RLIP76 protein knockdown attenuates obesity due to a high-fat diet.

Feeding a Western high-fat diet (HFD) to C57BL/6 mice induces obesity, associated with a chronic inflammatory state, lipid transport, and metabolic derangements, and organ system effects that particularly prominent in the kidneys. Here, we report that RLIP76 homozygous knock-out (RLIP76(-/-)) mice are highly resistant to obesity as well as these other features of metabolic syndrome caused by HFD. The normal increase in pro-inflammatory and fibrotic markers associated with HFD induced obesity in wild-type C57B mice was broadly and nearly completely abrogated in RLIP76(-/-) mice. This is a particularly striking finding because chemical markers of oxidative stress including lipid hydroperoxides and alkenals were significantly higher in RLIP76(-/-) mice. Whereas HFD caused marked suppression of AMPK in wild-type C57B mice, RLIP76(-/-) mice had baseline activation of AMP-activated protein kinase, which was not further affected by HFD. The baseline renal function was reduced in RLIP76(-/-) mice as compared with wild-type, but was unaffected by HFD, in marked contrast to severe renal impairment and glomerulopathy in the wild-type mice given HFD. Our findings confirm a fundamental role of RLIP76 in regulating the function of obesity-promoting pro-inflammatory cytokines, and provide a novel mechanism for targeted therapy of obesity and metabolic syndrome.

Preclinical Characterization and Phase I Trial Results of INBRX-109, A Third-Generation, Recombinant, Humanized, Death Receptor 5 Agonist Antibody, in Chondrosarcoma.

PURPOSE: Patients with unresectable/metastatic chondrosarcoma have poor prognoses; conventional chondrosarcoma is associated with a median progression-free survival (PFS) of <4 months after first-line chemotherapy. No standard targeted therapies are available. We present the preclinical characterization of INBRX-109, a third-generation death receptor 5 (DR5) agonist, and clinical findings from a phase I trial of INBRX-109 in unresectable/metastatic chondrosarcoma (NCT03715933). PATIENTS AND METHODS: INBRX-109 was first characterized preclinically as a DR5 agonist, with binding specificity and hepatotoxicity evaluated in vitro and antitumor activity evaluated both in vitro and in vivo. INBRX-109 (3 mg/kg every 3 weeks) was then evaluated in a phase I study of solid tumors, which included a cohort with any subtype of chondrosarcoma and a cohort with IDH1/IDH2-mutant conventional chondrosarcoma. The primary endpoint was safety. Efficacy was an exploratory endpoint, with measures including objective response, disease control rate, and PFS. RESULTS: In preclinical studies, INBRX-109 led to antitumor activity in vitro and in patient-derived xenograft models, with minimal hepatotoxicity. In the phase I study, INBRX-109 was well tolerated and demonstrated antitumor activity in unresectable/metastatic chondrosarcoma. INBRX-109 led to a disease control rate of 87.1% [27/31; durable clinical benefit, 40.7% (11/27)], including two partial responses, and median PFS of 7.6 months. Most treatment-related adverse events, including liver-related events, were low grade (grade ≥3 events in chondrosarcoma cohorts, 5.7%). CONCLUSIONS: INBRX-109 demonstrated encouraging antitumor activity with a favorable safety profile in patients with unresectable/metastatic chondrosarcoma. A randomized, placebo-controlled, phase II trial (ChonDRAgon, NCT04950075) will further evaluate INBRX-109 in conventional chondrosarcoma.

MRTX-500 Phase 2 Trial: Sitravatinib With Nivolumab in Patients With Nonsquamous NSCLC Progressing On or After Checkpoint Inhibitor Therapy or Chemotherapy.

INTRODUCTION: Sitravatinib, a receptor tyrosine kinase inhibitor targeting TYRO3, AXL, MERTK receptors, and vascular epithelial growth factor receptor 2, can shift the tumor microenvironment toward an immunostimulatory state. Combining sitravatinib with checkpoint inhibitors (CPIs) may augment antitumor activity. METHODS: The phase 2 MRTX-500 study evaluated sitravatinib (120 mg daily) with nivolumab (every 2 or 4 wk) in patients with advanced nonsquamous NSCLC who progressed on or after previous CPI (CPI-experienced) or chemotherapy (CPI-naive). CPI-experienced patients had a previous clinical benefit (PCB) (complete response, partial response, or stable disease for at least 12 weeks then disease progression) or no PCB (NPCB) from CPI. The primary end point was objective response rate (ORR); secondary objectives included safety and secondary efficacy end points. RESULTS: Overall, 124 CPI-experienced (NPCB, n = 35; PCB, n = 89) and 32 CPI-naive patients were treated. Investigator-assessed ORR was 11.4% in patients with NPCB, 16.9% with PCB, and 25.0% in CPI-naive. The median progression-free survival was 3.7, 5.6, and 7.1 months with NPCB, PCB, and CPI-naive, respectively; the median overall survival was 7.9 and 13.6 months with NPCB and PCB, respectively (not reached in CPI-naive patients; median follow-up 20.4 mo). Overall, (N = 156), any grade treatment-related adverse events (TRAEs) occurred in 93.6%; grade 3/4 in 58.3%. One grade 5 TRAE occurred in a CPI-naive patient. TRAEs led to treatment discontinuation in 14.1% and dose reduction or interruption in 42.9%. Biomarker analyses supported an immunostimulatory mechanism of action. CONCLUSIONS: Sitravatinib with nivolumab had a manageable safety profile. Although ORR was not met, this combination exhibited antitumor activity and encouraged survival in CPI-experienced patients with nonsquamous NSCLC.

Targeting the growth factors and angiogenesis pathways: small molecules in solid tumors.

Research over the last decades has provided us with a better understanding of the biology of solid tumors. In some solid tumor malignancies single molecular events leading to malignant transformation have been identified, whilst in others multiple pathways seem to play a role simultaneously. The delineation of such pathways has led to the identification of therapeutic targets. Multiple compounds are available now to treat those molecular aberrations. With the wider use of newer biologic therapies, the understanding of biologic characteristics predicting a clinical response has broadened. This review focuses on the clinical management of gastrointestinal stromal tumors as well as hepatocellular, non small cell lung, renal cell and breast cancer with small molecule tyrosine kinase inhibitors, integrating some of the clinical advances with monoclonal antibody therapies in solid tumor malignancies. It highlights recent advances in relevant biomarkers to predict efficacy of biologic therapies and elaborates on their relevance in the clinical management of patients with solid tumor malignancies.

Randomized Phase 2 Studies of Checkpoint Inhibitors Alone or in Combination With Pegilodecakin in Patients With Metastatic NSCLC (CYPRESS 1 and CYPRESS 2).

INTRODUCTION: Checkpoint inhibitors (CPIs) have been approved to treat metastatic NSCLC. Pegilodecakin + CPI suggested promising efficacy in phase 1 IVY, providing rationale for randomized phase 2 trials CYPRESS 1 and CYPRESS 2. METHODS: CYPRESS 1 (N = 101) and CYPRESS 2 (N = 52) included Eastern Cooperative Oncology Group performance status of 0 to 1 and first-line/second-line metastatic NSCLC, respectively, without known EGFR/ALK mutations. Patients were randomized 1:1; control arms received pembrolizumab (CYPRESS 1) or nivolumab (CYPRESS 2); experimental arms received pegilodecakin + CPI. Patients had programmed death-ligand 1 tumor proportion score of greater than or equal to 50% (CYPRESS 1) or 0% to 49% (CYPRESS 2). Primary end point was objective response rate (ORR) per investigator. Secondary end points included progression-free survival (PFS), overall survival (OS), and safety. Exploratory end points included immune activation biomarkers. RESULTS: Median follow-up for CYPRESS 1 and CYPRESS 2 was 10.0 and 11.6 months, respectively. Results for pegilodecakin + pembrolizumab versus pembrolizumab were as follows: ORR per investigator 47% versus 44% (OR = 1.1, 95% confidence interval [CI]: 0.5-2.5); median PFS 6.3 versus 6.1 months (hazard ratio [HR] = 0.937, 95% CI: 0.54-1.625); and median OS 16.3 months versus not reached (HR = 1.507, 95% CI: 0.708-3.209). Results per blinded independent central review were consistent. Treatment discontinuation rate owing to adverse events (AEs) doubled in the experimental arm (32% versus 15%). AEs with grade greater than or equal to 3 treatment-related AEs (62% versus 19%) included anemia (20% versus 0%) and thrombocytopenia (12% versus 2%). Results for pegilodecakin + nivolumab versus nivolumab were as follows: ORR per investigator 15% versus 12% (OR = 1.2, 95% CI: 0.3-5.9); median PFS 1.9 versus 1.9 months (HR = 1.006, 95% CI: 0.519-1.951); and median OS 6.7 versus 10.7 months (HR = 1.871, 95% CI: 0.772-4.532). AEs with grade greater than or equal to 3 treatment-related AEs (70.4% versus 16.7%) included anemia (40.7% versus 0%), fatigue (18% versus 0%), and thrombocytopenia (14.8% versus 0%). Biomarker data suggested activation of immunostimulatory signals of interleukin-10R pathway in pegilodecakin-containing arms. CONCLUSIONS: Despite evidence of biological effect in peripheral blood, adding pegilodecakin to CPI did not improve ORR, PFS, or OS, in first-line/second-line NSCLC. Pegilodecakin + CPI has been found to have overall higher toxicity compared with CPI alone, leading to doubling of treatment discontinuation rate owing to AEs.

Heterogeneity of non-cycling and cycling synchronized murine hematopoietic stem/progenitor cells.

Purified long-term multilineage repopulating marrow stem cells have been considered to be homogenous, but functionally these cells are heterogeneous. Many investigators urge clonal studies to define stem cells but, if stem cells are truly heterogeneous, clonal studies can only define heterogeneity. We have determined the colony growth and differentiation of individual lineage negative, rhodamine low, Hoechst low (LRH) stem cells at various times in cytokine culture, corresponding to specific cell cycle stages. These highly purified and cycle synchronized (98% in S phase at 40 h of culture) stem cells were exposed to two cytokine cocktails for 0, 18, 32, or 40 h and clonal differentiation assessed 14 days later. Total heterogeneity as to gross colony morphology and differentiation stage was demonstrated. This heterogeneity showed patterns of differentiation at different cycle times. These data hearken to previous suggestions that stem cells might be similar to radioactive isotopes; decay rate of a population of radioisotopes being highly predictable, while the decay of individual nuclei is heterogeneous and unpredictable (Till et al., 1964). Marrow stem cells may be most adequately defined on a population basis; stem cells existing in a continuum of reversible change rather than a hierarchy.

Pneumonectomy after neoadjuvant chemotherapy and radiation for advanced-stage lung cancer.

BACKGROUND: Intergroup 0139 Trial suggests an increase in mortality after pneumonectomy in patients receiving preoperative chemotherapy and radiation. We evaluate our outcomes with pneumonectomy after neoadjuvant chemotherapy and radiation. METHODS: Neoadjuvant chemotherapy and radiation consisted of cisplatin 50 mg/m2 on days 1, 8, 29, and 36 and etoposide 50 mg/m2 on days 1-5 and 29-33 given concurrently with 5,040 cGy radiation. From a prospective database, results after pneumonectomy were compared between patients receiving and not receiving neoadjuvant chemotherapy and radiation during the same time period. RESULTS: Over 7 years, 50 pneumonectomies were performed for non-small-cell carcinoma; 18 received neoadjuvant chemotherapy and radiation (group A) and 32 did not (group B). Comparing group A with group B, there was no significant difference in patient demographics, blood loss, transfusion requirements or pneumonectomy side. Group A had more patients with stage III disease [17/ 18 (94%) versus 15/32 (47%), P = 0.001] and also more often had vascularized flap for bronchial stump coverage [17/18 (94%) versus 4/32 (13%), P < 0.001]. There was no significant difference in operative morbidity or mortality. Mortality for group A was 0/18 and for group B was 2/32 (6.3%) (P = 0.530). Group A patients with IIIA(N2) disease (n = 13) had median recurrence-free survival of 12.4 months, median overall survival of 25 months, and 3- year overall survival of 22.2%. CONCLUSIONS: Using a multidisciplinary team approach at a tertiary care center, pneumonectomy can be performed successfully after neoadjuvant chemotherapy and radiation for advanced-stage lung cancer. Vascularized flap for bronchial stump coverage may be important in this regard.

The neoadjuvant therapy of colorectal hepatic metastases and the role of biologic sensitizing and resistance factors.

Liver metastasis represents a common systemic complication of colorectal cancers (CRCs). Partial liver resection has been demonstrated to result in long-term survival in certain well-selected patients with otherwise well-controlled systemic disease. Neoadjuvant therapy has been demonstrated to result in improved resectability and potentially longer survival in patients with liver metastases from CRC. The addition of biologic agents to chemotherapy has been shown to improve response rates and overall survival in patients with metastatic CRC. Here, we are discussing the role of biologic agents in the treatment of patients with liver metastases from CRC. We also discuss the role of biomarkers for response and resistance to such novel therapies.

Rlip Depletion Suppresses Growth of Breast Cancer.

RLIP76 (RAL-binding protein-1, Rlip) is a stress-protective mercapturic-acid-pathway transporter protein that also plays a key role in regulating clathrin-dependent endocytosis as a Ral effector. Targeted inhibition or depletion of Rlip causes regression of xenografts of many cancers and is capable of abrogating tumor formation in p53-null mice. This is associated with the reversion of the abnormal methylomic profile of p53-null mice to wild-type. In a query of The Cancer Genome Atlas (TCGA) databases, we found that Rlip expression was associated with poor survival and with significant differences in the frequencies of PIK3CA mutation, MYC amplification, and CDKN2A/B deletion, which were the most commonly mutated, amplified, and deleted genes, respectively, among TCGA breast cancer patients. We conducted the present study to further examine the effects of Rlip inhibition and to evaluate the in vitro and in vivo efficacy in breast cancer. Using immunogold electron microscopy, we found that plasma-membrane Rlip was accessible to cell-surface antibodies in the MCF7 (ER+) breast cancer cell line. Rlip depletion resulted in decreased survival of MCF7 and MDA-MB-231 cells and increased terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) positivity and DNA laddering, indicating apoptotic cell death. Additionally, in vitro knockdown of Rlip inhibited EGF endocytosis and WNT/MAPK signaling. Xenograft studies in nude mice showed regression of breast cancer via antisense-mediated depletion of Rlip mRNA as well as by anti-Rlip antibody. Finally, knockdown of Rlip by antisense locked nucleic acid oligonucleotides increased markers for apoptotic signaling and decreased markers for proliferation, angiogenesis, and cell cycling in MCF7 and MDA-MB-231luc xenografts. Our findings validate Rlip as an attractive target in breast cancer.

Safety, Efficacy, and Pharmacokinetics of Almonertinib (HS-10296) in Pretreated Patients With EGFR-Mutated Advanced NSCLC: A Multicenter, Open-label, Phase 1 Trial.

INTRODUCTION: Almonertinib (HS-10296) is a novel, third-generation EGFR tyrosine kinase inhibitor (EGFR TKI) that targets both EGFR-sensitizing and T790M resistance mutations. This first-in-human trial aimed to evaluate the safety, efficacy, and pharmacokinetics of almonertinib in patients with locally advanced or metastatic EGFR mutation-positive NSCLC that had progressed after pevious EGFR TKI therapy. METHODS: This phase 1, open-label, multicenter clinical trial (NCT0298110) included dose-escalation (55, 110, 220, and 260 mg) and dose-expansion cohorts (55, 110, and 220 mg) with once daily oral administration of almonertinib. In each expansion cohort, tumor biopsies were obtained for the determination of EGFR T790M status. The safety, tolerability, antitumor activity, and pharmacokinetics of almonertinib were evaluated. RESULTS: A total of 120 patients (26 patients in the dose-escalation cohort and 94 patients in the dose-expansion cohort) were enrolled. The maximum tolerated dose was not defined in the dose-escalation phase; the 260 mg regimen was not further evaluated in the dose-expansion phase owing to safety concerns and saturation of exposure. The most common treatment-related grade greater than or equal to 3 adverse events were increased blood creatine phosphokinase (10%) and increased alanine aminotransferase (3%). Among 94 patients with the EGFR T790M mutation in the dose-expansion cohort, the investigator-assessed objective response rate and disease control rate were 52% (95% confidence interval [CI]: 42-63) and 92% (95% CI: 84-96), respectively. Median progression-free survival was 11.0 months (95% CI: 9.5-not reached) months. CONCLUSIONS: Almonertinib is safe, tolerable and effective for patients with locally advanced or metastatic NSCLC harboring the EGFR T790M mutation who were pretreated with EGFR TKIs.

Nonengraftment haploidentical cellular immunotherapy for refractory malignancies: tumor responses without chimerism.

Allogeneic bone marrow transplantation relies on immunosuppression, which controls graft-versus-host disease (GVHD) and allows engraftment at the expense of diminished graft versus-tumor (GVT) activity. Advances in hematologic transplantation have prompted the development of effective, less-toxic regimens that attempt to balance GVH and GVT immunoreactions. We analyzed the safety and efficacy of haploidentical transplantation in a Phase I/II nonimmunosuppressive, nonmyeloablative setting. A total of 41 patients with relapsed refractory cancer received 100 cGy of total body irradiation (TBI), along with an infusion of 1 x 10(6) to 2 x 10(8) CD3+ cells/kg; 29 patients received the highest dose. A postinfusional cellular graft rejection syndrome resembling engraftment syndrome was noted at the 2 highest CD3+ infusion cohorts. There were 26 patients with hematologic malignancies with 14 responses, 9 of which were major. Two of 6 patients with lymphoma remained free of disease at 76 months and 82 months, respectively; there were 5 durable complete responses and 4 partial responses in 13 patients with acute myelogenous leukemia (AML). All responses occurred outside of donor chimerism. TBI at 100 cGy followed by HLA-haploidentical immunotherapy is a biologically active therapy for patients with refractory AML and lymphoma. Possible mechanisms contributing to its effectiveness include initial GVT kill, breaking of host tolerance to tumor through cross-reactive alloreactive responses, persistent nondetectable microchimerism, or some combination of these.

'Weighing in' on screening mammography.

BACKGROUND: Obesity is associated with increased post-menopausal breast cancer risk. Overweight and obese women also tend to have a poorer prognosis when diagnosed with breast cancer compared with their matched normal weight peers. In previous studies obesity was associated with decreased utilization of screening mammography. We present a study examining the association between Body Mass Index (BMI) and compliance with recommended mammographic screening using data from the 2004 Behavioral Risk Factor Surveillance Survey (BRFSS). PATIENTS AND METHODS: We included 130,185 female participants, aged 40 and older, who were randomly selected to participate in the world largest telephone survey. After weighted analysis, this is representative of 56,226,220 non-institutionalized US women. The primary outcome was the proportion of women who underwent screening mammography within the last 2 years preceding the survey stratified by BMI. The mammography screening behavior of normal weight women (BMI 18.5-24.99) was compared with underweight (<18.5), overweight (25-29.99), and women with obesity class I (30-34.99), class II (35-39.99), and class III (>or=40) using logistic regression analysis and weighted to provide estimates of women in the United States (US). RESULTS: Our sample included 1.91% underweight, 37.91% normal weight, 30.15% overweight and 14.36%, 5.44%, and 3.49% women with obesity classes' I-III respectively. Approximately 7% of women age 40 and older had insufficient information to calculate their BMI. Adjusting for age, race, smoking status, general health perception, level of education, and income level, underweight women had lower odds of complying with regular screening mammography (OR 0.57; 95% CI, 0.48-0.68). Women with obesity class III (OR 0.97; 95% CI, 0.84-1.13) showed a trend towards underutilization of screening mammograms which was not clinically significant. In contrary, in overweight women a significantly higher association with appropriate mammography utilization was identified OR 1.08 (95% CI, 1.01-1.15). Although not statistically significant, women with class I and II obesity showed a trend towards a higher utilization 1.08 (95% CI, 0.99-1.18) and 1.10 (95% CI, 0.98-1.25) respectively, when compared to women at desired weight. CONCLUSION: We present a weighted analysis of the BRFSS, evaluating the association of BMI and appropriate screening mammography among women 40 years and older. These results are generalizable to the US population of women in this age range. Underweight women had significantly lower odds of utilizing screening mammography appropriately when compared with women at desired weight. Results from previous studies reporting underutilization of screening mammography in high risk, obese, and overweighed women were not confirmed in this largest population based analysis performed to date.

Disparities in Compliance With National Guidelines for the Treatment of Malignant Pleural Mesothelioma.

BACKGROUND: Current guidelines support cancer-directed surgery, chemotherapy, or active surveillance for clinical stages 1 to 3 of epithelial malignant pleural mesothelioma (MPM). Definitive chemotherapy is recommended for sarcomatoid/biphasic histologies. Our objective is to assess compliance with recommendations, measuring their impact on overall survival. METHODS: The National Cancer Database participant user file (2004 to 2014) was queried for patients diagnosed with MPM clinical stages 1 to 3. Multivariable logistic regression model identified factors independently associated with guideline compliance. Kaplan-Meier analysis and Cox proportional hazards were used for overall survival comparison with histologic subgroup analysis. RESULTS: A total of 3419 patients with clinical stages 1 to 3 met criteria for analysis and comprised epithelial (68.5%), sarcomatoid (17.2%), and biphasic subtypes (14.3%). Cancer-directed surgery was significantly underutilized in epithelial MPM, with 29.3% having no treatment. On multivariable analysis, insurance status and facility type were the strongest predictors of guideline compliance. High-volume hospitals were the most compliant with guidelines (odds ratio 3.58, 95% confidence interval (CI), 2.34 to 5.49, P < .001). Median survival estimates for no treatment, chemotherapy alone, surgery plus chemotherapy, and trimodal therapy were 10.2, 15.4, 21.1, and 21.7 months, respectively (log rank P < .001). In epithelial MPM, a significant increase in overall survival was observed in surgery plus chemotherapy (hazard ratio 0.62, 95% CI, 0.53 to 0.73, P < .001) and trimodality (hazard ratio 0.61, 95% CI, 0.49 to 0.76, P < .001; reference: no treatment). CONCLUSIONS: There is a suboptimal compliance with national guidelines for the treatment of MPM, particularly in low-volume nonacademic settings. Adherence to recommended surgery-based multimodal therapy is associated with an overall survival improvement.

Anticancer activity of 2'-hydroxyflavanone towards lung cancer.

In previous studies, we found that 2'-hydroxyflavonone (2HF), a citrus flavonoid, inhibits the growth of renal cell carcinoma in a VHL-dependent manner. This was associated with the inhibition of glutathione S-transferases (GSTs), the first step enzyme of the mercapturic acid pathway that catalyzes formation of glutathione-electrophile conjugates (GS-E). We studied 2HF in small cell (SCLC) and non-small cell (NSCLC) lung cancer cell lines for sensitivity to 2HF antineoplastic activity and to determine the role of the GS-E transporter Rlip (Ral-interacting protein; RLIP76; RALBP1) in the mechanism of action of 2HF. Our results show that 2HF induced apoptosis in both histological types of lung cancer and inhibited proliferation and growth through suppression of CDK4, CCNB1, PIK3CA, AKT and RPS6KB1 (P70S6K) signaling. Increased E-cadherin and reduced fibronectin and vimentin indicated inhibition of epithelial-mesenchymal transition. Additionally, 2HF inhibited efflux of doxorubicin and increased its accumulation in the cells, but did not add to the transport inhibitory effect of anti-Rlip antibodies alone. Binding of Rlip to 2HF was evident from successful purification of Rlip by 2HF affinity chromatography. Consistent with increased drug accumulation, combined treatment with 1-chloro-2, 4-dinitrobenzene, reduced the GI50 of 2HF by an order of magnitude. Results of in-vivo nude mouse xenograft studies of SCLC and NSCLC, which showed that orally administered 2HF inhibited growth of both histological types of lung cancer, confirmed in-vitro study results. Our result suggest that Rlip inhibition is likely a mechanism of action. Our findings are basis of proposing 2HF as therapeutic or preventative drug for lung cancer.

Longitudinal monitoring of ctDNA EGFR mutation burden from urine correlates with patient response to EGFR TKIs: A case series.

Targetable, somatic EGFR mutations are highly prevalent in patients with non-small cell lung cancer (NSCLC), making them eligible for tyrosine kinase inhibitor (TKI) therapy. Circulating tumor DNA (ctDNA), isolated from blood or urine, has been demonstrated to reliably identify somatic tumor associated EGFR mutations, specifically in patients with inconclusive biopsy. When conventional imaging modalities are inconclusive, quantitative assessment of systemic ctDNA burden has the potential to assess therapeutic response. We report on the clinical use of non-invasive, urinary ctDNA liquid biopsies for the ultrasensitive detection and longitudinal monitoring of ctDNA EGFR systemic mutation burden in five patients with NSCLC treated with EGFR TKIs. Urinary ctDNA-based quantitative assessment of systemic EGFR mutant allele burden is a non-invasive molecular diagnostic testing modality that has the potential to be utilized as an ancillary tool to assess disease burden and response to therapy.

Mutation-Enrichment Next-Generation Sequencing for Quantitative Detection of KRAS Mutations in Urine Cell-Free DNA from Patients with Advanced Cancers.

Purpose: Tumor-derived cell-free DNA (cfDNA) from urine of patients with cancer offers noninvasive biological material for detection of cancer-related molecular abnormalities such as mutations in Exon 2 of KRASExperimental Design: A quantitative, mutation-enrichment next-generation sequencing test for detecting KRASG12/G13 mutations in urine cfDNA was developed, and results were compared with clinical testing of archival tumor tissue and plasma cfDNA from patients with advanced cancer.Results: With 90 to 110 mL of urine, the KRASG12/G13 cfDNA test had an analytical sensitivity of 0.002% to 0.006% mutant copies in wild-type background. In 71 patients, the concordance between urine cfDNA and tumor was 73% (sensitivity, 63%; specificity, 96%) for all patients and 89% (sensitivity, 80%; specificity, 100%) for patients with urine samples of 90 to 110 mL. Patients had significantly fewer KRASG12/G13 copies in urine cfDNA during systemic therapy than at baseline or disease progression (P = 0.002). Compared with no changes or increases in urine cfDNA KRASG12/G13 copies during therapy, decreases in these measures were associated with longer median time to treatment failure (P = 0.03).Conclusions: A quantitative, mutation-enrichment next-generation sequencing test for detecting KRASG12/G13 mutations in urine cfDNA had good concordance with testing of archival tumor tissue. Changes in mutated urine cfDNA were associated with time to treatment failure. Clin Cancer Res; 23(14); 3657-66. ©2017 AACR.

A Highly Sensitive and Quantitative Test Platform for Detection of NSCLC EGFR Mutations in Urine and Plasma.

INTRODUCTION: In approximately 60% of patients with NSCLC who are receiving EGFR tyrosine kinase inhibitors, resistance develops through the acquisition of EGFR T790M mutation. We aimed to demonstrate that a highly sensitive and quantitative next-generation sequencing analysis of EGFR mutations from urine and plasma specimens is feasible. METHODS: Short footprint mutation enrichment next-generation sequencing assays were used to interrogate EGFR activating mutations and the T790M resistance mutation in urine or plasma specimens from patients enrolled in TIGER-X (NCT01526928), a phase 1/2 clinical study of rociletinib in previously treated patients with EGFR mutant-positive advanced NSCLC. RESULTS: Of 63 patients, 60 had evaluable tissue specimens. When the tissue result was used as a reference, the sensitivity of EGFR mutation detection in urine was 72% (34 of 47 specimens) for T790M, 75% (12 of 16) for L858R, and 67% (28 of 42) for exon 19 deletions. With specimens that met a recommended volume of 90 to 100 mL, the sensitivity was 93% (13 of 14 specimens) for T790M, 80% (four of five) for L858R, and 83% (10 of 12) for exon 19 deletions. A comparable sensitivity of EGFR mutation detection was observed in plasma: 93% (38 of 41 specimens) for T790M, 100% (17 of 17) for L858R, and 87% (34 of 39) for exon 19 deletions. Together, urine and plasma testing identified 12 additional T790M-positive cases that were either undetectable or inadequate by tissue test. In nine patients monitored while receiving treatment with rociletinib, a rapid decrease in urine T790M levels was observed by day 21. CONCLUSIONS: DNA derived from NSCLC tumors can be detected with high sensitivity in urine and plasma, enabling diagnostic detection and monitoring of therapeutic response from these noninvasive "liquid biopsy" samples.