Top 10 Challenges in Cancer Immunotherapy.

Cancer immunotherapy is a validated and critically important approach for treating patients with cancer. Given the vast research and clinical investigation efforts dedicated to advancing both endogenous and synthetic immunotherapy approaches, there is a need to focus on crucial questions and define roadblocks to the basic understanding and clinical progress. Here, we define ten key challenges facing cancer immunotherapy, which range from lack of confidence in translating pre-clinical findings to identifying optimal combinations of immune-based therapies for any given patient. Addressing these challenges will require the combined efforts of basic researchers and clinicians, and the focusing of resources to accelerate understanding of the complex interactions between cancer and the immune system and the development of improved treatment options for patients with cancer.

TGFß attenuates tumour response to PD-L1 blockade by contributing to exclusion of T cells.

Therapeutic antibodies that block the programmed death-1 (PD-1)-programmed death-ligand 1 (PD-L1) pathway can induce robust and durable responses in patients with various cancers, including metastatic urothelial cancer. However, these responses only occur in a subset of patients. Elucidating the determinants of response and resistance is key to improving outcomes and developing new treatment strategies. Here we examined tumours from a large cohort of patients with metastatic urothelial cancer who were treated with an anti-PD-L1 agent (atezolizumab) and identified major determinants of clinical outcome. Response to treatment was associated with CD8+ T-effector cell phenotype and, to an even greater extent, high neoantigen or tumour mutation burden. Lack of response was associated with a signature of transforming growth factor ß (TGFß) signalling in fibroblasts. This occurred particularly in patients with tumours, which showed exclusion of CD8+ T cells from the tumour parenchyma that were instead found in the fibroblast- and collagen-rich peritumoural stroma; a common phenotype among patients with metastatic urothelial cancer. Using a mouse model that recapitulates this immune-excluded phenotype, we found that therapeutic co-administration of TGFß-blocking and anti-PD-L1 antibodies reduced TGFß signalling in stromal cells, facilitated T-cell penetration into the centre of tumours, and provoked vigorous anti-tumour immunity and tumour regression. Integration of these three independent biological features provides the best basis for understanding patient outcome in this setting and suggests that TGFß shapes the tumour microenvironment to restrain anti-tumour immunity by restricting T-cell infiltration.

Genomic Profiling Reveals Differences in Primary Central Nervous System Lymphoma and Large B-Cell Lymphoma, With Subtyping Suggesting Sensitivity to BTK Inhibition.

BACKGROUND: B-cell primary central nervous system (CNS) lymphoma (PCL) is diffuse large B-cell lymphoma (DLBCL) confined to the CNS. Less than 50% of patients with PCL achieve complete remission with current therapies. We describe the findings from comprehensive genomic profiling (CGP) of a cohort of 69 patients with PCL, 36 cases of secondary CNS lymphoma (SCL), and 969 cases of DLBCL to highlight their differences and characterize the PCL cohort. In addition, we highlight the differences in frequency of germinal center B-cell like (GCB) and non-GCB subtypes and molecular subtypes, particularly MCD and EZH subtypes, between PCL and DLBCL. MATERIALS AND METHODS: Sixty-nine cases of B-cell PCL, 36 cases of secondary CNS lymphoma (SCL), and 969 cases of DLBCL were evaluated by CGP of 405 genes via DNAseq and 265 genes via RNAseq for fusions (FoundationOne Heme). Tumor mutational burden (TMB) was calculated from 1.23 Mb of sequenced DNA. RESULTS: Genomic alterations with significant differences between PCL and DLBCL included MYD88, ETV6, PIM1, PRDM1, CXCR4, TP53, and CREBBP, while only MYD88 was significantly different between SCL and DLBCL. PCL cases were significantly enriched for the MCD molecular subtypes, which have an excellent response to BTKi. We report a patient with a durable complete response to BTKi consistent with their genomic profile. EBV status, CD274 amplification, and TMB status suggest that 38% of PCL patients may benefit from ICPI; however further study is warranted. CONCLUSION: CGP of PCLs reveals biomarkers, genomic alterations, and molecular classifications predictive of BTKi efficacy and potential ICPI efficacy. Given the limitations of standard of care for PCL, CGP is critical to identify potential therapeutic approaches for patients in this rare form of lymphoma.

A Novel HRD Signature Is Predictive of FOLFIRINOX Benefit in Metastatic Pancreatic Cancer.

BACKGROUND: Pancreatic cancer (PC) represents an aggressive disease with median overall survival (OS) of less than 1 year in the front-line setting. FOLFIRINOX and gemcitabine and paclitaxel (GP) are standard of care options for these patients; however, optimal selection of therapy is challenging. METHODS: Comprehensive genomic profiling was performed on 8358 PC patients. Outcomes were available for 1149 metastatic PC patients treated with 1L FOLFIRINOX or GP. A scar-based measure of HRD was called using a machine learning-based algorithm incorporating copy number and indel features. RESULTS: A scar-based HRD signature (HRDsig) was identified in 9% of patients. HRDsig significantly co-occurred with biallelic alterations in BRCA1/2, PALB2, BARD1, and RAD51C/D, but encompassed a larger population than that defined by BRCA1/BRCA2/PALB2 (9% vs. 6%). HRDsig was predictive of 1L FOLFIRNOX chemotherapy benefit with doubled OS relative to gemcitabine and paclitaxel (GP) (rwOS aHR 0.37 [0.22-0.62]), including 25% of the population with long-term (2 year+) survival in a real-world cohort of patients. Less benefit from FOLFIRINOX was observed in the HRDsig(-) population. Predictive value was seen in both the BRCA1/2/PALB2 mutant and wildtype populations, suggesting additional value to mutational profiling. CONCLUSION: A scar-based HRD biomarker was identified in a significant fraction of PC patients and is predictive of FOLFIRINOX benefit. Incorporating a biomarker like HRDsig could identify the right patients for platinum chemotherapy and potentially reduce FOLFIRINOX use by over 40%, minimizing toxicities with similar survival outcomes. Confirmatory studies should be performed.

Suppressing neutrophil-dependent angiogenesis abrogates resistance to anti-VEGF antibody in a genetic model of colorectal cancer.

We tested cis-ApcΔ716/Smad4+/- and cis-ApcΔ716/Smad4+/-KrasG12D mice, which recapitulate key genetic abnormalities accumulating during colorectal cancer (CRC) tumorigenesis in humans, for responsiveness to anti-VEGF therapy. We found that even tumors in cis-ApcΔ716/Smad4+/-KrasG12D mice, although highly aggressive, were suppressed by anti-VEGF treatment. We tested the hypothesis that inflammation, a major risk factor and trigger for CRC, may affect responsiveness to anti-VEGF. Chemically induced colitis (CIC) in cis-ApcΔ716/Smad4+/- and cis-ApcΔ716/Smad4+/-KrasG12D mice promoted development of colon tumors that were largely resistant to anti-VEGF treatment. The myeloid growth factor G-CSF was markedly increased in the serum after induction of colitis. Antibodies blocking G-CSF, or its target Bv8/PROK2, suppressed tumor progression and myeloid cell infiltration when combined with anti-VEGF in CIC-associated CRC and in anti-VEGF-resistant CRC liver metastasis models. In a series of CRC specimens, tumor-infiltrating neutrophils strongly expressed Bv8/PROK2. CRC patients had significantly higher plasma Bv8/PROK2 levels than healthy volunteers and high plasma Bv8/PROK2 levels were inversely correlated with overall survival. Our findings establish Bv8/PROK2 as a translational target in CRC, in combination with anti-VEGF agents.

Association of CD274 (PD-L1) Copy Number Changes with Immune Checkpoint Inhibitor Clinical Benefit in Non-Squamous Non-Small Cell Lung Cancer.

BACKGROUND: We sought to characterize response to immune checkpoint inhibitor (ICI) in non-squamous non-small cell lung cancer (NSCLC) across various CD274 copy number gain and loss thresholds and identify an optimal cutoff. MATERIALS AND METHODS: A de-identified nationwide (US) real-world clinico-genomic database was leveraged to study 621 non-squamous NSCLC patients treated with ICI. All patients received second-line ICI monotherapy and underwent comprehensive genomic profiling as part of routine clinical care. Overall survival (OS) from start of ICI, for CD274 copy number gain and loss cohorts across varying copy number thresholds, were assessed. RESULTS: Among the 621 patients, patients with a CD274 CN greater than or equal to specimen ploidy +2 (N = 29) had a significantly higher median (m) OS when compared with the rest of the cohort (N = 592; 16.1 [8.9-37.3] vs 8.6 [7.1-10.9] months, hazard ratio (HR) = 0.6 [0.4-1.0], P-value = .05). Patients with a CD274 copy number less than specimen ploidy (N = 299) trended toward a lower mOS when compared to the rest of the cohort (N = 322; 7.5 [5.9-11.3] vs 9.6 [7.9-12.8] months, HR = 0.9 [0.7-1.1], P-value = .3). CONCLUSION: This work shows that CD274 copy number gains at varying thresholds predict different response to ICI blockade in non-squamous NSCLC. Considering these data, prospective clinical trials should further validate these findings, specifically in the context of PD-L1 IHC test results.

Comprehensive Genomic Profiling (CGP)-Informed Personalized Molecular Residual Disease (MRD) Detection: An Exploratory Analysis from the PREDATOR Study of Metastatic Colorectal Cancer (mCRC) Patients Undergoing Surgical Resection.

A majority of patients with metastatic colorectal cancer (mCRC) experience recurrence post curative-intent surgery. The addition of adjuvant chemotherapy has shown to provide limited survival benefits when applied to all patients. Therefore, a biomarker to assess molecular residual disease (MRD) accurately and guide treatment selection is highly desirable for high-risk patients. This feasibility study evaluated the prognostic value of a tissue comprehensive genomic profiling (CGP)-informed, personalized circulating tumor DNA (ctDNA) assay (FoundationOne®Tracker) (Foundation Medicine, Inc., Cambridge, MA, USA) by correlating MRD status with clinical outcomes. ctDNA analysis was performed retrospectively on plasma samples from 69 patients with resected mCRC obtained at the MRD and the follow-up time point. Tissue CGP identified potentially actionable alterations in 54% (37/69) of patients. MRD-positivity was significantly associated with lower disease-free survival (DFS) (HR: 4.97, 95% CI: 2.67−9.24, p < 0.0001) and overall survival (OS) (HR: 27.05, 95% CI: 3.60−203.46, p < 0.0001). Similarly, ctDNA positive status at the follow-up time point correlated with a marked reduction in DFS (HR: 8.78, 95% CI: 3.59−21.49, p < 0.0001) and OS (HR: 20.06, 95% CI: 2.51−160.25, p < 0.0001). The overall sensitivity and specificity at the follow-up time point were 69% and 100%, respectively. Our results indicate that MRD detection using the tissue CGP-informed ctDNA assay is prognostic of survival outcomes in patients with resected mCRC. The concurrent MRD detection and identification of actionable alterations has the potential to guide perioperative clinical decision-making.

A pan-cancer analysis of PD-L1 immunohistochemistry and gene amplification, tumor mutation burden and microsatellite instability in 48,782 cases.

PD-L1 immunohistochemistry (IHC) currently has the most Food and Drug Administration (FDA) approvals as a companion diagnostic (CDx) for immunotherapies in specific tumor types; however, multiple other immunotherapy biomarkers exist. We performed this study to examine and report the prevalence of PD-L1 expression in a wide variety of tumor types and examine its relationship to microsatellite instability (MSI), tumor mutational burden (TMB), and CD274 (PD-L1) gene amplification. We performed a retrospective analysis of all cases in which both PD-L1 IHC (using the DAKO 22C3 IHC assay with either tumor proportion score (TPS) or combined positive score (CPS); or the VENTANA SP142 assay with infiltrating immune cell score (IC)) and comprehensive genomic profiling (CGP) were tested at Foundation Medicine between January 2016 and November 2019. Of note, PD-L1 positivity is defined per the CDx indication and tumor proportion score (TPS ≥ 1) for indications without a CDx claim; and TMB positivity is defined as ≥10 mutations/Mb. A total of 48,782 cases were tested for PD-L1 IHC and CGP. Immune cell expression of PD-L1 was more frequently identified than tumor cell expression of PD-L1. We saw a high correlation between PD-L1 expression and CD274 gene amplification (p < 0.0001), MSI and TMB (p < 0.0001), and PD-L1 and TMB (p < 0.0001). In addition, the combination of PD-L1 and TMB identified four unique disease subsets PD-L1-/TMB-, PD-L1+/TMB-, PD-L1-/TMB+, and PD-L1+/TMB+ with varying prevalence dependent on tumor type. Lastly, 50.3% (24527/48782) of the overall cohort was positive for at least one of the CDx or exploratory biomarkers described above. This is the largest pan-cancer analysis of relevant biomarkers associated with response to checkpoint inhibitors to date, including more than 48,000 cases. Additional clinical trials with treatment outcome data in individual tumor types are needed to determine whether the double positive PD-L1+/TMB+ disease subset would respond best to immunotherapy.

Differential regulation of PD-L1 expression by immune and tumor cells in NSCLC and the response to treatment with atezolizumab (anti-PD-L1).

Programmed death-ligand 1 (PD-L1) expression on tumor cells (TCs) by immunohistochemistry is rapidly gaining importance as a diagnostic for the selection or stratification of patients with non-small cell lung cancer (NSCLC) most likely to respond to single-agent checkpoint inhibitors. However, at least two distinct patterns of PD-L1 expression have been observed with potential biological and clinical relevance in NSCLC: expression on TC or on tumor-infiltrating immune cells (ICs). We investigated the molecular and cellular characteristics associated with PD-L1 expression in these distinct cell compartments in 4,549 cases of NSCLC. PD-L1 expression on IC was more prevalent and likely reflected IFN-γ-induced adaptive regulation accompanied by increased tumor-infiltrating lymphocytes and effector T cells. High PD-L1 expression on TC, however, reflected an epigenetic dysregulation of the PD-L1 gene and was associated with a distinct histology described by poor immune infiltration, sclerotic/desmoplastic stroma, and mesenchymal molecular features. Importantly, durable clinical responses to atezolizumab (anti-PD-L1) were observed in patients with tumors expressing high PD-L1 levels on either TC alone [40% objective response rate (ORR)] or IC alone (22% ORR). Thus, PD-L1 expression on TC or IC can independently attenuate anticancer immunity and emphasizes the functional importance of IC in regulating the antitumor T cell response.

MPDL3280A (anti-PD-L1) treatment leads to clinical activity in metastatic bladder cancer.

There have been no major advances for the treatment of metastatic urothelial bladder cancer (UBC) in the last 30 years. Chemotherapy is still the standard of care. Patient outcomes, especially for those in whom chemotherapy is not effective or is poorly tolerated, remain poor. One hallmark of UBC is the presence of high rates of somatic mutations. These alterations may enhance the ability of the host immune system to recognize tumour cells as foreign owing to an increased number of antigens. However, these cancers may also elude immune surveillance and eradication through the expression of programmed death-ligand 1 (PD-L1; also called CD274 or B7-H1) in the tumour microenvironment. Therefore, we examined the anti-PD-L1 antibody MPDL3280A, a systemic cancer immunotherapy, for the treatment of metastatic UBC. MPDL3280A is a high-affinity engineered human anti-PD-L1 monoclonal immunoglobulin-G1 antibody that inhibits the interaction of PD-L1 with PD-1 (PDCD1) and B7.1 (CD80). Because PD-L1 is expressed on activated T cells, MPDL3280A was engineered with a modification in the Fc domain that eliminates antibody-dependent cellular cytotoxicity at clinically relevant doses to prevent the depletion of T cells expressing PD-L1. Here we show that MPDL3280A has noteworthy activity in metastatic UBC. Responses were often rapid, with many occurring at the time of the first response assessment (6 weeks) and nearly all were ongoing at the data cutoff. This phase I expansion study, with an adaptive design that allowed for biomarker-positive enriched cohorts, demonstrated that tumours expressing PD-L1-positive tumour-infiltrating immune cells had particularly high response rates. Moreover, owing to the favourable toxicity profile, including a lack of renal toxicity, patients with UBC, who are often older and have a higher incidence of renal impairment, may be better able to tolerate MPDL3280A versus chemotherapy. These results suggest that MPDL3280A may have an important role in treating UBC-the drug received breakthrough designation status by the US Food and Drug Administration (FDA) in June 2014.

Predictive correlates of response to the anti-PD-L1 antibody MPDL3280A in cancer patients.

The development of human cancer is a multistep process characterized by the accumulation of genetic and epigenetic alterations that drive or reflect tumour progression. These changes distinguish cancer cells from their normal counterparts, allowing tumours to be recognized as foreign by the immune system. However, tumours are rarely rejected spontaneously, reflecting their ability to maintain an immunosuppressive microenvironment. Programmed death-ligand 1 (PD-L1; also called B7-H1 or CD274), which is expressed on many cancer and immune cells, plays an important part in blocking the 'cancer immunity cycle' by binding programmed death-1 (PD-1) and B7.1 (CD80), both of which are negative regulators of T-lymphocyte activation. Binding of PD-L1 to its receptors suppresses T-cell migration, proliferation and secretion of cytotoxic mediators, and restricts tumour cell killing. The PD-L1-PD-1 axis protects the host from overactive T-effector cells not only in cancer but also during microbial infections. Blocking PD-L1 should therefore enhance anticancer immunity, but little is known about predictive factors of efficacy. This study was designed to evaluate the safety, activity and biomarkers of PD-L1 inhibition using the engineered humanized antibody MPDL3280A. Here we show that across multiple cancer types, responses (as evaluated by Response Evaluation Criteria in Solid Tumours, version 1.1) were observed in patients with tumours expressing high levels of PD-L1, especially when PD-L1 was expressed by tumour-infiltrating immune cells. Furthermore, responses were associated with T-helper type 1 (TH1) gene expression, CTLA4 expression and the absence of fractalkine (CX3CL1) in baseline tumour specimens. Together, these data suggest that MPDL3280A is most effective in patients in which pre-existing immunity is suppressed by PD-L1, and is re-invigorated on antibody treatment.

Predictive markers of anti-VEGF and emerging role of angiogenesis inhibitors as immunotherapeutics.

The critical role of angiogenesis in promoting tumor growth and metastasis has been well established scientifically, and consequently blocking this pathway as a therapeutic strategy has demonstrated great clinical success for the treatment of cancer. The holy grail however, has been the identification of patients who derive significant survival benefit from this class of agents. Here we attempt to delineate the diverse mechanisms related to anti-VEGF including its role as an anti-vascular, anti-angiogenic or an anti-permeability factor and review the most promising predictive biomarkers interrogated in large clinical trials, that identify patients who may derive significant survival advantage with VEGF inhibition. Lastly, we describe the function of VEGF as an immunomodulator and illustrate the evidence for anti-VEGF in reprogramming the tumor milieu from an immunosuppressive to an immune permissive microenvironment in human cancers, thus elucidating the role of anti-VEGF as an optimal combination partner for immune checkpoint inhibitors.

Atezolizumab versus chemotherapy in patients with platinum-treated locally advanced or metastatic urothelial carcinoma (IMvigor211): a multicentre, open-label, phase 3 randomised controlled trial.

BACKGROUND: Few options exist for patients with locally advanced or metastatic urothelial carcinoma after progression with platinum-based chemotherapy. We aimed to assess the safety and efficacy of atezolizumab (anti-programmed death-ligand 1 [PD-L1]) versus chemotherapy in this patient population. METHODS: We conducted this multicentre, open-label, phase 3 randomised controlled trial (IMvigor211) at 217 academic medical centres and community oncology practices mainly in Europe, North America, and the Asia-Pacific region. Patients (aged ≥18 years) with metastatic urothelial carcinoma who had progressed after platinum-based chemotherapy were randomly assigned (1:1), via an interactive voice and web response system with a permuted block design (block size of four), to receive atezolizumab 1200 mg or chemotherapy (physician's choice: vinflunine 320 mg/m2, paclitaxel 175 mg/m2, or 75 mg/m2 docetaxel) intravenously every 3 weeks. Randomisation was stratified by PD-L1 expression (expression on <1% [IC0] or 1% to <5% [IC1] of tumour-infiltrating immune cells vs ≥5% of tumour-infiltrating immune cells [IC2/3]), chemotherapy type (vinflunine vs taxanes), liver metastases (yes vs no), and number of prognostic factors (none vs one, two, or three). Patients and investigators were aware of group allocation. Patients, investigators, and the sponsor were masked to PD-L1 expression status. The primary endpoint of overall survival was tested hierarchically in prespecified populations: IC2/3, followed by IC1/2/3, followed by the intention-to-treat population. This study, which is ongoing but not recruiting participants, is registered with ClinicalTrials.gov, number NCT02302807. FINDINGS: Between Jan 13, 2015, and Feb 15, 2016, we randomly assigned 931 patients from 198 sites to receive atezolizumab (n=467) or chemotherapy (n=464). In the IC2/3 population (n=234), overall survival did not differ significantly between patients in the atezolizumab group and those in the chemotherapy group (median 11·1 months [95% CI 8·6-15·5; n=116] vs 10·6 months [8·4-12·2; n=118]; stratified hazard ratio [HR] 0·87, 95% CI 0·63-1·21; p=0·41), thus precluding further formal statistical analysis. Confirmed objective response rates were similar between treatment groups in the IC2/3 population: 26 (23%) of 113 evaluable patients had an objective response in the atezolizumab group compared with 25 (22%) of 116 patients in the chemotherapy group. Duration of response was numerically longer in the atezolizumab group than in the chemotherapy group (median 15·9 months [95% CI 10·4 to not estimable] vs 8·3 months [5·6-13·2]; HR 0·57, 95% CI 0·26-1·26). In the intention-to-treat population, patients receiving atezolizumab had fewer grade 3-4 treatment-related adverse events than did those receiving chemotherapy (91 [20%] of 459 vs 189 [43%] of 443 patients), and fewer adverse events leading to treatment discontinuation (34 [7%] vs 78 [18%] patients). INTERPRETATION: Atezolizumab was not associated with significantly longer overall survival than chemotherapy in patients with platinum-refractory metastatic urothelial carcinoma overexpressing PD-L1 (IC2/3). However, the safety profile for atezolizumab was favourable compared with chemotherapy, Exploratory analysis of the intention-to-treat population showed well-tolerated, durable responses in line with previous phase 2 data for atezolizumab in this setting. FUNDING: F Hoffmann-La Roche, Genentech.

Atezolizumab as first-line treatment in cisplatin-ineligible patients with locally advanced and metastatic urothelial carcinoma: a single-arm, multicentre, phase 2 trial.

BACKGROUND: First-line chemotherapy for patients with cisplatin-ineligible locally advanced or metastatic urothelial carcinoma is associated with short response duration, poor survival, and high toxicity. This study assessed atezolizumab (anti-programmed death-ligand 1 [PD-L1]) as treatment for metastatic urothelial cancer in cisplatin-ineligible patients. METHODS: For this single-arm, multicentre, phase 2 study, in 47 academic medical centres and community oncology practices in seven countries in North America and Europe, we recruited previously untreated patients with locally advanced or metastatic urothelial cancer who were cisplatin ineligible. Patients were given 1200 mg intravenous atezolizumab every 21 days until progression. The primary endpoint was independently confirmed objective response rate per Response Evaluation Criteria in Solid Tumors version 1.1 (central review), assessed in prespecified subgroups based on PD-L1 expression and in all patients. All participants who received one or more doses of atezolizumab were included in the primary and safety analyses. This study was registered with ClinicalTrials.gov, number NCT02108652. FINDINGS: Between June 9, 2014, and March 30, 2015, we enrolled 123 patients, of whom 119 received one or more doses of atezolizumab. At 17·2 months' median follow-up, the objective response rate was 23% (95% CI 16 to 31), the complete response rate was 9% (n=11), and 19 of 27 responses were ongoing. Median response duration was not reached. Responses occurred across all PD-L1 and poor prognostic factor subgroups. Median progression-free survival was 2·7 months (2·1 to 4·2). Median overall survival was 15·9 months (10·4 to not estimable). Tumour mutation load was associated with response. Treatment-related adverse events that occurred in 10% or more of patients were fatigue (36 [30%] patients), diarrhoea (14 [12%] patients), and pruritus (13 [11%] patients). One treatment-related death (sepsis) occurred. Nine (8%) patients had an adverse event leading to treatment discontinuation. Immune-mediated events occurred in 14 (12%) patients. INTERPRETATION: Atezolizumab showed encouraging durable response rates, survival, and tolerability, supporting its therapeutic use in untreated metastatic urothelial cancer. FUNDING: F Hoffmann-La Roche, Genentech.

Randomized Phase II Trial of Parsatuzumab (Anti-EGFL7) or Placebo in Combination with Carboplatin, Paclitaxel, and Bevacizumab for First-Line Nonsquamous Non-Small Cell Lung Cancer.

LESSONS LEARNED: The lack of efficacy associated with anti-EGFL7 combined with standard bevacizumab and chemotherapy in this phase II trial in non-small cell lung carcinoma is consistent with the lack of benefit observed in colorectal carcinoma, highlighting the challenge of enhancing the efficacy of VEGF inhibition in unselected populations.Future efforts with agents like anti-EGFL7 should be guided by advances in pharmacodynamic and predictive biomarker development for antiangiogenic agents. BACKGROUND: Epidermal growth factor-like domain 7 (EGFL7) is an extracellular matrix-associated protein that is upregulated during angiogenesis and supports endothelial cell survival. This phase II trial evaluated the efficacy of the anti-EGFL7 antibody, parsatuzumab, in combination with bevacizumab plus platinum-based therapy for advanced or recurrent nonsquamous non-small cell lung cancer (NS-NSCLC). METHODS: Patients (n = 104) were randomized to either placebo or parsatuzumab (600 mg) in combination with bevacizumab (15 mg/kg) and carboplatin/paclitaxel, administered on day 1 of each 21-day cycle. Carboplatin and paclitaxel were administered for up to six cycles. Bevacizumab and parsatuzumab/placebo were administered for a maximum of 24 months. RESULTS: The progression-free survival (PFS) hazard ratio (HR) was 1.7 (95% confidence interval [CI], 1.0-2.8; p = .047). The median PFS was 6.7 months for the parsatuzumab arm versus 8.1 months for the placebo arm. The hazard ratio for overall survival (OS) was 1.1 (95% CI, 0.5-2.2; p = .847). The objective response rate (ORR) was 29% in the parsatuzumab arm and 56% in the placebo arm. Overall safety and tolerability were consistent with the established toxicity profile of bevacizumab. CONCLUSION: There was no evidence of efficacy for the addition of parsatuzumab to the combination of bevacizumab and chemotherapy for first-line NS-NSCLC.

Randomized Phase II Trial of Parsatuzumab (Anti-EGFL7) or Placebo in Combination with FOLFOX and Bevacizumab for First-Line Metastatic Colorectal Cancer.

LESSONS LEARNED: These negative phase II results for parsatuzumab highlight the challenges of developing an agent intended to enhance the efficacy of vascular endothelial growth factor inhibition without the benefit of validated pharmacodynamic biomarkers or strong predictive biomarker hypotheses.Any further clinical development of anti-EGFL7 is likely to require new mechanistic insights and biomarker development for antiangiogenic agents. BACKGROUND: EGFL7 (epidermal growth factor-like domain 7) is a tumor-enriched vascular extracellular matrix protein that supports endothelial cell survival. This phase II trial evaluated the efficacy of parsatuzumab (also known as MEGF0444A), a humanized anti-EGFL7 IgG1 monoclonal antibody, in combination with modified FOLFOX6 (mFOLFOX6) (folinic acid, 5-fluorouracil, and oxaliplatin) bevacizumab in patients with previously untreated metastatic colorectal cancer (mCRC). METHODS: One-hundred twenty-seven patients were randomly assigned to parsatuzumab, 400 mg, or placebo, in combination with mFOLFOX6 plus bevacizumab, 5 mg/kg. Treatment cycles were repeated every 2 weeks until disease progression or unacceptable toxicity for a maximum of 24 months, with the exception of oxaliplatin, which was administered for up to 8 cycles. RESULTS: The progression-free survival (PFS) hazard ratio was 1.17 (95% confidence interval [CI], 0.71-1.93; p = .548). The median PFS was 12 months for the experimental arm versus 11.9 months for the control arm. The hazard ratio for overall survival was 0.97 (95% CI, 0.46-2.1; p = .943). The overall response rate was 59% in the parsatuzumab arm and 64% in the placebo arm. The adverse event profile was similar in both arms. CONCLUSIONS: There was no evidence of efficacy for the addition of parsatuzumab to the combination of bevacizumab and chemotherapy for first-line mCRC. The Oncologist 2017;22:375-e30.

Profiling cancer gene mutations in clinical formalin-fixed, paraffin-embedded colorectal tumor specimens using targeted next-generation sequencing.

PURPOSE: The success of precision oncology relies on accurate and sensitive molecular profiling. The Ion AmpliSeq Cancer Panel, a targeted enrichment method for next-generation sequencing (NGS) using the Ion Torrent platform, provides a fast, easy, and cost-effective sequencing workflow for detecting genomic "hotspot" regions that are frequently mutated in human cancer genes. Most recently, the U.K. has launched the AmpliSeq sequencing test in its National Health Service. This study aimed to evaluate the clinical application of the AmpliSeq methodology. METHODS: We used 10 ng of genomic DNA from formalin-fixed, paraffin-embedded human colorectal cancer (CRC) tumor specimens to sequence 46 cancer genes using the AmpliSeq platform. In a validation study, we developed an orthogonal NGS-based resequencing approach (SimpliSeq) to assess the AmpliSeq variant calls. RESULTS: Validated mutational analyses revealed that AmpliSeq was effective in profiling gene mutations, and that the method correctly pinpointed "true-positive" gene mutations with variant frequency >5% and demonstrated high-level molecular heterogeneity in CRC. However, AmpliSeq enrichment and NGS also produced several recurrent "false-positive" calls in clinically druggable oncogenes such as PIK3CA. CONCLUSION: AmpliSeq provided highly sensitive and quantitative mutation detection for most of the genes on its cancer panel using limited DNA quantities from formalin-fixed, paraffin-embedded samples. For those genes with recurrent "false-positive" variant calls, caution should be used in data interpretation, and orthogonal verification of mutations is recommended for clinical decision making.

A phase I study of the human monoclonal anti-NRP1 antibody MNRP1685A in patients with advanced solid tumors.

The human monoclonal antibody MNRP1685A targets the VEGF binding domain of neuropilin-1 (NRP1), a multi-domain receptor necessary for neural development and blood vessel maturation. In nonclinical studies, MNRP1685A prevents vascular maturation by keeping blood vessels in an immature, highly VEGF-dependent state. We explored the safety and tolerability of MNRP1685A in patients with advanced solid tumors. Patients were treated with MNRP1685A given intravenously every 3 weeks using a 3 + 3 dose-escalation design with 7 dose-escalation cohorts. Twenty-four of 35 patients (69 %) experienced drug-related adverse events (AEs) of infusion-related reaction on the day of MNRP1685A administration. With premedication including dexamethasone, infusions were well-tolerated with main symptoms of pruritus and rash. Outside the day of infusion, most common (≥ 2 patients) related AEs were fatigue (17 %), pruritus (9 %), myalgia and thrombocytopenia (both 6 %) (all were Grade 1-2). MNRP1685A-related Grade ≥ 3 AEs consisted of one dose-limiting toxicity of Grade 3 upper gastrointestinal bleeding and one related Grade 3 thrombocytopenia, coinciding with unrelated Grade 3 fungemia and duodenal obstruction. MNRP1685A showed nonlinear PK with more-than-dose proportional increases in exposure, consistent with broad target expression. Transient platelet count reductions (≥ 30 % from predose) were observed in 56 % of evaluable patients. Nine patients were on study for ≥ 4 cycles, one colorectal cancer patient for one year. MNRP1685A was generally well-tolerated. The primary MNRP1685A-related AE was infusion-related reaction, which were attenuated by premedication including dexamethasone. Transient platelet count reductions were frequent but did not impact MNRP1685A dosing.

Circulating Tumor DNA Assessment for Treatment Monitoring Adds Value to PSA in Metastatic Castration Resistant Prostate Cancer.

PURPOSE: Enzalutamide after abiraterone progression is commonly used in metastatic castration resistant prostate cancer (mCRPC) despite a low rate of clinical benefit. Analyzing IMbassador250, a phase III trial assessing enzalutamide with or without atezolizumab after abiraterone, we hypothesized that baseline and early changes in circulating tumor DNA (ctDNA) tumor fraction (TF) may identify patients more likely to exhibit survival benefit from enzalutamide. EXPERIMENTAL DESIGN: ctDNA was quantified from plasma samples using a tissue-agnostic assay without buffy coat sequencing. Baseline ctDNA TF, changes in ctDNA TF from baseline to cycle 3 day 1 (C3D1), and detection at C3D1 alone, were compared vs overall response rate (ORR), radiographic progression-free survival (rPFS), median OS (mOS), and 50% reduction in PSA. RESULTS: ctDNA TF detection at baseline and/or C3D1 was associated with shorter rPFS and OS in 494 evaluable patients. Detection of ctDNA TF at C3D1, with or without detection at C1D1, was associated with worse rPFS and mOS than lack of detection. When ctDNA TF and PSA response at C3D1 were discordant, patients with [ctDNA TF undetected/PSA not reduced] had more favorable outcomes than [ctDNA TF detected/PSA reduced] (mOS 22.1 months vs. 16 months, p<0.001). CONCLUSIONS: In a large cohort of mCRPC patients receiving enzalutamide after abiraterone, we demonstrate the utility of a new tissue-agnostic assay for monitoring molecular response based on ctDNA TF detection and dynamics. CtDNA TF provides a minimally-invasive, complementary biomarker to PSA testing and may refine personalized treatment approaches.

Analytical validation of a novel comprehensive genomic profiling informed circulating tumor DNA monitoring assay for solid tumors.

Emerging technologies focused on the detection and quantification of circulating tumor DNA (ctDNA) in blood show extensive potential for managing patient treatment decisions, informing risk of recurrence, and predicting response to therapy. Currently available tissue-informed approaches are often limited by the need for additional sequencing of normal tissue or peripheral mononuclear cells to identify non-tumor-derived alterations while tissue-naïve approaches are often limited in sensitivity. Here we present the analytical validation for a novel ctDNA monitoring assay, FoundationOne®Tracker. The assay utilizes somatic alterations from comprehensive genomic profiling (CGP) of tumor tissue. A novel algorithm identifies monitorable alterations with a high probability of being somatic and computationally filters non-tumor-derived alterations such as germline or clonal hematopoiesis variants without the need for sequencing of additional samples. Monitorable alterations identified from tissue CGP are then quantified in blood using a multiplex polymerase chain reaction assay based on the validated SignateraTM assay. The analytical specificity of the plasma workflow is shown to be 99.6% at the sample level. Analytical sensitivity is shown to be >97.3% at ≥5 mean tumor molecules per mL of plasma (MTM/mL) when tested with the most conservative configuration using only two monitorable alterations. The assay also demonstrates high analytical accuracy when compared to liquid biopsy-based CGP as well as high qualitative (measured 100% PPA) and quantitative precision (<11.2% coefficient of variation).