A Cell-free DNA Blood-Based Test for Colorectal Cancer Screening.

BACKGROUND: Colorectal cancer is the third most diagnosed cancer in adults in the United States. Early detection could prevent more than 90% of colorectal cancer-related deaths, yet more than one third of the screening-eligible population is not up to date with screening despite multiple available tests. A blood-based test has the potential to improve screening adherence, detect colorectal cancer earlier, and reduce colorectal cancer-related mortality. METHODS: We assessed the performance characteristics of a cell-free DNA (cfDNA) blood-based test in a population eligible for colorectal cancer screening. The coprimary outcomes were sensitivity for colorectal cancer and specificity for advanced neoplasia (colorectal cancer or advanced precancerous lesions) relative to screening colonoscopy. The secondary outcome was sensitivity to detect advanced precancerous lesions. RESULTS: The clinical validation cohort included 10,258 persons, 7861 of whom met eligibility criteria and were evaluable. A total of 83.1% of the participants with colorectal cancer detected by colonoscopy had a positive cfDNA test and 16.9% had a negative test, which indicates a sensitivity of the cfDNA test for detection of colorectal cancer of 83.1% (95% confidence interval [CI], 72.2 to 90.3). Sensitivity for stage I, II, or III colorectal cancer was 87.5% (95% CI, 75.3 to 94.1), and sensitivity for advanced precancerous lesions was 13.2% (95% CI, 11.3 to 15.3). A total of 89.6% of the participants without any advanced colorectal neoplasia (colorectal cancer or advanced precancerous lesions) identified on colonoscopy had a negative cfDNA blood-based test, whereas 10.4% had a positive cfDNA blood-based test, which indicates a specificity for any advanced neoplasia of 89.6% (95% CI, 88.8 to 90.3). Specificity for negative colonoscopy (no colorectal cancer, advanced precancerous lesions, or nonadvanced precancerous lesions) was 89.9% (95% CI, 89.0 to 90.7). CONCLUSIONS: In an average-risk screening population, this cfDNA blood-based test had 83% sensitivity for colorectal cancer, 90% specificity for advanced neoplasia, and 13% sensitivity for advanced precancerous lesions. (Funded by Guardant Health; ECLIPSE ClinicalTrials.gov number, NCT04136002.).

Blood-based tumor mutational burden as a biomarker for atezolizumab in non-small cell lung cancer: the phase 2 B-F1RST trial.

Tumor mutational burden (TMB) in circulating tumor DNA (ctDNA) has shown promise in predicting benefit from PD-L1/PD-1 inhibitors in retrospective studies. Aiming to assess blood TMB (bTMB) prospectively, we conducted B-F1RST ( NCT02848651 ), an open-label, phase 2 trial that evaluated bTMB as a predictive biomarker for first-line atezolizumab monotherapy in locally advanced or metastatic stage IIIB-IVB non-small cell lung cancer (n = 152). The co-primary endpoints were investigator-assessed objective response rate (ORR) per RECIST version 1.1 and investigator-assessed progression-free survival (PFS) between high and low bTMB subgroups at the pre-defined bTMB ≥ 16 (14.5 mutations per megabase) cutoff. Secondary endpoints included investigator-assessed PFS, overall survival (OS) and duration of response at various bTMB cutoffs, as well as safety. Investigator-assessed PFS in the bTMB ≥ 16 versus bTMB < 16 groups was not statistically significant. However, bTMB ≥ 16 was associated with higher ORR, and ORR improved as bTMB cutoffs increased. No new safety signals were seen. In exploratory analyses, patients with maximum somatic allele frequency (MSAF) < 1% had higher ORR than patients with MSAF ≥ 1%. However, further analysis showed that this effect was driven by better baseline prognostics rather than by MSAF itself. At 36.5-month follow-up, an exploratory analysis of OS found that bTMB ≥ 16 was associated with longer OS than bTMB < 16. Further study and assay optimization will be required to develop bTMB as a predictive, standalone biomarker of immunotherapy or for use in conjunction with other biomarkers.

Oncogene-specific differences in tumor mutational burden, PD-L1 expression, and outcomes from immunotherapy in non-small cell lung cancer.

BACKGROUND: Non-small cell lung cancer (NSCLC) patients bearing targetable oncogene alterations typically derive limited benefit from immune checkpoint blockade (ICB), which has been attributed to low tumor mutation burden (TMB) and/or PD-L1 levels. We investigated oncogene-specific differences in these markers and clinical outcome. METHODS: Three cohorts of NSCLC patients with oncogene alterations (n=4189 total) were analyzed. Two clinical cohorts of advanced NSCLC patients treated with ICB monotherapy [MD Anderson (MDACC; n=172) and Flatiron Health-Foundation Medicine Clinico-Genomic Database (CGDB; n=894 patients)] were analyzed for clinical outcome. The FMI biomarker cohort (n=4017) was used to assess the association of oncogene alterations with TMB and PD-L1 expression. RESULTS: High PD-L1 expression (PD-L1 ≥50%) rate was 19%-20% in classic EGFR, EGFR exon 20 and HER2-mutant tumors, and 34%-55% in tumors with ALK, BRAF V600E, ROS1, RET, or MET alterations. Compared with KRAS-mutant tumors, BRAF non-V600E group had higher TMB (9.6 vs KRAS 7.8 mutations/Mb, p=0.003), while all other oncogene groups had lower TMB (p<0.001). In the two clinical cohorts treated with ICB, molecular groups with EGFR, HER2, ALK, ROS1, RET, or MET alterations had short progression-free survival (PFS; 1.8-3.7 months), while BRAF V600E group was associated with greater clinical benefit from ICB (CGDB cohort: PFS 9.8 months vs KRAS 3.7 months, HR 0.66, p=0.099; MDACC cohort: response rate 62% vs KRAS 24%; PFS 7.4 vs KRAS 2.8 months, HR 0.36, p=0.026). KRAS G12C and non-G12C subgroups had similar clinical benefit from ICB in both cohorts. In a multivariable analysis, BRAF V600E mutation (HR 0.58, p=0.041), PD-L1 expression (HR 0.57, p=0.022), and high TMB (HR 0.66, p<0.001) were associated with longer PFS. CONCLUSIONS: High TMB and PD-L1 expression are predictive for benefit from ICB treatment in oncogene-driven NSCLCs. NSCLC harboring BRAF mutations demonstrated superior benefit from ICB that may be attributed to higher TMB and higher PD-L1 expression in these tumors. Meanwhile EGFR and HER2 mutations and ALK, ROS1, RET, and MET fusions define NSCLC subsets with minimal benefit from ICB despite high PD-L1 expression in NSCLC harboring oncogene fusions. These findings indicate a TMB/PD-L1-independent impact on sensitivity to ICB for certain oncogene alterations.

Atezolizumab in patients with advanced non-small cell lung cancer and history of asymptomatic, treated brain metastases: Exploratory analyses of the phase III OAK study.

OBJECTIVES: To assess the safety and efficacy of atezolizumab and docetaxel in patients with and without a history of asymptomatic, treated brain metastases in the phase III OAK trial. MATERIALS AND METHODS: Patients received 1200 mg atezolizumab or 75 mg/m2 docetaxel every 3 weeks until unacceptable toxicity, disease progression, or loss of clinical atezolizumab benefit. Patients with asymptomatic, treated supratentorial metastases were eligible. Patients had brain scans before enrollment; follow-up brain scans and treatment were required when clinically indicated. RESULTS: Approximately 14% of patients in each arm had a history of asymptomatic, treated brain metastases (61/425 in the atezolizumab arm and 62/425 in the docetaxel arm). Fewer treatment-related adverse events (AEs), serious AEs, and treatment-related neurologic AEs were reported with atezolizumab than with docetaxel, regardless of history of asymptomatic, treated brain metastases. In patients with a history of asymptomatic, treated brain metastases, median overall survival (OS) was longer with atezolizumab than with docetaxel (16.0 vs 11.9 months; hazard ratio = 0.74; 95% CI: 0.49-1.13). Median OS was also longer with atezolizumab in patients without a history of asymptomatic, treated brain metastases (13.2 vs 9.3 months; hazard ratio = 0.74; 95% CI: 0.63-0.88). Landmark analyses showed that patients with a history of asymptomatic, treated brain metastases had a lower probability of developing new symptomatic brain lesions with atezolizumab vs docetaxel at 6-24 months. Patients without a history had a lower probability with atezolizumab at 18-24+ months. CONCLUSION: Atezolizumab had an acceptable neurologic safety profile, showed a trend toward an OS benefit, and led to a prolonged time to radiographic identification of new symptomatic brain lesions compared with docetaxel in patients who had a history of asymptomatic, treated brain metastases. Clinicaltrials.gov registration number: NCT02008227.

A randomized, phase II trial of standard triweekly compared with dose-dense biweekly capecitabine plus oxaliplatin plus bevacizumab as first-line treatment for metastatic colorectal cancer: XELOX-A-DVS (dense versus standard).

BACKGROUND: Capecitabine administered for 7 days biweekly with oxaliplatin (XELOX) biweekly has been reported to have activity and safety profiles similar to those of standard capecitabine given for 14 days triweekly. Multiple studies have shown that the addition of bevacizumab to 5-fluorouracil-based chemotherapy is active and well tolerated. METHODS: Patients with metastatic colorectal cancer (mCRC) were randomized to XELOX plus bevacizumab using a standard triweekly cycle (Q3W) or a dose-dense biweekly cycle (Q2W) schedule. The primary endpoint was the progression-free survival (PFS) interval. This trial is registered on ClinicalTrials.gov (identifier, NCT00159432). RESULTS: In total, 435 U.S. patients were randomized. The median PFS intervals were 9.6 months in the Q3W group and 9.1 months in the Q2W group. The median overall survival times were 28.4 months and 22.1 months and the median times to treatment failure were 5.5 months and 3.4 months, respectively. Overall, gastrointestinal disorders were the most common (93%) adverse event (AE). Grade 3 or 4 AEs occurred in 75% and 81% of patients in the Q3W and Q2W groups, respectively. Treatment discontinuation as a result of diarrhea (5% versus 10%) and hand-foot syndrome (2% versus 9%) was less common in the Q3W group than in the Q2W group, respectively. CONCLUSIONS: Based on these results, the first-line treatment of U.S. patients with mCRC using a biweekly combination of XELOX and bevacizumab at the doses studied cannot be recommended. XELOX Q3W remains the preferred schedule for the management of mCRC.

An apoA-I mimetic peptibody generates HDL-like particles and increases alpha-1 HDL subfraction in mice.

The aim of this study is to investigate the capability of an apoA-I mimetic with multiple amphipathic helices to form HDL-like particles in vitro and in vivo. To generate multivalent helices and to track the peptide mimetic, we have constructed a peptibody by fusing two tandem repeats of 4F peptide to the C terminus of a murine IgG Fc fragment. The resultant peptidbody, mFc-2X4F, dose-dependently promoted cholesterol efflux in vitro, and the efflux potency was superior to monomeric 4F peptide. Like apoA-I, mFc-2X4F stabilized ABCA1 in J774A.1 and THP1 cells. The peptibody formed larger HDL particles when incubated with cultured cells compared with those by apoA-I. Interestingly, when administered to mice, mFc-2X4F increased both pre-ß and α-1 HDL subfractions. The lipid-bound mFc-2X4F was mostly in the α-1 migrating subfraction. Most importantly, mFc-2X4F and apoA-I were found to coexist in the same HDL particles formed in vivo. These data suggest that the apoA-I mimetic peptibody is capable of mimicking apoA-I to generate HDL particles. The peptibody and apoA-I may work cooperatively to generate larger HDL particles in vivo, either at the cholesterol efflux stage and/or via fusion of HDL particles that were generated by the peptibody and apoA-I individually.

Association of age and overall survival in capecitabine-treated patients with metastatic breast cancer in clinical trials.

We sought to determine if an association exists between age and capecitabine efficacy among patients with metastatic breast cancer (MBC). Pooled analysis of five phase II or III registration trials of capecitabine 2,500-2,510 mg/m(2)/day for 2 weeks and 1 week off, or combination therapy was performed. Four trials enrolled patients previously exposed to other chemotherapy, generally a taxane. Of 570 patients, 193 (34%) were 18-49 years old, 246 (43%) were 50-64, and 131 (23%) were ≥ 65. Median average daily dose was 2,067 mg/m² in the 18- to 49-year-old group and 2,105 mg/m² in the 50-64 and ≥ 65 year groups. Overall survival (OS) in all groups was similar by log-rank test for the individual trials (P = 0.71-0.95) and Cox regression of the pooled trials. Univariate analysis demonstrated no difference in clinical benefit or objective response between groups. Treatment failure analysis showed 283 (50%) patients experienced progressive disease, while 114 (20%) withdrew for safety. Serious adverse events (AEs) occurred in 71 (36.8%), 85 (34.6%), and 59 (45.0%) patients in the 18-49, 50-64, and ≥ 65 years groups, respectively. There was no statistically significant association between age and OS, clinical benefit, or objective response in patients with MBC treated with capecitabine. Frequency of AEs and serious AEs was not related to age at treatment, although women ≥ 65 years were more likely to withdraw from treatment because of an AE than younger women.

Long-term inhibition of the glucagon receptor with a monoclonal antibody in mice causes sustained improvement in glycemic control, with reversible alpha-cell hyperplasia and hyperglucagonemia.

Uncontrolled hepatic glucose output (HGO) contributes significantly to the pathological hyperglycemic state of patients with type 2 diabetes. Glucagon, through action on its receptor, stimulates HGO, thereby leading to increased glycemia. Antagonizing the glucagon signaling pathway represents an attractive therapeutic approach for the treatment of type 2 diabetes. We previously reported the generation and characterization of several high-affinity monoclonal antibodies (mAbs) targeting the glucagon receptor (GCGR). In the present study, we demonstrate that a 5-week treatment of diet-induced obese mice with mAb effectively normalized nonfasting blood glucose. Similar treatment also reduced fasting blood glucose without inducing hypoglycemia or other undesirable metabolic perturbations. In addition, no hypoglycemia was found in db/db mice that were treated with a combination of insulin and mAb. Long-term treatment with the mAb caused dose-dependent hyperglucagonemia and minimal to mild alpha-cell hyperplasia in lean mice. There was no evidence of pancreatic alpha-cell neoplastic transformation in mice treated with mAb for as long as 18 weeks. Treatment-induced hyperglucagonemia and alpha-cell hyperplasia were reversible after treatment withdrawal for periods of 4 and 10 weeks, respectively. It is noteworthy that pancreatic beta-cell function was preserved, as demonstrated by improved glucose tolerance throughout the 18-week treatment period. Our studies further support the concept that long-term inhibition of GCGR signaling by a mAb could be an effective approach for controlling diabetic hyperglycemia.

Epratuzumab, a humanized monoclonal antibody targeting CD22: characterization of in vitro properties.

PURPOSE: Epratuzumab is a novel humanized antihuman CD22 IgG1 antibody that has recently shown promising clinical activity, both as a single agent and in combination with rituximab, in patients with non-Hodgkin's lymphomas (NHL). In an attempt to better understand the mode of action of epratuzumab, the antibody was tested in vitro in a variety of cell-based assays similar to those used to evaluate the biological activity of other therapeutic monoclonal antibodies, including rituximab. In this report, we present epratuzumab activities as they relate to binding, signaling, and internalization of the receptor CD22. METHODS: Chinese hamster ovary-expressed CD22 extracellular domain was used to measure epratuzumab affinity on Biacore. CD22 receptor density and internalization rate were measured indirectly using a monovalently labeled, noncompeting (with epratuzumab) anti-CD22 antibody on Burkitt lymphoma cell lines, primary B cells derived from fresh tonsils, and B cells separated from peripheral blood samples obtained from patients with chronic lymphocytic leukemia or healthy volunteers. Epratuzumab-induced CD22 phosphorylation was measured by immunoprecipitation/Western blot and compared with that induced by anti-IgM stimulation. RESULTS: Epratuzumab binds to CD22-extracellular domain, with an affinity of K(D) = 0.7 nM. Binding of epratuzumab to B cell lines, or primary B cells from healthy individuals and patients with NHL, results in rapid internalization of the CD22/antibody complex. Internalization appears to be faster at early time points in cell lines than in primary B cells and NHL patient-derived B cells, but the maximum internalization reached is comparable for all B cell populations after several hours of treatment and appears to reach saturation at antibody concentrations of 1-5 micro g/ml. Finally, epratuzumab binding results in modest but significant CD22 phosphorylation. CONCLUSIONS: Epratuzumab represents an excellent anti-CD22 ligating agent, highly efficacious in inducing CD22 internalization, and can induce phosphorylation. Although we cannot unequivocally demonstrate here that epratuzumab-induced internalization and signaling of CD22 directly contribute to its therapeutic efficacy, these properties are the fundamental characteristics of the target CD22 and its interaction with epratuzumab. Similar results were observed when epratuzumab was tested in vitro on Burkitt B cell lines as well as on primary normal B cells and neoplastic B cells separated from fresh peripheral blood samples from patients with chronic lymphocytic leukemia.

Enhancement of therapeutic protein in vivo activities through glycoengineering.

Delivery of protein therapeutics often requires frequent injections because of low activity or rapid clearance, thereby placing a burden on patients and caregivers. Using glycoengineering, we have increased and prolonged the activity of proteins, thus allowing reduced frequency of administration. Glycosylation analogs with new N-linked glycosylation consensus sequences introduced into the protein were screened for the presence of additional N-linked carbohydrates and retention of in vitro activity. Suitable consensus sequences were combined in one molecule, resulting in glycosylation analogs of rHuEPO, leptin, and Mpl ligand. All three molecules had substantially increased in vivo activity and prolonged duration of action. Because these proteins were of three different classes (rHuEPO is an N-linked glycoprotein, Mpl ligand an O-linked glycoprotein, and leptin contains no carbohydrate), glycoengineering may be generally applicable as a strategy for increasing the in vivo activity and duration of action of proteins. This strategy has been validated clinically for glycoengineered rHuEPO (darbopoetin alfa).