Circulating Tumor DNA Analysis Guiding Adjuvant Therapy in Stage II Colon Cancer.

BACKGROUND: The role of adjuvant chemotherapy in stage II colon cancer continues to be debated. The presence of circulating tumor DNA (ctDNA) after surgery predicts very poor recurrence-free survival, whereas its absence predicts a low risk of recurrence. The benefit of adjuvant chemotherapy for ctDNA-positive patients is not well understood. METHODS: We conducted a trial to assess whether a ctDNA-guided approach could reduce the use of adjuvant chemotherapy without compromising recurrence risk. Patients with stage II colon cancer were randomly assigned in a 2:1 ratio to have treatment decisions guided by either ctDNA results or standard clinicopathological features. For ctDNA-guided management, a ctDNA-positive result at 4 or 7 weeks after surgery prompted oxaliplatin-based or fluoropyrimidine chemotherapy. Patients who were ctDNA-negative were not treated. The primary efficacy end point was recurrence-free survival at 2 years. A key secondary end point was adjuvant chemotherapy use. RESULTS: Of the 455 patients who underwent randomization, 302 were assigned to ctDNA-guided management and 153 to standard management. The median follow-up was 37 months. A lower percentage of patients in the ctDNA-guided group than in the standard-management group received adjuvant chemotherapy (15% vs. 28%; relative risk, 1.82; 95% confidence interval [CI], 1.25 to 2.65). In the evaluation of 2-year recurrence-free survival, ctDNA-guided management was noninferior to standard management (93.5% and 92.4%, respectively; absolute difference, 1.1 percentage points; 95% CI, -4.1 to 6.2 [noninferiority margin, -8.5 percentage points]). Three-year recurrence-free survival was 86.4% among ctDNA-positive patients who received adjuvant chemotherapy and 92.5% among ctDNA-negative patients who did not. CONCLUSIONS: A ctDNA-guided approach to the treatment of stage II colon cancer reduced adjuvant chemotherapy use without compromising recurrence-free survival. (Supported by the Australian National Health and Medical Research Council and others; DYNAMIC Australian New Zealand Clinical Trials Registry number, ACTRN12615000381583.).

Circulating tumor DNA dynamics and recurrence risk in patients undergoing curative intent resection of colorectal cancer liver metastases: A prospective cohort study.

BACKGROUND: In patients with resectable colorectal liver metastases (CRLM), the role of pre- and postoperative systemic therapy continues to be debated. Previous studies have shown that circulating tumor DNA (ctDNA) analysis, as a marker of minimal residual disease, is a powerful prognostic factor in patients with nonmetastatic colorectal cancer (CRC). Serial analysis of ctDNA in patients with resectable CRLM could inform the optimal use of perioperative chemotherapy. Here, we performed a validation study to confirm the prognostic impact of postoperative ctDNA in resectable CRLM observed in a previous discovery study. METHODS AND FINDINGS: We prospectively collected plasma samples from patients with resectable CRLM, including presurgical and postsurgical samples, serial samples during any pre- or postoperative chemotherapy, and serial samples in follow-up. Via targeted sequencing of 15 genes commonly mutated in CRC, we identified at least 1 somatic mutation in each patient's tumor. We then designed a personalized assay to assess 1 mutation in plasma samples using the Safe-SeqS assay. A total of 380 plasma samples from 54 patients recruited from July 2011 to Dec 2014 were included in our analysis. Twenty-three (43%) patients received neoadjuvant chemotherapy, and 42 patients (78%) received adjuvant chemotherapy after surgery. Median follow-up was 51 months (interquartile range, 31 to 60 months). At least 1 somatic mutation was identified in all patients' tumor tissue. ctDNA was detectable in 46/54 (85%) patients prior to any treatment and 12/49 (24%) patients after surgery. There was a median 40.93-fold (19.10 to 87.73, P < 0.001) decrease in ctDNA mutant allele fraction with neoadjuvant chemotherapy, but ctDNA clearance during neoadjuvant chemotherapy was not associated with a better recurrence-free survival (RFS). Patients with detectable postoperative ctDNA experienced a significantly lower RFS (HR 6.3; 95% CI 2.58 to 15.2; P < 0.001) and overall survival (HR 4.2; 95% CI 1.5 to 11.8; P < 0.001) compared to patients with undetectable ctDNA. For the 11 patients with detectable postoperative ctDNA who had serial ctDNA sampling during adjuvant chemotherapy, ctDNA clearance was observed in 3 patients, 2 of whom remained disease-free. All 8 patients with persistently detectable ctDNA after adjuvant chemotherapy have recurred. End-of-treatment (surgery +/- adjuvant chemotherapy) ctDNA detection was associated with a 5-year RFS of 0% compared to 75.6% for patients with an undetectable end-of-treatment ctDNA (HR 14.9; 95% CI 4.94 to 44.7; P < 0.001). Key limitations of the study include the small sample size and the potential for false-positive findings with multiple hypothesis testing. CONCLUSIONS: We confirmed the prognostic impact of postsurgery and posttreatment ctDNA in patients with resected CRLM. The potential utility of serial ctDNA analysis during adjuvant chemotherapy as an early marker of treatment efficacy was also demonstrated. Further studies are required to define how to optimally integrate ctDNA analyses into decision-making regarding the use and timing of adjuvant therapy for resectable CRLM. TRIAL REGISTRATION: ACTRN12612000345886.

Dual blockade of IL-4 and IL-13 with dupilumab, an IL-4Rα antibody, is required to broadly inhibit type 2 inflammation.

BACKGROUND: Dupilumab, a fully human monoclonal antibody that binds IL-4Rα and inhibits signaling of both IL-4 and IL-13, has shown efficacy across multiple diseases with underlying type 2 signatures and is approved for treatment of asthma, atopic dermatitis, and chronic sinusitis with nasal polyposis. We sought to provide a comprehensive analysis of the redundant and distinct roles of IL-4 and IL-13 in type 2 inflammation and report dupilumab mechanisms of action. METHODS: Using primary cell assays and a mouse model of house dust mite-induced asthma, we compared IL-4 vs IL-13 vs IL-4Rα blockers. RESULTS: Intranasal administration of either IL-4 or IL-13 confers an asthma-like phenotype in mice by inducing immune cell lung infiltration, including eosinophils, increasing cytokine/chemokine expression and mucus production, thus demonstrating redundant functions of these cytokines. We further teased out their respective contributions using human in vitro culture systems. Then, in a mouse asthma model by comparing in head-to-head studies, either IL-4 or IL-13 inhibition to dual IL-4/IL-13 inhibition, we demonstrate that blockade of both IL-4 and IL-13 is required to broadly block type 2 inflammation, which translates to protection from allergen-induced lung function impairment. Notably, only dual IL-4/IL-13 blockade prevented eosinophil infiltration into lung tissue without affecting circulating eosinophils, demonstrating that tissue, but not circulating eosinophils, contributes to disease pathology. CONCLUSIONS: Overall, these data support IL-4 and IL-13 as key drivers of type 2 inflammation and help provide insight into the therapeutic mechanism of dupilumab, a dual IL-4/IL-13 blocker, in multiple type 2 diseases.

Antibody drug conjugates of cleavable amino-benzoyl-maytansinoids.

ADCs based on the natural product maytansine have been successfully employed clinically. In a previous report, ADCs based on hydrophilic non-cell permeable maytansinoids was presented. The authors in this report further explore the maytansine scaffold to develop tubulin inhibitors capable of cell permeation. The research resulted in amino-benzoyl-maytansinoid payloads that were further elaborated with linkers for conjugating to antibodies. This approach was applied to MUC16 tumor targeting antibodies for ovarian cancers. A positive control ADC was evaluated alongside the amino-benzoyl-maytansinoid ADC and the efficacy observed was equivalent while the isotype control ADCs had no effect.

Antibody drug conjugates of cleavable amino-alkyl and aryl maytansinoids.

Natural products have been used for many medicinal purposes for centuries. Antibody drug conjugates (ADCs) have utilized this rich source of small molecule therapeutics to produce several clinically useful treatments. ADCs based on the natural product maytansine have been successful clinically. The authors further the utility of the anti-cancer natural product maytansine by developing efficacious payloads and linker-payloads for conjugating to antibodies. The success of our approach was realized in the EGFRvIII targeting ADC EGFRvIII-16. The ADC was able to regress tumors in 2 tumor models (U251/EGFRvIII and MMT/EGFRvIII). When compared to a positive control ADC, the efficacy observed was similar or improved while the isotype control ADCs had no effect.

A phase II study of ipilimumab plus temozolomide in patients with metastatic melanoma.

Checkpoint blockade has revolutionized the treatment of melanoma; however, it benefits only the minority of patients. Several agents have been combined with immunotherapy to improve T-cell activation and persistence including growth factor, chemotherapy, and radiation. Preclinical data suggest that temozolomide, which metabolizes to the same active compound as dacarbazine, selectively depletes regulatory T cells. This potential immunomodulatory effect of temozolomide provides rationale for combination with ipilimumab. We performed an open-label single-arm phase II study of ipilimumab plus temozolomide in the frontline setting for patients with metastatic melanoma and LDH ≤2× upper limit of normal. Ipilimumab was given at 10 mg/kg on day 1 and temozolomide 200 mg/m2 orally days 1-4 every 3 weeks for four doses followed by maintenance ipilimumab every 12 weeks plus temozolomide every 4 weeks. The primary objective of the study was 6-month PFS. A total of 64 patients were enrolled and the 6-month PFS was 45% with median OS of 24.5 months. There were 10 (15.6%) confirmed partial responses and 10 (15.6%) confirmed complete responses. Duration of response amongst responders is 35 months with 10 patients demonstrating an ongoing response at median follow-up of 20 months. There were no deaths or unexpected toxicities on study. The most common gastrointestinal side effects were nausea and constipation rather than diarrhea or colitis. These results suggest that the combination of induction ipilimumab plus temozolomide could potentially be an effective strategy to enhance antitumor activity with a manageable toxicity profile. These findings warrant further evaluation in a large prospective study.

Mice with megabase humanization of their immunoglobulin genes generate antibodies as efficiently as normal mice.

Mice genetically engineered to be humanized for their Ig genes allow for human antibody responses within a mouse background (HumAb mice), providing a valuable platform for the generation of fully human therapeutic antibodies. Unfortunately, existing HumAb mice do not have fully functional immune systems, perhaps because of the manner in which their genetic humanization was carried out. Heretofore, HumAb mice have been generated by disrupting the endogenous mouse Ig genes and simultaneously introducing human Ig transgenes at a different and random location; KO-plus-transgenic humanization. As we describe in the companion paper, we attempted to make mice that more efficiently use human variable region segments in their humoral responses by precisely replacing 6 Mb of mouse Ig heavy and kappa light variable region germ-line gene segments with their human counterparts while leaving the mouse constant regions intact, using a unique in situ humanization approach. We reasoned the introduced human variable region gene segments would function indistinguishably in their new genetic location, whereas the retained mouse constant regions would allow for optimal interactions and selection of the resulting antibodies within the mouse environment. We show that these mice, termed VelocImmune mice because they were generated using VelociGene technology, efficiently produce human:mouse hybrid antibodies (that are rapidly convertible to fully human antibodies) and have fully functional humoral immune systems indistinguishable from those of WT mice. The efficiency of the VelocImmune approach is confirmed by the rapid progression of 10 different fully human antibodies into human clinical trials.

Myostatin deficiency but not anti-myostatin blockade induces marked proteomic changes in mouse skeletal muscle.

Pharmacologic blockade of the myostatin (Mstn)/activin receptor pathway is being pursued as a potential therapy for several muscle wasting disorders. The functional benefits of blocking this pathway are under investigation, in particular given the findings that greater muscle hypertrophy results from Mstn deficiency arising from genetic ablation compared to post-developmental Mstn blockade. Using high-resolution MS coupled with SILAC mouse technology, we quantitated the relative proteomic changes in gastrocnemius muscle from Mstn knockout (Mstn(-/-) ) and mice treated for 2-weeks with REGN1033, an anti-Mstn antibody. Relative to wild-type animals, Mstn(-/-) mice had a two-fold greater muscle mass and a >1.5-fold change in expression of 12.0% of 1137 quantified muscle proteins. In contrast, mice treated with REGN1033 had minimal changes in muscle proteome (0.7% of 1510 proteins >1.5-fold change, similar to biological difference 0.5% of 1310) even though the treatment induced significant 20% muscle mass increase. Functional annotation of the altered proteins in Mstn(-/-) mice corroborates the mutiple physiological changes including slow-to-fast fiber type switch. Thus, the proteome-wide protein expression differs between Mstn(-/-) mice and mice subjected to specific Mstn blockade post-developmentally, providing molecular-level insights to inform mechanistic hypotheses to explain the observed functional differences.

Aflibercept exhibits VEGF binding stoichiometry distinct from bevacizumab and does not support formation of immune-like complexes.

Anti-vascular endothelial growth factor (VEGF) therapies have improved clinical outcomes for patients with cancers and retinal vascular diseases. Three anti-VEGF agents, pegaptanib, ranibizumab, and aflibercept, are approved for ophthalmic indications, while bevacizumab is approved to treat colorectal, lung, and renal cancers, but is also used off-label to treat ocular vascular diseases. The efficacy of bevacizumab relative to ranibizumab in treating neovascular age-related macular degeneration has been assessed in several trials. However, questions persist regarding its safety, as bevacizumab can form large complexes with dimeric VEGF165, resulting in multimerization of the Fc domain and platelet activation. Here, we compare binding stoichiometry, Fcγ receptor affinity, platelet activation, and binding to epithelial and endothelial cells in vitro for bevacizumab and aflibercept, in the absence or presence of VEGF. In contrast to bevacizumab, aflibercept forms a homogenous 1:1 complex with each VEGF dimer. Unlike multimeric bevacizumab:VEGF complexes, the monomeric aflibercept:VEGF complex does not exhibit increased affinity for low-affinity Fcγ receptors, does not activate platelets, nor does it bind to the surface of epithelial or endothelial cells to a greater degree than unbound aflibercept or control Fc. The latter finding reflects the fact that aflibercept binds VEGF in a unique manner, distinct from antibodies not only blocking the amino acids necessary for VEGFR1/R2 binding but also occluding the heparin-binding site on VEGF165.

Clinical responses to selumetinib (AZD6244; ARRY-142886)-based combination therapy stratified by gene mutations in patients with metastatic melanoma.

BACKGROUND: The high prevalence of v-raf murine sarcoma viral oncogene homolog B1 (BRAF) and neuroblastoma v-ras oncogene homolog (NRAS) mutations in melanoma provides a strong rationale to test the clinical efficacy of mitogen-activated protein kinase kinase (MEK) inhibition in this disease. The authors hypothesized that the presence of BRAF or NRAS mutations would correlate with clinical benefit among patients who received treatment with combination regimens that included the MEK inhibitor selumetinib. METHODS: BRAF and NRAS mutation status was determined retrospectively in available tissue specimens from patients with melanoma who were enrolled in a phase 1 trial of selumetinib in combination with 1 of 4 drugs (dacarbazine, docetaxel, temsirolimus, or erlotinib). The clinical response rate and the time to progression (TTP) were assessed as a function of BRAF and NRAS mutation status. RESULTS: Among 18 patients analyzed, 9 patients (50%) harbored a BRAF mutation (8 had a valine-to-glutamic acid substitution at residue 600 [V600E]; 1 had an arginine nonsense mutation at residue 603 [R603]), 4 patients (22%) harbored an NRAS mutation (2 had a glutamine-to-arginine substitution at residue 61 [Q61R], 1 had a glutamine-to-lysine substitution at residue 61 [Q61K], and 1 had a glycine-to-lysine substitution at residue 12 [G12S]), and 5 patient (28%) had the wild type of both genes. These mutations were mutually exclusive. Among the 9 patients who had BRAF mutations, 5 patients (56%) achieved a partial response, and 4 patients (44%) achieved stable disease for at least 6 weeks. No patient with the wild-type BRAF gene achieved a clinical response (P = .01 vs patients with BRAF mutations). The presence of an NRAS mutation did not correlate with the clinical response rate. The presence of a BRAF mutation was correlated significantly with the TTP in a multivariate model (hazard ratio, 0.22; P = .02 vs wild-type BRAF). CONCLUSIONS: Higher response rates and longer TTP were observed with selumetinib-containing regimens in patients who had tumors that harbored a BRAF mutation compared with patients who had wild-type BRAF.

The Dll4/Notch pathway controls postangiogenic blood vessel remodeling and regression by modulating vasoconstriction and blood flow.

Blood vessel remodeling is crucial to the formation of the definitive vasculature, but little is known about the mechanisms controlling this process. We show that Delta-like ligand 4 (Dll4)/Notch pathway regulates vessel regression in normal pathologic conditions. Genetic and pharmacologic inhibition of Dll4/Notch prevented retinal capillary regression in the oxygen-induced retinopathy (OIR) model and during normal development. Deletion of the Notch-regulated ankyrin repeat protein, a negative regulator of the Notch pathway, produced an opposite phenotype. Inhibition of Dll4/Notch reduced vessel occlusion, maintaining blood flow that is essential for survival of microvessels. Dll4/Notch inhibition up-regulated the expression of vasodilators adrenomedullin and suppressed the expression of vasoconstrictor angiotensinogen. Angiotensin II induced rapid nonperfusion and regression of developing retinal capillaries, whereas Ace1 and AT1 inhibitors and adrenomedullin attenuated vasoobliteration in OIR, indicating that both pathways are involved in modulating vessel remodeling. In contrast, inhibition of vascular endothelial growth factor-A (VEGF-A) did not result in a pervasive loss of retinal capillaries, demonstrating that reduced expression of VEGF-A is not the proximate cause of capillary regression in OIR. Modulation of VEGF-A and Dll4/Notch signaling produced distinct changes in blood vessel morphology and gene expression, indicating that these pathways can have largely independent functions in vascular remodeling.

BRAF, NRAS and KIT sequencing analysis of spindle cell melanoma.

BACKGROUND: Spindle cell melanoma represents a rare but distinct subset of melanoma, and its genomic spectrum has not been fully defined. METHODS: We searched our institutional database for patients with a diagnosis of pure spindle cell-type melanoma whose tumors had been analyzed for BRAF, NRAS, and KIT mutations using pyrosequencing technique. RESULTS: We identified 24 patients with spindle cell melanoma, including 10 patients with desmoplastic melanoma, whose tumors had been analyzed for at least one of the three genes. The median Breslow thickness was 2.6 mm, and the most common site of the primary melanoma was the trunk, followed by the head and neck region. BRAF, NRAS and KIT genomic sequencing was performed successfully in 20, 18 and 14 patients, respectively. Among the 20 melanomas with completed BRAF-sequencing analysis, 6 (30%) harbored a mutation, of which 5 (83%) had a V600E mutation and 1 (17%) had a V600R mutation. None of the melanomas harbored NRAS or KIT mutations. CONCLUSION: As has been reported in other common types of melanoma, V600 BRAF mutation is the most common mutation of those tested in spindle cell melanoma. NRAS or KIT mutation appears to be rare, if not completely absent.

Blockade of Dll4 inhibits tumour growth by promoting non-productive angiogenesis.

Tumour growth requires accompanying expansion of the host vasculature, with tumour progression often correlated with vascular density. Vascular endothelial growth factor (VEGF) is the best-characterized inducer of tumour angiogenesis. We report that VEGF dynamically regulates tumour endothelial expression of Delta-like ligand 4 (Dll4), which was previously shown to be absolutely required for normal embryonic vascular development. To define Dll4 function in tumour angiogenesis, we manipulated this pathway in murine tumour models using several approaches. Here we show that blockade resulted in markedly increased tumour vascularity, associated with enhanced angiogenic sprouting and branching. Paradoxically, this increased vascularity was non-productive-as shown by poor perfusion and increased hypoxia, and most importantly, by decreased tumour growth-even for tumours resistant to anti-VEGF therapy. Thus, VEGF-induced Dll4 acts as a negative regulator of tumour angiogenesis; its blockade results in a striking uncoupling of tumour growth from vessel density, presenting a novel therapeutic approach even for tumours resistant to anti-VEGF therapies.

Comparison of two dosing schedules of palonosetron for the prevention of nausea and vomiting due to interleukin-2-based biochemotherapy.

BACKGROUND: Treatment of metastatic melanoma with interleukin-2-based biochemotherapy involves administration of a combination of moderately and highly emetogenic chemotherapies over 5 days. Corticosteroids for the prevention of biochemotherapy-induced nausea and vomiting (CINV) are contraindicated because they cause lysis of LAK cells produced in response to interleukin-2. Palonosetron is a long-acting, highly potent, second-generation serotonin receptor antagonist. The recommended dosing schedule of palonosetron for the control of CINV due to biochemotherapy is not known. METHODS: In a phase II design, treatment-naïve patients with metastatic melanoma undergoing the first cycle of biochemotherapy were randomized to receive palonosetron 0.25 mg intravenously for CINV prophylaxis on either days 1 and 4 (schedule 1) or days 1, 3, and 5 (schedule 2). All patients received dacarbazine on day 1, cisplatin, vinblastine, and interleukin-2 on days 1-4, and interferon alpha-2b on days 1-5. We evaluated and compared, by palonosetron dosing schedule, the pattern and the severity of CINV during the first 7 days of treatment and the duration of the 21-day cycle as well as the impact on daily function with the Functional Living Index-Emesis. RESULTS: Thirty patients (median age 53 years) were enrolled. Eighteen (60%) were men. A consistent trend of a better control of both nausea and vomiting favoring schedule 2 was observed during the first 7 days and throughout the cycle. Significantly more patients experienced nausea on any day during the first 7 days on schedule 1 (mean number of episodes 8.1 ± 1.5) than on schedule 2 (mean number of episodes 5.6 ± 2.3, p = 0.028). The impact on daily function was similar between the two groups. CONCLUSIONS: Both dosing schedules of palonosetron were tolerated well. Alternate day dosing of palonosetron was more effective in controlling CINV in this patient population.

Targeting public neoantigens for cancer immunotherapy.

Several current immunotherapy approaches target private neoantigens derived from mutations that are unique to individual patients' tumors. However, immunotherapeutic agents can also be developed against public neoantigens derived from recurrent mutations in cancer driver genes. The latter approaches target proteins that are indispensable for tumor growth, and each therapeutic agent can be applied to numerous patients. Here we review the opportunities and challenges involved in the identification of suitable public neoantigen targets and the development of therapeutic agents targeting them.

Autologous tumor-derived heat-shock protein peptide complex-96 (HSPPC-96) in patients with metastatic melanoma.

BACKGROUND: Glycoprotein-96, a non-polymorphic heat-shock protein, associates with intracellular peptides. Autologous tumor-derived heat shock protein-peptide complex 96 (HSPPC-96) can elicit potent tumor-specific T cell responses and protective immunity in animal models. We sought to investigate the feasibility, safety, and antitumor activity of HSPPC-96 vaccines prepared from tumor specimens of patients with metastatic melanoma. METHODS: Patients with a Karnofsky Performance Status >70% and stage III or stage IV melanoma had to have a metastasis >3 cm in diameter resectable as part of routine clinical management. HSPPC-96 tumor-derived vaccines were prepared in one of three dose levels (2.5, 25, or 100 microg/dose) and administered as an intradermal injection weekly for 4 consecutive weeks. In vivo induction of immunity was evaluated using delayed-type hypersensitivity (DTH) to HSPPC-96, irradiated tumor, and dinitrochlorobenzene (DNCB). The gamma-interferon (IFNgamma) ELISPOT assay was used to measure induction of a peripheral blood mononuclear cell response against autologous tumor cells at baseline and at the beginning of weeks 3, 4, and 8. RESULTS: Among 36 patients enrolled, 72% had stage IV melanoma and 83% had received prior systemic therapy. The smallest tumor specimen from which HSPPC-96 was prepared weighed 2 g. Twelve patients (including 9 with stage IV and indicator lesions) had a negative DNCB skin test result at baseline. All 36 patients were treated and evaluable for toxicity and response. There were no serious toxicities. There were no observed DTH responses to HSPPC-96 or to autologous tumor cells before or during treatment. The IFNgamma-producing cell count rose modestly in 5 of 26 patients and returned to baseline by week 8, with no discernible association with HSPPC-96 dosing or clinical parameters. There were no objective responses among 16 patients with stage IV disease and indicator lesions. Among 20 patients treated in the adjuvant setting, 11 with stage IV melanoma at baseline had a progression-free and overall survival of 45% and 82%, respectively, with a median follow-up of 10 years. CONCLUSION: Treatment with autologous tumor-derived HSPPC-96 was feasible and safe at all doses tested. Observed immunological effects and antitumor activity were modest, precluding selection of a biologically active dose. Nevertheless, the 25-microg dose level was shown to be practical for further study.

VEGF Trap complex formation measures production rates of VEGF, providing a biomarker for predicting efficacious angiogenic blockade.

VEGF is the best characterized mediator of tumor angiogenesis. Anti-VEGF agents have recently demonstrated impressive efficacy in human cancer trials, but the optimal dosing of such agents must still be determined empirically, because biomarkers to guide dosing have yet to be established. The widely accepted (but unverified) assumption that VEGF production is quite low in normal adults led to the notion that increased systemic VEGF levels might quantitatively reflect tumor mass and angiogenic activity. We describe an approach to determine host and tumor production of VEGF, using a high-affinity and long-lived VEGF antagonist now in clinical trials, the VEGF Trap. Unlike antibody complexes that are usually rapidly cleared, the VEGF Trap forms inert complexes with tissue- and tumor-derived VEGF that remain stably in the systemic circulation, where they are readily assayable, providing unprecedented capability to accurately measure VEGF production. We report that VEGF production is surprisingly high in non-tumor-bearing rodents and humans, challenging the notion that systemic VEGF levels can serve as a sensitive surrogate for tumor load; tumor VEGF contribution becomes significant only with very large tumor loads. These findings have the important corollary that anti-VEGF therapies must be sufficiently dosed to avoid diversion by host-derived VEGF. We further show that our assay can indicate when VEGF is optimally blocked; such biomarkers to guide dosing do not exist for other anti-VEGF agents. Based on this assay, VEGF Trap doses currently being assessed in clinical trials are in the efficacious range.

A phase IB trial of intravenous INO-1001 plus oral temozolomide in subjects with unresectable stage-III or IV melanoma.

INO-1001 is a PARP-1 inhibitor that interrupts the repair process of N-methylpurines generated by temozolomide. We evaluated the pharmacokinetics of INO-1001 and determined its safety when used with temozolomide at 200 mg/m(2)/day x 5 days every 4 weeks. We enrolled 12 adult patients, in cohorts of 3-6 patients, into the study. INO-1001 at doses of 100, 200 and 400 mg was given intravenous for 1 hr q 12 hr for 10 doses. INO-1001 had a moderate clearance, volume of distribution and a relatively short terminal half-life. Myelosuppression and elevation of liver transaminases were dose-limiting toxicities (DLTs) of INO-1001 at 400 mg.

Anti-VEGF drug interference with VEGF quantitation in the R&D systems human quantikine VEGF ELISA kit.

AIM: To evaluate the accuracy of the Quantikine Human VEGF Immunoassay (R&D Systems) in the presence of VEGF inhibitors. MATERIALS & METHODS: Quantikine VEGF ELISA (R&D), anti-VEGF165 mAb (R&D), VEGF165 and aflibercept (Regeneron), ranibizumab and bevacizumab (Genentech). RESULTS: Binding affinity of anti-VEGF165 mAb for VEGF was threefold weaker than aflibercept, but 33- and 40-fold stronger than ranibizumab or bevacizumab. Extended incubation of VEGF complexed with inhibitors led to VEGF dissociation from ranibizumab and bevacizumab, but not aflibercept, and subsequent binding by the immunoassay capture antibody. The immunoassay also detected VEGF:ranibizumab and VEGF:bevacizumab complexes but not VEGF:aflibercept complexes. CONCLUSION: The immunoassay cannot accurately quantitate VEGF in the presence of these VEGF inhibitors as they interfere with the capture and detection of free VEGF.

Circulating Tumor DNA Analyses as Markers of Recurrence Risk and Benefit of Adjuvant Therapy for Stage III Colon Cancer.

Importance: Adjuvant chemotherapy in patients with stage III colon cancer prevents recurrence by eradicating minimal residual disease. However, which patients remain at high risk of recurrence after completing standard adjuvant treatment cannot currently be determined. Postsurgical circulating tumor DNA (ctDNA) analysis can detect minimal residual disease and is associated with recurrence in colorectal cancers. Objective: To determine whether serial postsurgical and postchemotherapy ctDNA analysis could provide a real-time indication of adjuvant therapy efficacy in stage III colon cancer. Design, Setting, and Participants: This multicenter, Australian, population-based cohort biomarker study recruited 100 consecutive patients with newly diagnosed stage III colon cancer planned for 24 weeks of adjuvant chemotherapy from November 1, 2014, through May 31, 2017. Patients with another malignant neoplasm diagnosed within the last 3 years were excluded. Median duration of follow-up was 28.9 months (range, 11.6-46.4 months). Physicians were blinded to ctDNA results. Data were analyzed from December 10, 2018, through June 23, 2019. Exposures: Serial plasma samples were collected after surgery and after chemotherapy. Somatic mutations in individual patients' tumors were identified via massively parallel sequencing of 15 genes commonly mutated in colorectal cancer. Personalized assays were designed to quantify ctDNA. Main Outcomes and Measures: Detection of ctDNA and recurrence-free interval (RFI). Results: After 4 exclusions, 96 eligible patients were eligible; median patient age was 64 years (range, 26-82 years); 49 (51%) were men. At least 1 somatic mutation was identified in the tumor tissue of all 96 evaluable patients. Circulating tumor DNA was detectable in 20 of 96 (21%) postsurgical samples and was associated with inferior recurrence-free survival (hazard ratio [HR], 3.8; 95% CI, 2.4-21.0; P < .001). Circulating tumor DNA was detectable in 15 of 88 (17%) postchemotherapy samples. The estimated 3-year RFI was 30% when ctDNA was detectable after chemotherapy and 77% when ctDNA was undetectable (HR, 6.8; 95% CI, 11.0-157.0; P < .001). Postsurgical ctDNA status remained independently associated with RFI after adjusting for known clinicopathologic risk factors (HR, 7.5; 95% CI, 3.5-16.1; P < .001). Conclusions and Relevance: Results suggest that ctDNA analysis after surgery is a promising prognostic marker in stage III colon cancer. Postchemotherapy ctDNA analysis may define a patient subset that remains at high risk of recurrence despite completing standard adjuvant treatment. This high-risk population presents a unique opportunity to explore additional therapeutic approaches.