Supplementary Tables 1-2, Figures 1-4. Supplementary Table 1: List of assay primer sets used in this study. Supplementary Table 2. Table of co-regulation among the 14 genes using a Kendall tau analysis. Supplementary Figure 1: Forest plots showing associations of gene expression levels with OS in KRAS WT (A) and KRAS Mut (B) pts. All assayed genes are shown. Supplementary Figure 2: Forest plots showing associations of gene expression levels with PFS in KRAS WT (A) and KRAS Mut (B) pts. Supplementary Figure 3: Forest plots showing associations of gene expression levels and treatment group with OS in KRAS WT (A) and KRAS Mut (B) pts. Supplementary Figure 4: Forest plots showing associations of gene expression levels and treatment group with PFS in KRAS WT (A) and KRAS Mut (B) pts. All assayed genes are shown.

Figure S1, consort diagram. Table S1, pts characteristics. Table S2, baseline levels. Table S3, prognostic markers. Table S4, adjusted prognostic markers. Table S5, early changes. Table S6, late changes at disease progression.

Both transcriptomics and metabolomics hold promise for identifying acute coronary syndrome (ACS) but they have not been used in combination, nor have dynamic changes in levels been assessed as a diagnostic tool. We assessed integrated analysis of peripheral blood miRNA and metabolite analytes to distinguish patients with myocardial ischemia on cardiac stress testing. We isolated and quantified miRNA and metabolites before and after stress testing from seven patients with myocardial ischemia and 1:1 matched controls. The combined miRNA and metabolomic data were analyzed jointly in a supervised, dimension-reducing discriminant analysis. We implemented a baseline model (T0) and a stress-delta model. This novel integrative analysis of the baseline levels of metabolites and miRNA expression showed modest performance for distinguishing cases from controls. The stress-delta model showed worse performance. This pilot study shows potential for an integrated precision medicine approach to cardiac stress testing.

Abstract Purpose: CALGB 80405 compared the combination of first-line chemotherapy with cetuximab or bevacizumab in the treatment of advanced or metastatic colorectal cancer (mCRC). Although similar clinical outcomes were observed in the cetuximab-chemotherapy group and the bevacizumab-chemotherapy group, biomarkers could identify patients deriving more benefit from either biologic agent. Patients and Methods: In this exploratory analysis, the Angiome, a panel of 24 soluble protein biomarkers were measured in baseline plasma samples in CALGB 80405. Prognostic biomarkers were determined using univariate Cox proportional hazards models. Predictive biomarkers were identified using multivariable Cox regression models including interaction between biomarker level and treatment. Results: In the total population, high plasma levels of Ang-2, CD73, HGF, ICAM-1, IL6, OPN, TIMP-1, TSP-2, VCAM-1, and VEGF-R3 were identified as prognostic of worse progression-free survival (PFS) and overall survival (OS). PlGF was identified as predictive of lack of PFS benefit from bevacizumab [bevacizumab HR, 1.51; 95% confidence interval (CI), 1.10–2.06; cetuximab HR, 0.94; 95% CI, 0.71–1.25; Pinteraction = 0.0298] in the combined FOLFIRI/FOLFOX regimens. High levels of VEGF-D were predictive of lack of PFS benefit from bevacizumab in patients receiving FOLFOX regimen only (FOLFOX/bevacizumab HR, 1.70; 95% CI, 1.19–2.42; FOLFOX/cetuximab HR, 0.92; 95% CI, 0.68–1.24; Pinteraction = 0.0097). Conclusions: In this exploratory, hypothesis-generating analysis, the Angiome identified multiple prognostic biomarkers and two potential predictive biomarkers for patients with mCRC enrolled in CALGB 80405. PlGF and VEGF-D predicted lack of benefit from bevacizumab in a chemo-dependent manner. See related commentaries by Mishkin and Kohn, p. 2722 and George and Bertagnolli, p. 2725

Trial Information ClinicalTrials.gov Identifier: Sponsor: Duke University Principal Investigator: John H. Strickler IRB Approved: Yes Lessons Learned Antitumor activity was observed in the study population. Dose modifications of cabozantinib improve long-term tolerability. Biomarkers are needed to identify patient populations most likely to benefit. Further study of cabozantinib with or without panitumumab in patients with metastatic colorectal cancer is warranted. Background The epidermal growth factor receptor (EGFR) antibody panitumumab is active in patients with RAS wild-type (WT) metastatic colorectal cancer (mCRC), but nearly all patients experience resistance. MET amplification is a driver of panitumumab resistance. Cabozantinib is an inhibitor of multiple kinases, including vascular endothelial growth factor receptor 2 (VEGFR2) and c-MET, and may delay or reverse anti-EGFR resistance. Methods In this phase Ib clinical trial, we established the maximum tolerated dose (MTD) and recommended phase II dose (RP2D) of cabozantinib and panitumumab. We then treated an expansion cohort to further describe the tolerability and clinical activity of the RP2D. Eligibility included patients with KRAS WT mCRC (later amended to include only RAS WT mCRC) who had received prior treatment with a fluoropyrimidine, oxaliplatin, irinotecan, and bevacizumab. Results Twenty-five patients were enrolled and treated. The MTD/RP2D was cabozantinib 60 mg p.o. daily and panitumumab 6 mg/kg I.V. every 2 weeks. The objective response rate (ORR) was 16%. Median progression free survival (PFS) was 3.7 months (90% confidence interval [CI], 2.3-7.1). Median overall survival (OS) was 12.1 months (90% CI, 7.5-14.3). Five patients (20%) discontinued treatment due to toxicity, and 18 patients (72%) required a dose reduction of cabozantinib. Conclusion The combination of cabozantinib and panitumumab has activity. Dose reductions of cabozantinib improve tolerability.

The radiologic appearance of locally advanced lung cancer may be linked to molecular changes of the disease during treatment, but characteristics of this phenomenon are poorly understood. Radiomics, liquid biopsy of cell-free DNA (cfDNA), and next-generation sequencing of circulating tumor DNA (ctDNA) encode tumor-specific radiogenomic expression patterns that can be probed to study this problem. Preliminary findings are reported from a radiogenomic analysis of CT imaging, cfDNA, and ctDNA in 24 patients (median age, 64 years; range, 49-74 years) with stage III lung cancer undergoing chemoradiation on a prospective pilot study (NCT00921739) between September 2009 and September 2014. Unsupervised clustering of radiomic signatures resulted in two clusters that were associated with ctDNA TP53 mutations (P = .03) and changes in cfDNA concentration after 2 weeks of chemoradiation (P = .02). The radiomic features dissimilarity (hazard ratio [HR] = 0.56; P = .05), joint entropy (HR = 0.56; P = .04), sum entropy (HR = 0.53; P = .02), and normalized inverse difference (HR = 1.77; P = .05) were associated with overall survival. These results suggest heterogeneous and low-attenuating disease without a detectable ctDNA TP53 mutation was associated with early surges of cfDNA concentration in response to therapy and a generally better prognosis. Keywords: CT-Quantitative, Radiation Therapy, Lung, Computer Applications-3D, Oncology, Tumor Response, Outcomes Analysis Clinical trial registration no. NCT00921739 Supplemental material is available for this article. © RSNA, 2021.

Abstract Regorafenib is a tyrosine kinase inhibitor approved by the FDA for the treatment of patients with chemotherapy refractory metastatic colorectal cancer (mCRC). Regorafenib inhibits signaling through multiple receptors associated with angiogenesis, metastasis, and tumor immunity. Here, we report biomarker results from LCCC1029, a randomized, placebo-controlled, phase II trial of chemotherapy ± regorafenib in patients with second-line mCRC. A panel of 20 soluble protein biomarkers (termed the Angiome) was assessed in the plasma of 149 patients from the LCCC1029 trial both at baseline and along the treatment continuum. Baseline protein levels were analyzed for prognostic and predictive value for progression-free survival (PFS) and overall survival (OS). Changes in protein levels during treatment were analyzed for potential pharmacodynamic effects. Six markers (HGF, IL6, PlGF, VEGF-R1, OPN, and IL6R) were found to be prognostic for PFS. Nine markers (IL6, TIMP-1, PlGF, VCAM-1, ICAM-1, OPN, TSP-2, HGF, and VEGF-R1) were prognostic for OS. Higher baseline levels of OPN (Pintx = 0.0167), VCAM-1 (Pintx = 0.0216), and PDGF-AA (Pintx = 0.0435) appeared to predict for PFS benefit from regorafenib compared with placebo. VCAM-1 was also potentially predictive of OS benefit from regorafenib compared with placebo (Pintx = 0.0124). On-treatment changes of six markers reflected potential on-target effect of regorafenib. Consistent results were observed in an Italian cohort where 105 patients with late-stage mCRC received regorafenib monotherapy. The key findings of this study suggest that VCAM-1 may be a predictive biomarker for regorafenib benefit, while multiple protein markers may be prognostic of outcome in patients with mCRC.

PURPOSE:Concurrent chemoradiation therapy (CRT) is the principal treatment modality for locally advanced lung cancer. Cell death due to CRT leads to the release of cell-free DNA (cfDNA) and circulating tumor DNA (ctDNA) into the bloodstream, but the kinetics and characteristics of this process are poorly understood. We hypothesized that there could be clinically meaningful changes in cfDNA and ctDNA during a course of CRT for lung cancer. METHODS AND MATERIALS:Multiple samples of plasma were obtained from 24 patients treated with CRT for locally advanced lung cancer to a mean dose of 66 Gy (range, 58-74 Gy) at the following intervals: before CRT, at weeks 2 and 5 during CRT, and 6 weeks after treatment. cfDNA was quantified, and a novel next generation sequencing (NGS) technique using enhanced tagged/targeted-amplicon sequencing was performed to analyze ctDNA. RESULTS:Patients for whom specific mutations in ctDNA were undetectable at the baseline time point had improved survival, and potentially etiologic driver mutations could be tracked throughout the course of CRT via NGS in multiple patients. We quantified the levels of cfDNA from patients before CRT, at week 2, week 5, and at 6 weeks after treatment. No differences were observed at weeks 2 and 5 of therapy, but we noted a significant increase in cfDNA in the posttreatment follow-up samples compared with samples collected before CRT (P = .05). CONCLUSIONS:Dynamic changes in both cfDNA and ctDNA were observed throughout the course of CRT in patients with locally advanced lung cancer. Specific mutations with therapeutic implications can be identified and tracked using NGS methodologies. Further work is required to characterize the changes in cfDNA and ctDNA over time in patients treated with CRT and to assess the predictive and prognostic potential of this powerful technology.

Background Patients with chemotherapy refractory metastatic colorectal cancer (CRC) have a poor prognosis and limited therapeutic options. In this phase Ib/II clinical trial, we established the maximum tolerated dose (MTD) and recommended phase II dose (RPTD) for the combination of capecitabine and ziv-aflibercept, and then we evaluated the efficacy of the combination in patients with chemotherapy refractory metastatic CRC. Methods All patients were required to have a Karnofsky Performance Status > 70% and adequate organ function. The phase Ib dose escalation cohort included patients with advanced solid tumors who had progressed on all standard therapies. Using a standard 3 + 3 design, we identified the MTD and RPTD for the combination. Fifty patients with metastatic CRC who had progressed on or were intolerant of a fluoropyrimidine, oxaliplatin, irinotecan, and bevacizumab were then enrolled in a single-arm phase II expansion cohort, and were treated at the RPTD. Prior EGFR antibody therapy was required for subjects with RAS wildtype tumors. The primary endpoint for the expansion cohort was progression-free survival (PFS) at two months. Secondary endpoints included objective response rate (ORR) and overall survival (OS). Results A total of 63 patients were enrolled and evaluable for toxicity (13 dose escalation; 50 expansion). The MTD and RPTD were: capecitabine 850 mg/m2, P.O. bid, days 1–14, and ziv-aflibercept 6 mg/kg I.V., day 1, of each 21-day cycle. In the expansion cohort, 72% of patients were progression-free at two months (95% confidence interval [CI], 60–84%). Median PFS and OS were 3.9 months (95% CI, 2.3–4.5) and 7.1 months (95% CI: 5.8–10.0), respectively. Among all patients evaluable for toxicity, the most common treatment related adverse events (all grade [%]; grade ≥ 3 [%]) included palmar-plantar erythrodysesthesia (41%; 6%), hypertension (33%; 22%), and mucositis (19%; 5%). RNA was isolated from archived tumor specimens and gene expression analyses revealed no association between angiogenic biomarkers and clinical outcomes. Conclusion The combination of capecitabine and ziv-aflibercept at the RPTD demonstrated acceptable safety and tolerability. PFS at 2 months in patients with chemotherapy refractory metastatic CRC was significantly greater than that in historical controls, indicating that this combination warrants further study. Trial registration This clinical trial was registered in the www.clinicaltrials.gov system as NCT01661972 on July 31, 2012.

Supplemental Digital Content is available in the text. Objectives One of the standard of care regimens for advanced pancreatic cancer is gemcitabine-based chemotherapy. The efficacy of gemcitabine is limited by dose-limiting hematologic toxicities especially neutropenia. Uncovering the variability of these toxicities attributed to germline DNA variation is of great importance. Patients and methods CALGB 80303 was a randomized study in advanced pancreatic cancer patients treated with gemcitabine with or without bevacizumab. The study protocol included genotyping of genes of gemcitabine disposition (CDA, DCTD, SLC29A1, SLC28A1, and SLC29A2), as well as a genome-wide analysis. The clinical phenotype was time to early high-grade neutropenia event accounting for progression or death or other treatment-terminating adverse events as competing for informative events. The inference was carried out on the basis of the association between genotype and cause-specific hazard of a neutropenic event. Results The primary analyses were carried out on the basis of 294 genetically estimated European pancreatic cancer patients. For CDA rs2072671 (A>C), AC and CC patients had a lower risk of neutropenia than AA patients (P=0.01, hazard ratio: 0.61, 95% confidence interval: 0.41–0.89). For SLC28A1 rs3825876 (G>A), AA patients have a higher risk of neutropenia than GA and GG patients (P=0.02, hazard ratio: 1.51, 95% confidence interval: 1.06–2.16). CDA rs2072671 was associated with increased mRNA expression in whole blood in three studies (P=2.7e−14, 6.61e−62, and 9.70e−65). In the genome-wide analysis, variants in TGFB2 were among the top hits (lowest P=1.62e−06) but had no effect in luciferase assays. Conclusion This is the first genetic analysis of gemcitabine-induced neutropenia using a competing risk model in a prospective randomized clinical study has proposed a potentially novel mechanism of the protective effect of the CDA rs2072671 variant. Further confirmation is needed.

588 Background: CALGB/SWOG 80405 found no difference in overall survival (OS) or progression-free survival (PFS) in 1137 patients with KRAS wild-type metastatic colorectal cancer (mCRC) treated with either the anti-VEGF agent bevacizumab (Bev) or the anti-EGFR agent cetuximab (Cet) in combination with chemotherapy (FOLFOX or FOLFIRI) as first-line therapy. Additional molecular biomarkers are required for the optimization of treatment choices for patients with mCRC. Methods: Levels of 24 proteins were assayed in EDTA-plasma samples using multiplex ELISA techniques. Plasma samples were collected at baseline and cycle 2 day 1 (C2D1). Analyses of baseline markers have been presented previously. Changes from baseline to C2D1 were expressed as an L-ratio (LR, log2 of C2D1 divided by baseline measure). LRs dichotomized at 0 were analyzed for potential prognostic and predictive effects on OS and PFS using Cox models while adjusting for confounders (post-hoc optimized cutpoints were used for PLGF, TIMP1, VCAM1, and VEGFR3 due to skew in the distributions of these markers). P-values are not corrected for multiple testing. Results: Baseline and matched C2D1 samples from 446 patients (212 Bev/234 Cet) were available for analysis. Thirteen markers were differentially modulated between arms (p < 0.05). Early increases in CD73 (p = 0.001), ICAM1 (p = 0.022), TIMP1 (p = 0.001), TSP2 (p = 0.024), and VEGFR3 (p = 0.006) were prognostic for shorter OS. Increases in ICAM1 (p = 0.008), TIMP1 (p = 0.042), and TGFB1 (p = 0.039) were prognostic for shorter PFS. Changes in VEGFR3 (pint = 0.097) and TGFBR3 (pint = 0.052) were predictive for OS and PFS, respectively. No other markers were predictive for OS or PFS (p < 0.1). Conclusions: Changes in circulating plasma protein biomarkers observed in patients enrolled in CALGB/SWOG 80405 were both prognostic and predictive for OS and PFS and warrant further study. Such changes could provide valuable information and help guide treatment decisions. Support: U10CA180821, U10CA180882, U10CA180888, U10CA180830; ClinicalTrials.gov: NCT00265850. [Table: see text]

Colorectal cancer (CRC) is the third most frequent neoplastic disorder and is a main cause of tumor-related mortality as many patients progress to stage IV metastatic CRC. Standard care consists of combination chemotherapy (FOLFIRI or FOLFOX). Patients with WT KRAS typing are eligible to receive anti-EGFR therapy combined with chemotherapy. Unfortunately, predicting efficacy of CRC anti-EGFR therapy has remained challenging. Here we uncover that the EGFR-pathway component RasGRP1 acts as CRC tumor suppressor in the context of aberrant Wnt signaling. We find that RasGRP1 suppresses EGF-driven proliferation of colonic epithelial organoids. Having established that RasGRP1 dosage levels impacts biology, we focused on CRC patients next. Mining five different data platforms, we establish that RasGRP1 expression levels decrease with CRC progression and predict poor clinical outcome of patients. Lastly, deletion of one or two Rasgrp1 alleles makes CRC spheroids more susceptible to EGFR inhibition. Retrospective analysis of the CALGB80203 clinical trial shows that addition of anti-EGFR therapy to chemotherapy significantly improves outcome for CRC patients when tumors express low RasGRP1 suppressor levels. In sum, RasGRP1 is a unique biomarker positioned in the EGFR pathway and of potential relevance to anti-EGFR therapy for CRC patients.

587 Background: The LCCC1029 trial demonstrated that addition of the multitargeted kinase inhibitor regorafenib (Rego) to FOLFIRI in metastatic colorectal cancer (mCRC) patients (pts) modestly prolonged progression-free survival (PFS). In this preplanned analysis, circulating angiogenic and inflammatory proteins were explored as potential prognostic and predictive biomarkers of Rego benefit. Methods: Plasma samples from 149 mCRC pts (107 in Rego + FOLFIRI and 42 in placebo + FOLFIRI) were evaluated for 20 markers at baseline (n = 149) and cycle 1 day 21 (C1D21, n = 81). Predictive and prognostic values of each marker at baseline were analyzed for both PFS and overall survival (OS) using Cox proportional hazard models. On-treatment changes were quantified as fold change [log2(C1D21/baseline)] and differences between arms were evaluated using the Mann-Whitney test. Results: The primary objective of this study was to determine whether any marker was predictive of benefit with Rego for PFS. Although no treatment by marker interactions were significant after adjusting for multiple testing, the top three markers of interest were OPN (unadjusted p-values of 0.02), VCAM-1 (0.02), and PDGF-AA (0.04). VCAM-1 was also predictive for OS benefit in pts treated with Rego (unadjusted p = 0.01). Baseline levels of multiple markers (including HGF, IL-6, PlGF, VEGF-R1, OPN) were prognostic for both PFS and OS. Higher levels of these markers were associated with worse survival. Biomarker changes in response to treatment were explored and compared between arms. Fold change of three markers (PlGF, VEGF-A, VCAM-1) were significantly different between arms (p < 0.0001), all being markedly up-regulated in the Rego arm compared to the placebo after treatment. Conclusions: In this hypothesis generating report, VCAM-1, OPN, and PDGF-AA were the top biomarkers when analyzing the potential predictive association with PFS, where a lower hazard was observed for pts receiving Rego. Candidate prognostic markers were identified, including PlGF and VEGF-R1, key factors in VEGF biology. Biomarker changes observed here may offer insights into potential combinatorial strategies with Rego for future studies.

PURPOSE:This study evaluated the maximum tolerated dose or recommended phase II dose (RPTD) and safety and tolerability of the ganitumab and everolimus doublet regimen followed by the ganitumab, everolimus, and panitumumab triplet regimen.MATERIALS AND METHODS:This was a standard 3 + 3 dose escalation trial. Doublet therapy consisted of ganitumab at 12 mg/kg every 2 weeks; doses of everolimus were adjusted according to dose-limiting toxicities (DLTs). Panitumumab at 4.8 mg/kg every 2 weeks was added to the RPTD of ganitumab and everolimus. DLTs were assessed in cycle 1; toxicity evaluation was closely monitored throughout treatment. Treatment continued until disease progression or undesirable toxicity. Pretreatment and on-treatment skin biopsies were collected to assess insulin-like growth factor 1 receptor and mammalian target of rapamycin (mTOR) target modulation.RESULTS:Forty-three subjects were enrolled. In the doublet regimen, two DLTs were observed in cohort 1, no DLTs in cohort -1, and one in cohort -1B. The triplet combination was discontinued because of unacceptable toxicity. Common adverse events were thrombocytopenia/neutropenia, skin rash, mucositis, fatigue, and hyperglycemia. In the doublet regimen, two patients with refractory non-small cell lung cancer (NSCLC) achieved prolonged complete responses ranging from 18 to >60 months; one treatment-naïve patient with chondrosarcoma achieved prolonged stable disease >24 months. In dermal granulation tissue, the insulin-like growth factor receptor and mTOR pathways were potently and specifically inhibited by ganitumab and everolimus, respectively.CONCLUSION:The triplet regimen of ganitumab, everolimus, and panitumumab was associated with unacceptable toxicity. However, the doublet of ganitumab at 12 mg/kg every 2 weeks and everolimus five times weekly had an acceptable safety profile and demonstrated notable clinical activity in patients with refractory NSCLC and sarcoma.IMPLICATIONS FOR PRACTICE:This trial evaluated the maximum tolerated dose or recommended phase II dose and safety and tolerability of the ganitumab and everolimus doublet regimen followed by the ganitumab, everolimus, and panitumumab triplet regimen. Although the triplet regimen of ganitumab, everolimus, and panitumumab was associated with unacceptable toxicity, the doublet of ganitumab at 12 mg/kg every 2 weeks and everolimus at five times weekly had an acceptable safety profile and demonstrated notable clinical activity in patients with refractory non-small cell lung cancer and sarcoma.

Background: One of the standard of care regimens for advanced pancreatic cancer is gemcitabine-based chemotherapy. The efficacy of gemcitabine is reduced by dose-limiting hematologic toxicities, especially neutropenia. Uncovering the variability of these toxicities attributed to germline DNA variation is of great importance. Methods: CALGB 80303 was a randomized study in advanced pancreatic cancer patients treated with gemcitabine with or without bevacizumab. The study protocol included genotyping of genes of gemcitabine disposition (CDA, DCTD, SLC29A1, SLC28A1, SLC29A2), as well as a genome-wide analysis. The clinical phenotype was time to high-grade early neutropenia event accounting for progression or death, with other treatment-terminating adverse events as competing informative events. The inference was conducted on the basis of the association between genotype and cause-specific hazard of a neutropenic event. Results: The primary analyses were conducted on the basis of 294 genetically estimated Europeans. For CDA rs2072671 (A>C), AC and CC patients had a lower risk of neutropenia than AA patients (unadjusted P-value 0.01, HR 0.61, 95% CI 0.41-0.89). For SLC28A1 rs3825876 (G>A), AA patients have a higher risk of neutropenia than GA and GG patients (unadjusted P-value 0.02, HR 1.51, 95% CI 1.06-2.16). The C allele of CDA rs2072671 was associated with increased mRNA expression in whole blood in three studies (unadjusted P-values 2.7e-14, 6.61e-62, 9.70e-65). In the genome-wide analysis, variants in TGFB2 were among the top hits (unadjusted lowest P-value 1.62e-06) but had no effect in luciferase assays. Conclusions: The first genetic analysis of gemcitabine-induced neutropenia using a competing risk model in a prospective randomized clinical study has proposed a potentially novel mechanism of the protective effect of the CDA rs2072671 variant. We hypothesize that the rs2072671 (A>C) variant acts through a local effect in either the bone marrow or in circulating neutrophils (or both), by protecting neutrophils from the anti-proliferative effects of gemcitabine. Further confirmation is needed. Clinical trial identification: NCI-2012-02960; NCT00088894. Legal entity responsible for the study: Alliance for Clinical Trials in Oncology. Funding: NIH. Disclosure: All authors have declared no conflicts of interest.

Angiogenesis is essential in tumor biology and is regulated by vascular endothelial growth factor (VEGF) ligands and receptors. Here we aimed to discover genetic variants associated with levels of circulating angiogenic proteins in cancer patients. Plasma was collected at baseline in 216 pancreatic and 114 colorectal cancer patients. Thirty-one angiogenic proteins were measured by ELISA. 484,523 Single Nucleotide Polymorphisms (SNP) were tested for association with plasma levels for each protein in pancreatic cancer patients. Three top-ranked hits were then genotyped in colorectal cancer patients, where associations with the same proteins were measured. The results demonstrated rs2284284 and MCP1 (P-value = 6.7e–08), rs7504372 and VEGF-C (P-value = 9.8e–09), and rs7767396 and VEGF-A (P-value = 5.8e–09) were SNP-protein pairs identified in pancreatic cancer patients. In colorectal cancer patients, only rs7767396 (A > G) and VEGF-A was validated (P-value = 5.18e–05). The AA genotype of rs7767396 exhibited 2.04–2.3 and 2.7–3.4-fold higher VEGF-A levels than those with AG and GG genotypes. The G allele of rs7767396 reduces binding of the NF-AT1 transcription factor. In conclusion, a common genetic variant predicts the plasma levels of VEGF-A in cancer patients through altered binding of NF-AT1.

Importance Epidermal growth factor receptor (EGFR) (HER1) signaling depends on ligand binding and dimerization with itself or other HER receptors. We previously showed in a randomized trial that high EGFR ligand expression is predictive of panitumumab benefit in advanced colorectal cancer. Tumor expression of HER3 may further refine the RAS wild-type (wt) population benefitting from anti-EGFR agents. Objective To examine HER3 messenger RNA expression as a prognostic and predictive biomarker for anti-EGFR therapy in a randomized clinical trial of panitumumab. Design, Setting, and Participants The study was a prospectively planned retrospective biomarker study of pretreatment samples from the PICCOLO trial that tested the addition of panitumumab to irinotecan therapy in patients with KRAS wt advanced colorectal cancer who experienced failure with prior fluoropyrimidine treatment. HER3 was assessed as a prognostic marker, then as a predictive biomarker in patients with RAS wt, first as a continuous variable and then as a binary (high vs low) variable. Relationship with MEK-AKT pathway mutations and EGFR ligands epiregulin and amphiregulin (EREG/AREG) were also assessed. Main Outcomes and Measures Primary end point was progression-free survival (PFS); secondary end points were response rate and overall survival (OS). Results In 308 patients (mean age at randomization, 61.6 years; 193 men) higher HER3 was weakly prognostic for OS (hazard ratio [HR] per 2-fold change, 0.91; 95% CI, 0.83-0.99; P = .04) but not PFS (HR, 0.93; 95% CI, 0.83-1.05; P = .25). Higher HER3 was predictive, being associated with prolonged PFS on irinotecan plus panitumumab (IrPan) (HR, 0.71; 95% CI, 0.61-0.82; P < .001), but not irinotecan (HR, 0.96; 95% CI, 0.82-1.13; P = .65) in patients with RAS wt, with significant interaction between biomarker and treatment (P = .001). Similar interaction was seen for OS (P = .004). In an exploratory binary model, dividing the population at the 66th percentile, HER3 was predictive of panitumumab benefit: in patients with high HER3 expression, median PFS was 8.2 months (IrPan) vs 4.4 months (irinotecan) (HR, 0.33; 95% CI, 0.19-0.58; P < .001). Patients with low HER3 expression gained no benefit in PFS: 3.3 months (IrPan) vs 4.3 months (irinotecan) (HR, 0.96; 95% CI, 0.67-1.38; P = .84), with significant interaction (P = .002). The binary model was also predictive for OS, with significant interaction (P = .01). Combining HER3 and ligand data, patients with HER3-high, AREG/EREG-high tumors gained markedly from panitumumab (PFS HR, 0.24; 95% CI, 0.11-0.51; P < .005 and OS HR, 0.36; 95% CI, 0.18-0.73; P = .004). Conversely, patients with HER3-low, AREG/EREG-low tumors did not benefit (PFS HR, 1.14; 95% CI, 0.73-1.79; P = .57 and OS HR, 1.44; 95% CI, 0.92-2.26; P = .11). Conclusions and Relevance High HER3 expression identified patients with RAS wt who gained markedly from panitumumab, and those who did not, with statistically significant biomarker-treatment interactions for PFS and OS. This finding provides insight into the mechanism of anti-EGFR agents and is of potential clinical utility.

Production of lipid-derived inositol phosphates including IP4 and IP5 is an evolutionarily conserved process essential for cellular adaptive responses that is dependent on both phospholipase C and the inositol phosphate multikinase Ipk2 (also known as Arg82 and IPMK). Studies of Ipk2, along with Arg82 prior to demonstrating its IP kinase activity, have provided an important link between control of gene expression and IP metabolism as both kinase dependent and independent functions are required for proper transcriptional complex function that enables cellular adaptation in response to extracellular queues such as nutrient availability. Here we define a promoter sequence cis-element, 5′-CCCTAAAAGG-3′, that mediates both kinase-dependent and independent functions of Ipk2. Using a synthetic biological strategy, we show that proper gene expression in cells lacking Ipk2 may be restored through add-back of two components: IP4/IP5 production and overproduction of the MADS box DNA binding protein, Mcm1. Our results are consistent with a mechanism by which Ipk2 harbors a dual functionality that stabilizes transcription factor levels and enzymatically produces a small molecule code, which together coordinate control of biological processes and gene expression.

Cell-free DNA (cfDNA), of which circulating tumor DNA (ctDNA) is a subset, is a promising potential biomarker. Tumor cells apoptose or necrose and release DNA into the circulation which can be readily quantified, and cfDNA possesses a wide dynamic range which correlates with tumor burden. The release of cfDNA during a course of fractionated radiotherapy has not been systematically characterized, and to our knowledge, no studies have examined cfDNA during chemoradiation (CRT) for locally advanced thoracic malignancies. We hypothesized that there would be quantifiable change in cfDNA during a course of CRT for non-small cell lung cancer (NSCLC). We obtained patient plasma on an Institutional Review Board-approved trial from 24 patients treated with concurrent cisplatin/etoposide-based CRT for locally advanced NSCLC to a mean dose of 66 Gy (range 58-74 Gy). Plasma was obtained prior to therapy, at week 2, week 5, and at 6-week post-treatment follow-up, unless patients refused phlebotomy. Samples were single-spun at acquisition and subsequently processed via the Maxwell RSC ccfDNA Plasma Kit and quantified using the QuantiFluor ONE dsDNA system (Promega) according to the manufacturer’s protocols. A novel next generation sequencing (NGS) technique employing targeted-amplicon sequencing (InVision) was performed on a subset of samples. The cfDNA concentration was normalized by plasma volume and statistical associations were assessed via ANOVA and two sample t-test. Ninety total samples were examined. The median time between the baseline and post-treatment follow-up sample acquisition was 92 days. The mean normalized concentration of cfDNA isolated from baseline, week 2, week 5, and post-treatment follow-up was 10.1 pg/μl (σ = 8.9), 11.1 pg/μl (σ = 10.0), 11.1 pg/μl (σ = 7.2), and 18.7 pg/μl (σ = 18.5), respectively. There was a statistically significant increase in cfDNA between the baseline and post-treatment follow-up sample (P = 0.05). NGS was performed on a subset of samples to monitor specific genomic alterations in ctDNA, and it was possible to track putatively etiologic driver mutations. For example, in one patient a missense mutation was detected in the tumor suppressor gene p53 at a known pathologic site, V143M, and additional mutations were observed at subsequent time points. The concentration of cfDNA increases after CRT in patients with locally advanced NSCLC, and mutations specific to a tumor and present in ctDNA can be identified and tracked with NGS. Further work is required to determine whether the kinetics of cfDNA change over longer periods of time; whether NGS can be used to track the efficacy of CRT; or whether cfDNA-related endpoints correlate with patient outcomes. The system that we have established could potentially be adopted for future prospective clinical trials.

3597 Background: CALGB 80405 was a randomized phase III trial comparing FOLFOX or FOLFIRI + Bev, Cetux or both. Results showed no significant differences between Bev and Cetux in progression-free survival (PFS) or overall survival (OS). The primary objective of our study was to validate 7 biomarkers previously identified to be predictive for outcome for either Bev or Cetux. Methods: Baselineplasma samples were analyzed via multiplex ELISA technology for Ang-2, sCD73, sHER3, HGF, IL-6, SDF-1, and VEGF-D. The markers were correlated with PFS and OS using univariate Cox proportional hazards models stratified by chemotherapy (chemo); predictive markers were tested by adding a treatment by analyte interaction term to the model. Exploratory analyses included a chemo by biologic by marker term. Results: Baseline samples were available from 715 KRAS wild type pts treated with Bev or Cetux. No prespecified biomarkers were predictive for OS or PFS after multiple testing correction. However, lower levels of VEGF-D were associated with greater benefit from Bev for PFS (uncorrected interaction p = 0.049; HR 0.69 [95% CI 0.48-1.00]). Based on exploratory analyses, a potential 3-way interaction between chemo, biologic, and VEGF-D levels with respect to PFS was observed (p = 0.028). In subset analyses, VEGF-D was found to predict for benefit from Bev in pts treated with FOLFOX (p = 0.0028; HR 0.50 [95% CI 0.31-0.79]), but not in pts treated with FOLFIRI (p = 0.53; HR 1.27 [95% CI 0.59-2.73]). IL-6 was strongly prognostic for PFS and OS (p < 0.0001). Conclusions: While the pre-specified criteria for validation of these markers was not met, these data suggest a role for VEGF-D in mediating resistance to Bev, particularly for pts receiving FOLFOX. These findings are consistent with prior observations and highlight the potential importance of alternate VEGF ligands in response to anti-VEGF therapeutics. In addition, IL-6 was found to be strongly prognostic, highlighting the importance of inflammation in mCRC. Additional analyses are ongoing. Support: U10CA180821, U10CA180882. ClinicalTrials.gov Id: NCT00265850