatigue, anorexia, vomiting and constipation. DLTs included muscular weakness, hyponatremia, neutropenia, delirium and confusional state. Risk factors for severe toxicities include pre-existing neurological dysfunction or advanced gynecologic cancer after pelvic surgery. Rigosertib pharmacokinetics showed rapid plasma distribution phases and urinary excretion. Elevations in plasma Cmax and AUC due to decreases in plasma clearance were associated with acute grade ≥3 toxicities. Of 22 evaluable patients, 9 (41%) achieved a best overall response of stable disease; all other patients (n=13; 59%) progressed. The median progression-free survival time was 50 days (95% confidence interval [CI]: 37-80 days). Nine (41%) patients survived for over 1 y. In summary, prolonged IV infusions of rigosertib were generally well tolerated. Nine (41%) patients achieved stable disease and 9 (41%) patients survived for over 1 year. The RPTD appears to be 850 mg/m(2)/24hr CI x 3 days. (ClinicalTrials.gov identifier: NCT01538537).

e13584 Background: ON 01910.Na is a novel multitargeted inhibitor of several regulatory pathways including polo-like kinase 1 (Plk1) and PI-3 kinases, with synergistic nonclinical activity when combined with oxaliplatin, prompting phase I clinical evaluation. Methods: Patients with advanced cancer received escalating doses of ON 01910.Na as 24hr weekly continuous infusions. Oxaliplatin was administered biweekly as a 85mg/m2, 2 hr infusion. The ON 01910.Na dose cohorts ranged from 250-1,350 mg/m2 based on a modified Fibonacci escalation algorithm using the accelerated titration scheme (NCI). Intrapatient dose escalation was allowed. Results: Thirty patients (pts) (22 females; ages: 29-80 yrs received an overall mean 9.4 (range=1-30) number of weeks of combination therapy: ON 01910.Na mg/m2: 250 n=10; 650 n=9; 1,050 n=8; 1,350 n=3. No dose-limiting toxicity was observed in any patient. Grade 3+ toxicities included pain (3 pts), lethargy and weakness (1 pt), pneumonia (1 pt), hyponatremia (1pt), anemia (1pt), transient ischemic attack (1pt), vomiting (1pt), urinary tract infection (1pt), shortness of breath and myocardial infarction (1pt). Confirmed partial response was observed in 1 pt with chemotherapy- refractory ovarian cancer (duration = 11 wks) and in another pt with metastatic breast cancer (duration = 13 wks). Stable disease was observed in 4 pts with colon cancer (42 wks and 12 wks), ovarian and renal cancer (12 wks). Conclusions: ON 01910.Na administered as a 24hr infusion in combination with oxaliplatin was well tolerated and resulted in objective tumor responses in advanced ovarian, breast, colon and renal cancer.

OBJECTIVE:To define the safety [dose limiting toxicity (DLT)] and recommended phase II dose of the combination of sorafenib plus gemcitabine and capecitabine for advanced renal cell carcinoma (RCC).METHODS:In this phase I dose-escalation study, cohorts of 3 to 6 patients with metastatic RCC received sorafenib (200 or 400 mg po BID), gemcitabine (750 or 1000 mg/m(2) intravenous on days 1 and 8), and capecitabine (415 or 622 mg/m(2) po BID days 1-14) every 21 days using a standard 3+3 design.RESULTS:Fifteen patients with advanced RCC (93% with clear cell histology and 87% treatment naive) received treatment. The recommended phase II doses for the combination were sorafenib 200 mg/m(2) BID continuously plus gemcitabine 750 mg/m(2) intravenous days 1 and 8 and capecitabine 415 mg/m(2) BID days 1 to 14, every 21 days. Of the 15 patients, 3 developed dose-limiting hand-foot syndrome during the first 2 cycles; 2 additional DLT's were grade 3 mucositis and transaminase elevation. Four of 14 evaluable patients had a partial response by response evaluation criteria in solid tumors (29%; 95% confidence interval (CI): 8, 58%). Median progression-free survival was 7.5 months (95% CI-0, 18.7), and median overall survival has not been reached at a median follow-up of 28.8 months. The median number of treatment cycles given was 7 (range, 2-38+).CONCLUSIONS:The combination of sorafenib plus gemcitabine and capecitabine is tolerable, but requires attenuation of sorafenib and capecitabine dosing because of the overlapping toxicity of hand-foot syndrome. Antitumor activity was observed leading to an ongoing phase II trial.

PURPOSE:To determine the clinical and biologic effects of bevacizumab, an anti-vascular endothelial growth factor (VEGF) monoclonal antibody, in unresectable hepatocellular carcinoma (HCC).PATIENTS AND METHODS:Adults with organ-confined HCC, Eastern Cooperative Oncology Group performance status of 0 to 2, and compensated liver disease were eligible. Patients received bevacizumab 5 mg/kg (n = 12) or 10 mg/kg (n = 34) every 2 weeks until disease progression or treatment-limiting toxicity. The primary objective was to determine whether bevacizumab improved the 6-month progression-free survival (PFS) rate from 40% to 60%. Secondary end points included determining the effects of bevacizumab on arterial enhancement and on plasma cytokine levels and the capacity of patients' plasma to support angiogenesis via an in vitro assay.RESULTS:The study included 46 patients, of whom six had objective responses (13%; 95% CI, 3% to 23%), and 65% were progression free at 6 months. Median PFS time was 6.9 months (95% CI, 6.5 to 9.1 months); overall survival rate was 53% at 1 year, 28% at 2 years, and 23% at 3 years. Grade 3 to 4 adverse events included hypertension (15%) and thrombosis (6%, including 4% with arterial thrombosis). Grade 3 or higher hemorrhage occurred in 11% of patients, including one fatal variceal bleed. Bevacizumab was associated with significant reductions in tumor enhancement by dynamic contrast-enhanced magnetic resonance imaging and reductions in circulating VEGF-A and stromal-derived factor-1 levels. Functional angiogenic activity was associated with VEGF-A levels in patient plasma.CONCLUSION:We observed significant clinical and biologic activity for bevacizumab in nonmetastatic HCC and achieved the primary study end point. Serious bleeding complications occurred in 11% of patients. Further evaluation is warranted in carefully selected patients.

16023 Background: While RCC is often considered chemo-resistant, the combination of GEM and 5-FU or CAP has activity. S improves progression free survival and is well-tolerated, but has limited response rates and patients (pts) progress at a median of 6 months. We chose to study the combination of S plus GEM and CAP. Methods: Cohorts of 3–6 pts were treated with S, GEM and CAP to establish dose limiting toxicity (DLT) and the maximum tolerated dose (MTD). After 2 of the initial 4 pts developed DLT (grade 3 hand/foot), the protocol was amended and the initial CAP dose was lowered. A subsequent amendment allowed one prior anti-angiogenic therapy. Results: 15 pts (median age 56) were enrolled with the following characteristics: 7 ECOG PS 0, 4 PS 1, 4 PS 2; 5 MSK good risk, 6 intermediate, and 4 poor risk; 14 of 15 clear cell variant (3 with sarcomatoid differentiation), 1 papillary variant; 40% with liver involvement and 40% with bone metastases; 13 pts were treatment naïve, 1 progressed on cytokines and 1 on sunitinib. Two of 5 pts experienced DLT at dose level B including one gr 3 ALT and one gr 3 mucositis. The MTD for the combination is S 200 mg/m2 BID d1–21, GEM 750 mg/m2 d1&8, and CAP 415 mg/m2 BID D1–14, with only 1 of 6 pts experiencing a DLT at this dose level (gr 3 hand/foot). Grade 3/4 toxicities were as follows: neutropenia 3 (20%), anemia 1, thrombocytopenia 1, hand/foot 3 (20%), fatigue 2 (13%), ALT 1, hypoalbuminemia 1, abdominal pain 1, pneumonia 1, proteinuria 1, thrombosis (catheter related) 1. Of 14 pts evaluable for response by RECIST, 4 with PR, 8 with SD, and 2 PD. Twelve of 14 had some improvement in measurable disease. 5 pts received at least 10 cycles; 4 phase I pts remain on therapy. Median progression free survival was 39.0 weeks [95% CI 20.2, 57.8] and median overall survival was 97.4 weeks [52.1, 142.8]. Conclusions: The combination of S plus GEM and CAP is tolerated with encouraging anti-tumor activity seen. The phase II component is open at the MTD to pts who have received up to 1 previous anti-angiogenic therapy and accrual is ongoing. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration Pfizer Pfizer, sanofi-aventis

PURPOSE:To evaluate thalidomide in advanced hepatocellular carcinoma (HCC) and to evaluate combined thalidomide and low-dose interferon-alpha2a (IFN-alpha2a) after tumor progression on thalidomide. Systemic therapy is minimally effective in HCC and tumor angiogenesis is a potential therapeutic target.PATIENTS AND METHODS:Patients with unresectable HCC were eligible if they had preserved hepatic and renal function. The initial thalidomide dosage was 200 mg daily and was adjusted for toxicity. Upon progression, patients could continue thalidomide with additional low-dosage (one million units twice daily) IFN-alpha2a.RESULTS:Thirty-eight enrolled patients were predominantly hepatitis C virus infected (53%), Child-Pugh class A (79%), and Eastern Cooperative Oncology Group performance status 0-1 (92%); 60% had extrahepatic metastasis. Confirmed disease control was seen in seven patients (18%) and included one complete and one partial response (5% response rate). The median progression-free survival was 2.1 months, and median overall survival was 5.5 months. Tumor invasion of the portal vein or vena cava, large (>10 cm) tumor, and younger age were associated with shorter overall survival. Toxicity included fatigue in 74% of patients. Six patients stopped therapy because of side effects, including two patients (5%) with grade 4 arteriothrombotic events. Five patients continued thalidomide upon progression with the addition of IFN-alpha2a; there was no disease control and 80% had grade 3 toxicity.CONCLUSIONS:Thalidomide is not well tolerated and confers limited disease control in advanced HCC. Combination thalidomide and low-dose IFN-alpha2a is neither safe nor efficacious in this population.

4144 Background: HCC is a vascular tumor in which angiogenesis contributes to pathogenesis. VEGF-inhibition has not been previously attempted in cirrhosis or HCC. Methods: We enrolled patients with unresectable HCC to treatment with bevacizumab at 5 mg/kg or 10 mg/kg every 14 days to assess safety and preliminary efficacy. Inclusion criteria were BR < 3.0 mg/dl, transaminases 75K and PT within 4 seconds of normal. Additional criteria included no history of myocardial infarction, stroke, or condition requiring ongoing antiplatelet or antithrombotic therapy. Results: Initial safety assessments for patients treated for at least 8 weeks indicate that 10 of 11 patients treated at 5 mg/kg and 7 of 8 patients treated at 10mg/kg tolerated therapy with acceptable toxicity. Of the first 28 patients treated at either dose, four have had therapy discontinued because of adverse events (including one grade 3 transient ischemic attack). Three patients had serious esophageal bleeding which was likely secondary to progression of HCC and liver disease (although contribution of drug to these events cannot be excluded). The protocol has been modified to identify and treat esophageal varices prior to enrollment. Other toxicities have included grade 2 fatigue, abdominal pain, gastric ulcer, hypertension, hyperbilirubinemia, rash and proteinuria and grade 1 gingivitis, diarrhea, vomiting and epistaxis. Of the initial 25 patients evaluable for efficacy, 5 had progressive disease within 16 weeks. Two patients had PR and 18 had SD. Median time-to-progression was 6.5 months (range 3.9–24.2). Nineteen of 25 (76%) patients had HCC that progressed following transplant, surgery, RFA or embolization procedures. Conclusions: Bevacizumab can be given safely at both 5 mg/kg and 10 mg/kg in HCC patients with localized, unresectable HCC, preserved liver function and no significant esophageal varices. Preliminary results suggest significant disease-modifying effect (80% disease-control rate); median time-to-progression to-date has exceeded 6-months. [Table: see text]

Twenty consecutive patients with unresectable, locally advanced pancreatic cancer were treated with split courses Of radiotherapy (RT) and simultaneous multidrug chemotherapy consisting of 5 fluorouracil, continuous infusion, streptozotocin, and cisplatin. A separate, retrospective study identified a group of 28 contemporary patients with less advanced pancreatic cancers, all of which were successfully resected. The survival rate of the two groups were similar over the first 2 years, although it initially favored the unresectable group. This pattern of survival among patients treated with combined modality therapy provides a basis for new studies. At the two clinical extremes, these include treatment of unresectable tumors previously considered ineligible for this treatment and initial treatment before resection of stage I tumors.