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Identification of circulating tumor DNA (ctDNA) following curative intent therapies is a surrogate for microscopic residual disease for patients with metastatic colorectal cancer (mCRC). Preclinically, in micrometastatic microsatellite stable (MSS) colorectal cancer, increased TGFβ signaling results in exclusion of antitumor cytotoxic T cells from the tumor microenvironment. Bintrafusp alfa (BA) is a bifunctional fusion protein composed of the extracellular domain of the TGFβRII receptor (“TGFβ trap”) and anti-PD-L1 antibody. Patients with liver-limited, MSS mCRC and with detected ctDNA after complete resection of all known tumors and standard-of-care therapy were treated with 1,200 mg of BA intravenously every 14 days for six doses. The primary endpoint was ctDNA clearance. Radiographic characteristics at recurrence were compared using independent t tests to historical data from a similar cohort of patients with liver-limited mCRC who underwent observation. Only 4 of 15 planned patients received BA before the study was stopped early for loss of equipoise. There was no grade ≥3 adverse event. None of the patients cleared ctDNA. All patients developed radiographic recurrence by the first planned restaging. Although not detectable at prior to treatment, TGFβ3 was found in circulation in all patients at cycle 2 day 1. Compared with a historical cohort, patients administered BA developed more metastases (15 vs. 2, P = 0.005) and greater tumor volumes (9 cm vs. 2 cm, P = 0.05). Treatment with BA in patients with ctDNA-detected, liver-limited mCRC did not clear ctDNA and was associated with large-volume recurrence, highlighting the potential context-specific complexity of dual TGFβ and PD-L1 inhibition. Significance: Use of ctDNA to identify patients with micrometastatic disease for therapeutic intervention is feasible. Treatment with BA in patients with liver-limited mCRC and with detectable ctDNA after resection generated rapid progression. Approaches targeting TGFβ signaling must consider its pathway complexity in future immunotherapy combination strategies.

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Identification of circulating tumor DNA (ctDNA) following curative intent therapies is a surrogate for microscopic residual disease for patients with metastatic colorectal cancer (mCRC). Preclinically, in micrometastatic microsatellite stable (MSS) colorectal cancer, increased TGFβ signaling results in exclusion of antitumor cytotoxic T cells from the tumor microenvironment. Bintrafusp alfa (BA) is a bifunctional fusion protein composed of the extracellular domain of the TGFβRII receptor (“TGFβ trap”) and anti-PD-L1 antibody. Patients with liver-limited, MSS mCRC and with detected ctDNA after complete resection of all known tumors and standard-of-care therapy were treated with 1,200 mg of BA intravenously every 14 days for six doses. The primary endpoint was ctDNA clearance. Radiographic characteristics at recurrence were compared using independent t tests to historical data from a similar cohort of patients with liver-limited mCRC who underwent observation. Only 4 of 15 planned patients received BA before the study was stopped early for loss of equipoise. There was no grade ≥3 adverse event. None of the patients cleared ctDNA. All patients developed radiographic recurrence by the first planned restaging. Although not detectable at prior to treatment, TGFβ3 was found in circulation in all patients at cycle 2 day 1. Compared with a historical cohort, patients administered BA developed more metastases (15 vs. 2, P = 0.005) and greater tumor volumes (9 cm vs. 2 cm, P = 0.05). Treatment with BA in patients with ctDNA-detected, liver-limited mCRC did not clear ctDNA and was associated with large-volume recurrence, highlighting the potential context-specific complexity of dual TGFβ and PD-L1 inhibition.Significance:Use of ctDNA to identify patients with micrometastatic disease for therapeutic intervention is feasible. Treatment with BA in patients with liver-limited mCRC and with detectable ctDNA after resection generated rapid progression. Approaches targeting TGFβ signaling must consider its pathway complexity in future immunotherapy combination strategies.

Purpose: Aberrant activation of the intracellular tyrosine kinase Src has been implicated as a mechanism of acquired chemotherapy resistance in metastatic colorectal cancer (mCRC). Here, the oral tyrosine kinase Src inhibitor, dasatinib, was investigated in combination with FOLFOX and cetuximab.Experimental Design: We performed a phase IB/II study of 77 patients with previously treated mCRC. Primary objectives were to determine the maximum tolerated dose, dose-limiting toxicities (DLT), pharmacodynamics, and efficacy. Using a 3 + 3 design, patients received FOLFOX6 with cetuximab and escalating doses of dasatinib (100, 150, 200 mg daily), followed by a 12-patient expansion cohort at 150 mg. Phase II studies evaluated FOLFOX plus dasatinib 100 mg in KRAS c12/13mut patients or in combination with cetuximab if KRAS c12/13WT. FAK and paxillin were utilized as surrogate blood biomarkers of Src inhibition, and paired biopsies of liver metastases were obtained in patients in the expansion cohort.Results: In phase IB, the DLTs were grade 3/4 fatigue (20%) and neutropenia (23%). In phase II, grade 3/4 fatigue (23%) and pleural effusions (11%) were present. Response rates were 20% (6 of 30) in the phase IB escalation and expansion cohort and 13% (3 of 24) and 0% (0 of 23) in the KRAS c12/13WT and mutant cohorts of phase II, respectively. Median progression-free survival was 4.6, 2.3, and 2.3 months, respectively. There was no evidence of Src inhibition based on surrogate blood biomarkers or paired tumor biopsies.Conclusions: The combination of dasatinib plus FOLFOX with or without cetuximab showed only modest clinical activity in refractory colorectal cancer. This appears to be primarily due to a failure to fully inhibit Src at the achievable doses of dasatinib. The combination of dasatinib plus FOLFOX with or without cetuximab did not show meaningful clinical activity in refractory colorectal cancer due to failure to fully inhibit Src. Clin Cancer Res; 23(15); 4146–54. ©2017 AACR.

Validation of peripheral blood mononuclear cells as a biomarker of Src inhibition. Freshly collected peripheral blood mononuclear cells from a healthy volunteer were treated ex-vivo with dasatinib 300 nM or hydrogen peroxide 0.05 mM. The peripheral blood mononuclear cell lysates were generated and immunoblotted for phosphoFAK [Y861] and phophopaxillin [Y118].

Operating characteristics. A summary of the probability of stopping the trial early for the indicated true response rates.

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Lack of correlation between Src activity in PBMCs and hepatic tumors. For the patients in the expansion cohort, the change in the expression of pSrc and total Src was not correlated with a change in the phosphorlyation of the Src substrate paxillin. Patients with no change or a decrease in the expression of pSrc or Src from baseline to C2D8 were included in the "no increase" category.

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Purpose: Research biopsies are frequently incorporated within clinical trials in oncology and are often a mandatory requirement for trial enrollment. However, limited information is available regarding the extent and completeness of research biopsy reporting.Experimental Design: We identified a cohort of therapeutic clinical trials where research biopsies were performed between January 2005 and October 2010 from an IR database at our institution. Clinical trial protocols were compared with the highest level of corresponding publication as a manuscript or registry report.Results: A total of 866 research biopsies were performed across 46 clinical trials, with a median of 8 patients biopsied/trial and 19 biopsies collected/trial. After a median follow-up time of 4.3 years from trial completion, 36 of 46 trials (78%) reported trial results: published manuscripts (n = 35), or registry report (n = 1). A total of 635 conducted biopsies were reported in 18 of the 46 trials (39%). Six (33%) of these 18 trials underreported the number of biopsies performed. Of 33 trials with mandatory research biopsies, 13 (39%) trials reported on these biopsies. Biopsy complications occurred in 8 trials [n = 39 patients, 6 grade 3/4 adverse events (AE)] but only 1 trial reported these. Factors associated with biopsy reporting included a larger number of biopsies (P ≤ 0.001) and serial biopsies (P < 0.001). Twelve of 16 (75%) trials with >12 biopsies performed reported on these biopsies compared with only 20% (6/30) that performed ≤12 biopsies.Conclusions: Despite ethical obligations to report research biopsies, the majority (61%) of trials do not report results from research biopsies. Complications are rarely reported in these studies. Improved reporting of results and AEs from research biopsies is needed. Clin Cancer Res; 23(21); 6450–7. ©2017 AACR.

Characteristics and Reporting by Trial

Factors Correlating with Research Biopsy Reporting from Published Manuscripts

Prespecified dose modifications for adverse events related to study drug in Phase IB and Phase II.

Symptom-specific dose adjustment tables.

PURPOSE Acquired resistance to anti–epidermal growth factor receptor (EGFR) inhibitor (EGFRi) therapy in colorectal cancer (CRC) has previously been explained by the model of acquiring new mutations in KRAS/NRAS/EGFR, among other MAPK-pathway members. However, this was primarily on the basis of single-agent EGFRi trials and little is known about the resistance mechanisms of EGFRi combined with effective cytotoxic chemotherapy in previously untreated patients. METHODS We analyzed paired plasma samples from patients with RAS/BRAF/EGFR wild-type metastatic CRC enrolled in three large randomized trials evaluating EGFRi in the first line in combination with chemotherapy and as a single agent in third line. The mutational signature of the alterations acquired with therapy was evaluated. CRC cell lines with resistance to cetuximab, infusional fluorouracil, leucovorin, and oxaliplatin, and SN38 were developed, and transcriptional changes profiled. RESULTS Patients whose tumors were treated with and responded to EGFRi alone were more likely to develop acquired mutations (46%) compared with those treated in combination with cytotoxic chemotherapy (9%). Furthermore, contrary to the generally accepted hypothesis of the clonal evolution of acquired resistance, we demonstrate that baseline resistant subclonal mutations rarely expanded to become clonal at progression, and most remained subclonal or disappeared. Consistent with this clinical finding, preclinical models with acquired resistance to either cetuximab or chemotherapy were cross-resistant to the alternate agents, with transcriptomic profiles consistent with epithelial-to-mesenchymal transition. By contrast, commonly acquired resistance alterations in the MAPK pathway do not affect sensitivity to cytotoxic chemotherapy. CONCLUSION These findings support a model of resistance whereby transcriptomic mechanisms of resistance predominate in the presence of active cytotoxic chemotherapy combined with EGFRi, with a greater predominance of acquired MAPK mutations after single-agent EGFRi. The proposed model has implications for prospective studies evaluating EGFRi rechallenge strategies guided by acquired MAPK mutations, and highlights the need to address transcriptional mechanisms of resistance.

Acquired resistance to anti-epidermal growth factor receptor (EGFR) inhibitor (EGFRi) therapy in colorectal cancer (CRC) has previously been explained by the model of acquiring new mutations inWe analyzed paired plasma samples from patients withPatients whose tumors were treated with and responded to EGFRi alone were more likely to develop acquired mutations (46%) compared with those treated in combination with cytotoxic chemotherapy (9%). Furthermore, contrary to the generally accepted hypothesis of the clonal evolution of acquired resistance, we demonstrate that baseline resistant subclonal mutations rarely expanded to become clonal at progression, and most remained subclonal or disappeared. Consistent with this clinical finding, preclinical models with acquired resistance to either cetuximab or chemotherapy were cross-resistant to the alternate agents, with transcriptomic profiles consistent with epithelial-to-mesenchymal transition. By contrast, commonly acquired resistance alterations in the MAPK pathway do not affect sensitivity to cytotoxic chemotherapy.These findings support a model of resistance whereby transcriptomic mechanisms of resistance predominate in the presence of active cytotoxic chemotherapy combined with EGFRi, with a greater predominance of acquired MAPK mutations after single-agent EGFRi. The proposed model has implications for prospective studies evaluating EGFRi rechallenge strategies guided by acquired MAPK mutations, and highlights the need to address transcriptional mechanisms of resistance.

BackgroundMonotherapy with immune checkpoint blockade is ineffective for patients (pts) with microsatellite stable (MSS) metastatic colorectal cancer (mCRC). This study investigates whether the combination of trametinib (T) with durvalumab (D) can alter the immune tumor microenvironment (TME) by successfully priming and activating T-cells.MethodsOpen-label, single-center, phase II trial with primary endpoint of immune-related response rate for combination of T+D in refractory MSS mCRC pts (NCT03428126). T is 2 mg/day orally starting 1 week prior to D, which is given 1500 mg intravenously every 4 weeks. Simon 2-stage design used to enroll 29 pts into first stage, requiring a response in two or more pts to proceed to stage 2. Tumor biopsies were collected at baseline (BL) and early on-treatment (OT) at week 4.ResultsTwenty nine treated pts include 48% females, median age 48 years (range 28–75), and median prior therapies 2 (range 1–5). No grade (G) 4 or 5 treatment-related adverse events (TRAE). The most common TRAE of any grade was acneiform rash, 17% being G3. One of 29 pts had confirmed partial response (PR) lasting 9.3 months (mo) for an overall response rate of 3.4%. Seven pts had stable disease (SD) and five pts (1 PR, 4 SD) demonstrated decrease in total carcinoembryonic antigen ng/mL (best percentage reduction: 94%, 95%, 42%, 34%, and 22%, respectively). Median progression-free survival was 3.2 mo (range 1.1–9.3 months). Three pts with both liver and lung metastases demonstrated discrepant responses in which clinical benefit was present in the lung metastases but not liver metastases. Comparison of BL and 4-week OT tumor tissue flow cytometry demonstrated no changes in T-cell infiltration but upregulation expression of PD-1 and Tim3 on CD8 T cells. However, expression of PD-1 and Tim3 as single markers and as coexpressed markers was observed to increase OT relative to BL (p=0.03, p=0.06 and p=0.06, respectively).ConclusionsT+D demonstrated acceptable tolerability in pts with refractory MSS mCRC. The response rate in the first stage of the study did not meet efficacy criteria to proceed to the second stage. Specific site of metastatic disease may impact outcomes in novel immunotherapy combination trials.Trial registration numberNCT03428126.