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The objective of this study was to study the performance of two available home spirometers used by people with Cystic Fibrosis (PwCF) over a short-term period and to assess user experience.This was a prospective observational study. Participants age 6 years and older were recruited to participate if they could complete acceptable spirometry in the clinic setting.Participants used either the NuvoAir Air Next or the ZEPHYRx MIR Spirobank Smart spirometer. They underwent a one-time virtual training session, then completed 2 weeks of daily spirometry followed by 2 months of weekly spirometry. Participants responded to surveys and completed a debrief interview to understand user experience. Statistical analyses examined feasibility, reliability, and accuracy of each spirometer in an unsupervised, real-world setting.We report high adherence (80% [95% CI 61%-92%]) to our study protocol in all session attempts, but lower rates of adherence after discarding sessions performed with inadequate technique (47% [95% CI 28%-66%] to 63% [95% CI 44%-80%]). We found high reliability of each device by analyzing day-to-day variability and good concordance to recent in-clinic testing (NuvoAir r = 0.91 [0.82-0.93]; ZEPHYRx r = 0.70 [0.45-0.84]). Patient experience in this cohort was favorable with most reporting ease of use and reassurance knowing lung function was being tracked over time.This real-world study showed good performance of two different available home spirometers used by children and adults with CF. While overall adherence was high, suboptimal technique reduced the total interpretable data, possibly limiting feasibility. Future work should focus on developing sustainable training and coaching programs to support the success of home spirometry in a CF chronic care model.

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KINOMEscan selectivity profiling of JBJ-04-125-02 and IC50 of EAI045, JBJ-02-112-05 and JBJ-04-125-02 in BaF3 cells.

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JBJ-04-125-02 is more potent than EAI045 and JBJ-02-112-05 in the presence of cetuximab and is effective in inhibiting EGFR-dependent proliferation and phosphorylation in L858R/ C797S Ba/F3 cells.

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mp3 file (7.8 MB). In the January edition of the Cancer Discovery podcast, Executive Editor Mark Landis talks with David Weinstock about his paper, in which analysis of a donor-recipient pair of sisters with follicular lymphoma reveals the time-course of somatic mutations acquired during lymphomagenesis.

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Allosteric kinase inhibitors offer a potentially complementary therapeutic strategy to ATP-competitive kinase inhibitors due to their distinct sites of target binding. In this study, we identify and study a mutant-selective EGFR allosteric inhibitor, JBJ-04-125-02, which as a single agent can inhibit cell proliferation and EGFRL858R/T790M/C797S signaling in vitro and in vivo. However, increased EGFR dimer formation limits treatment efficacy and leads to drug resistance. Remarkably, osimertinib, an ATP-competitive covalent EGFR inhibitor, uniquely and significantly enhances the binding of JBJ-04-125-02 for mutant EGFR. The combination of osimertinib and JBJ-04-125-02 results in an increase in apoptosis, a more effective inhibition of cellular growth, and an increased efficacy in vitro and in vivo compared with either single agent alone. Collectively, our findings suggest that the combination of a covalent mutant–selective ATP-competitive inhibitor and an allosteric EGFR inhibitor may be an effective therapeutic approach for patients with EGFR-mutant lung cancer.Significance:The clinical efficacy of EGFR tyrosine kinase inhibitors (TKI) in EGFR-mutant lung cancer is limited by acquired drug resistance, thus highlighting the need for alternative strategies to inhibit EGFR. Here, we identify a mutant EGFR allosteric inhibitor that is effective as a single agent and in combination with the EGFR TKI osimertinib.This article is highlighted in the In This Issue feature, p. 813

Supplemental methods for structural modeling of inhibitor binding, pharmacokinetic assays, in vivo studies, crosslinking and biotinylated drug pull-down assays.

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Supplementary Legends

Supplemental Table 1: Identified NF1 variants; Supplemental Table 2: Tumors with multiple NF1 mutations include splice site, missense, nonsense, and frameshift mutations; Supplemental Table 3: KRAS mutations identified by next-generation sequencing.

Supplementary Figure Legend

Thirteen patients were enrolled and underwent at least one cycle of therapy.

Screening with drugs targeting sunitinib related receptor tyrosine kinases (RTK) or MAPK pathway were performed in PC9TMEM87A-RASGRF1 clone 1. Relative viability compared with control (treated with DMSO) was shown. A modest inhibitory effect was seen for gilteritinib without control osimertinib, suggesting the off-target toxic effect of the drug rather than on-target inhibition.

Purpose:We pursued genomic analysis of an exceptional responder with non–small cell lung cancer (NSCLC) through a multi-platform effort to discover novel oncogenic targets.Experimental Design:In this open-label, single-arm phase II study (NCT01829217), an enriched cohort of patients with advanced NSCLC was treated with the multi-kinase inhibitor sunitinib. The primary endpoint was objective response rate. Tissue was collected for multi-platform genomic analysis of responders, and a candidate oncogene was validated using in vitro models edited by CRISPR-Cas9.Results:Of 13 patients enrolled, 1 patient (8%), a never smoker, had a partial response lasting 33 months. Genomic analysis of the responder identified no oncogenic variant using multi-platform DNA analysis including hotspot allelotyping, massively parallel hybrid-capture next-generation sequencing, and whole-exome sequencing. However, bulk RNA-sequencing (RNA-seq) revealed a novel fusion, TMEM87A–RASGRF1, with high overexpression of the fusion partners. RASGRF1 encodes a guanine exchange factor which activates RAS from GDP-RAS to GTP-RAS. Oncogenicity was demonstrated in NIH/3T3 models with intrinsic TMEM87A–RASGRF1 fusion. In addition, activation of MAPK was shown in PC9 models edited to express this fusion, although sensitivity to MAPK inhibition was seen without apparent sensitivity to sunitinib.Conclusions:Sunitinib exhibited limited activity in this enriched cohort of patients with advanced NSCLC. Nonetheless, we find that RNA-seq of exceptional responders represents a potentially underutilized opportunity to identify novel oncogenic targets including oncogenic activation of RASGRF1.

Supplementary Table S3. List of LKB1 mutant lung adenocarcinoma specimens by OncoMap tested for LKB1 IHC.

Post-baseline toxicity incidence.

Supplemental Figure 3. Tumor volumes (TV; mean +/- SEM) over time of PDX DFCI 359 and PDX 315 dosed with neratinib (40mg/kg daily), TDM1 (20 mg/kg weekly), and their combinations (n = 8 mice/cohort). The data for the vehicle and neratinib arms for DFCI 315 are from the same experiment as in Figure 5D.

Supplementary Table S1. KRAS mutant NSCLC patients' characteristics (n=514)

Supplemental Figure 2. A, KRAS G12C mutation is the most frequently mutated in smokers while G12D is most common in never-smokers. B and C, Never-smokers were significantly more likely than former or current smokers to have a transition mutation (G>A) rather than transversion mutations known to be smoking-related (G>T or G>C).

Shown is the schema of study design and enrollment.(30) As noted, the cut-off for data analysis was May 15, 2015.

Supplementary Table S2. Panel of cancer cell lines with known LKB1 status tested for LKB1 IHC.

Supplementary Table S1

Table S1: Inclusion and exclusion criteria for study eligibility; Table S2: PCR primer and probe sequences; Table S3: Patient and tumor characteristics.

Purpose: Genotype-directed therapy is the standard of care for advanced non–small cell lung cancer (NSCLC), but obtaining tumor tissue for genotyping remains a challenge. Circulating tumor cell (CTC) or cell-free DNA (cfDNA) analysis may allow for noninvasive evaluation. This prospective trial evaluated CTCs and cfDNA in EGFR-mutant NSCLC patients treated with erlotinib until progression.Experimental Design: EGFR-mutant NSCLC patients were enrolled in a phase II trial of erlotinib. Blood was collected at baseline, every 2 months on study, and at disease progression. Plasma genotyping was performed by droplet digital PCR for EGFR19del, L858R, and T790M. CTCs were isolated by CellSave, enumerated, and analyzed by immunofluorescence for CD45 and pan-cytokeratin and EGFR and MET FISH were also performed. Rebiopsy was performed at disease progression.Results: Sixty patients were enrolled; 44 patients discontinued therapy for disease progression. Rebiopsy occurred in 35 of 44 patients (80%), with paired CTC/cfDNA analysis in 41 of 44 samples at baseline and 36 of 44 samples at progression. T790M was identified in 23 of 35 (66%) tissue biopsies and 9 of 39 (23%) cfDNA samples. CTC analysis at progression identified MET amplification in 3 samples in which tissue analysis could not be performed. cfDNA analysis identified T790M in 2 samples in which rebiopsy was not possible. At diagnosis, high levels of cfDNA but not high levels of CTCs correlated with progression-free survival.Conclusions: cfDNA and CTCs are complementary, noninvasive assays for evaluation of acquired resistance to first-line EGFR TKIs and may expand the number of patients in whom actionable genetic information can be obtained at acquired resistance. Serial cfDNA monitoring may offer greater clinical utility than serial monitoring of CTCs. Clin Cancer Res; 22(24); 6010–20. ©2016 AACR.

Purpose:Evaluating drug responses using primary patient-derived cells ex vivo represents a potentially rapid and efficient approach to screening for new treatment approaches. Here, we sought to identify neratinib combinations in HER2 mutant non–small cell lung cancer (NSCLC) patient xenograft-derived organotypic spheroids (XDOTS) using a short-term ex vivo system.Experimental Design:We generated two HER2-mutant NSCLC PDX models [DFCI359 (HER2 exon19 755_757LREdelinsRP) and DFCI315 (HER2 exon20 V777_G778insGSP)] and used the PDX tumors to generate XDOTS. Tumor spheroids were grown in a microfluidic device and treated ex vivo with neratinib-based drug combinations. Live/dead quantification was performed by dual-labeling deconvolution fluorescence microscopy. The most efficacious ex vivo combination was subsequently validated in vivo using the DFCI359 and DFCI315 PDXs and a HER2YVMA genetically engineered mouse model.Results:Both neratinib and afatinib, but not gefitinib, induced cell death in DFCI359 XDOTS. The combinations of neratinib/trastuzumab and neratinib/temsirolimus enhanced the therapeutic benefit of neratinib alone in DFCI315 and DFCI359. The combination of neratinib and trastuzumab in vivo was more effective compared with single-agent neratinib or trastuzumab and was associated with more robust inhibition of HER2 and downstream signaling.Conclusions:The XDOTS platform can be used to evaluate therapies and therapeutic combinations ex vivo using PDX tumors. This approach may accelerate the identification and clinical development of therapies for targets with no or few existing models and/or therapies.

Table S4. LKB1 patient characteristics in KRAS mutant patients (n=148)*

Supplemental Legend

Supplemental Figure 1. A, OS in stage IV KRAS-mutant NSCLC according to KRAS tranversion or transition mutations and LKB1 status. B, OS by different KRAS mutations (G12C vs. G12D)

Vitamin D deficiency is prevalent in patients with inflammatory bowel disease (IBD). The goal of this study was to assess the efficacy and safety of high-dose, interval cholecalciferol administration in patients with IBD receiving infliximab.This prospective, longitudinal, open-label study enrolled pediatric and young adult patients with IBD and vitamin D deficiency. Subjects received 50,000 IU every 4 to 5 weeks (n = 11) or 100,000 IU every 6 to 8 weeks (n = 32) of oral cholecalciferol for 1 year. Dosing was directly observed and administered in conjunction with infliximab infusions. The primary endpoint was vitamin D sufficiency, defined as a 25-hydroxy-vitamin D (25-OHD) level ≥30 ng/mL.Forty-three participants constituted the primary analysis population. 25-OHD levels reached steady-state after the third dose, and mean increases in 25-OHD levels were 8 vs. 4.5 ng/mL in the 100,000 IU vs. 50,000 IU treatment groups, respectively. Only 43.8% of patients receiving 100,000 IU and 18.2% of patients receiving 50,000 IU achieved sufficiency. There was no difference in the 25-OHD level responsiveness in patients with Crohn disease versus those with ulcerative colitis (P = 0.72). There was no correlation between 25-OHD levels and clinical disease activity in patients with Crohn disease (P = 0.85) or ulcerative colitis (P = 0.24).Supplementation with cholecalciferol was well-tolerated and direct observation is a promising paradigm for ensuring compliance with therapy. Patients with IBD, however, appear to require high doses of cholecalciferol, with less than half of patients (37% overall) achieving vitamin D sufficiency. Additional studies are necessary to determine the optimal treatment regimens.

Mesothelioma is a rare and fatal cancer with limited therapeutic options until the recent approval of combination immune checkpoint blockade. Here we report the results of the phase 2 PrE0505 trial (NCT02899195) of the anti-PD-L1 antibody durvalumab plus platinum-pemetrexed chemotherapy for 55 patients with previously untreated, unresectable pleural mesothelioma. The primary endpoint was overall survival compared to historical control with cisplatin and pemetrexed chemotherapy; secondary and exploratory endpoints included safety, progression-free survival and biomarkers of response. The combination of durvalumab with chemotherapy met the pre-specified primary endpoint, reaching a median survival of 20.4 months versus 12.1 months with historical control. Treatment-emergent adverse events were consistent with known side effects of chemotherapy, and all adverse events due to immunotherapy were grade 2 or lower. Integrated genomic and immune cell repertoire analyses revealed that a higher immunogenic mutation burden coupled with a more diverse T cell repertoire was linked to favorable clinical outcome. Structural genome-wide analyses showed a higher degree of genomic instability in responding tumors of epithelioid histology. Patients with germline alterations in cancer predisposing genes, especially those involved in DNA repair, were more likely to achieve long-term survival. Our findings indicate that concurrent durvalumab with platinum-based chemotherapy has promising clinical activity and that responses are driven by the complex genomic background of malignant pleural mesothelioma., In a phase 2 trial, the combination of chemotherapy with durvalumab, an anti-PD-L1 antibody, exhibited promising clinical activity in patients with previously untreated, unresectable mesothelioma, with additional analyses providing insights into genomic and immunologic features potentially associated with response.

Introduction Smoking cessation has been reported to benefit patients even after a diagnosis of lung cancer. We studied the smoking behavior of patients who participated in a phase 3 trial of adjuvant therapy following resection of stages IB–IIIA NSCLC. Methods The ECOG-ACRIN 1505 was conducted to determine whether the addition of bevacizumab to adjuvant chemotherapy would improve overall survival (OS) for patients with early-stage NSCLC. Studying the association between smoking status and OS was a secondary end point. Patients completed a questionnaire on their smoking habits at baseline, 3, 6, 9, and 12 months. Results A total of 1501 patients were enrolled, and 99.8%, 95%, 94%, 93%, and 93% responded to the questionnaire at baseline, 3, 6, 9, and 12 months, respectively. A total of 90% reported a current or previous history of cigarette smoking. In addition, 60% of nonsmokers at enrollment reported smoking after diagnosis (before randomization); however, 1% of them reported smoking at 12 months. Furthermore, 94% of the respondents smoked none/fewer cigarettes daily at 12 months. The incidence of grades 3–5 toxicity on treatment was 68%, 76%, and 72% in never, former, and current smokers, respectively (p = 0.05). The disease-free survival for never-smokers relative to current and former smokers was (hazard ratio [HR] 0.93, p = 0.64 and HR 1.05, p = 0.72), and OS was (adjusted HR for death 0.54, p = 0.005 and adjusted HR for death 0.68, p = 0.03), respectively. Conclusions This is the first comprehensive, prospective report of smoking habits in patients with NSCLC patients from a phase III early-stage trial. There was a high rate of smoking reduction and cessation following study entry. The disease-free survival did not differ significantly between smokers and never smokers, though there were less grade 3–5 toxicities and more favorable OS in never-smokers.

To determine bone mineral density (BMD) of transgender girls before pubertal blockade, and correlate with lifestyle and clinical variables.Six transfemale peri-pubertal girls had knee magnetic resonance imaging (MRI) with T1-weighted images and single-voxel proton magnetic resonance spectroscopy (MRS). BMD measurements were obtained via dual-energy X-ray absorptiometry. Questionnaires about physical activity, diet, and the Eating Attitudes Test (EAT-26) were completed. The T2 relaxation rate of water (R2 = 1/T2 in s -1) was correlated with scores on surveys.Three participants (50 %) had a low bone mineral density for age based on total body less head Z-score less than -2; two participants (33 %) had a low BMD for age at lumbar spine. All had EAT-26 scores below threshold for clinical concern. All participants self-reported regular exercise. Bone marrow MR variables (T1, fat fraction, unsaturation index and R2 of water) were not correlated with DXA measures.Participants had low BMD on beginning pubertal blockade. Clinicians should consider monitoring BMD among youth AMAB, a group at potential risk for poor bone health.

Study Objectives Hypothalamic amenorrhea is common in adolescents and young adults (AYAs) with anorexia nervosa (AN), and ovarian reserve is not routinely assessed. AN increases rates of fertility problems, but how or when AN negatively influences future fertility is unclear. We sought to determine whether biomarkers of ovarian reserve were impacted in AYA with AN. Design Cross-sectional study. Setting Tertiary care center. Participants Females with AN and amenorrhea (n = 97) at the pre-intervention visit of a clinical trial, n = 19 females without an eating disorder or menstrual dysfunction. Main Outcome Measures Serum anti-Mullerian hormone (AMH) concentrations. Results AMH levels were higher in AYA with AN than unaffected adolescents (4.7 vs. 3.2 ng/mL; P = .03). Neither FSH nor inhibin B differed between groups. In 19.6% of participants with AN, AMH levels were elevated above the normal range (>6.78 ng/mL). These subjects had a longer disease duration than those with normal AMH levels (9 vs. 3 mos; P = .03); age or degree of malnutrition did not differ between AN subjects with normal or elevated AMH. Conclusions AMH levels appear to be normal or elevated in AYA with AN. Low AMH in a patient with AN should raise clinical concern regarding ovarian reserve, and should not be attributed to degree of malnutrition alone. Currently, AMH is not regularly assessed during routine AN clinical care. However, our findings suggest some clinical utility in identifying those patients with reduced ovarian reserve. Potential links between the hypothalamic amenorrhea suffered by patients with AN and PCOS should be explored.

Purpose: We pursued genomic analysis of an exceptional responder with non–small cell lung cancer (NSCLC) through a multi-platform effort to discover novel oncogenic targets. Experimental Design: In this open-label, single-arm phase II study (NCT01829217), an enriched cohort of patients with advanced NSCLC was treated with the multi-kinase inhibitor sunitinib. The primary endpoint was objective response rate. Tissue was collected for multi-platform genomic analysis of responders, and a candidate oncogene was validated using in vitro models edited by CRISPR-Cas9. Results: Of 13 patients enrolled, 1 patient (8%), a never smoker, had a partial response lasting 33 months. Genomic analysis of the responder identified no oncogenic variant using multi-platform DNA analysis including hotspot allelotyping, massively parallel hybrid-capture next-generation sequencing, and whole-exome sequencing. However, bulk RNA-sequencing (RNA-seq) revealed a novel fusion, TMEM87A–RASGRF1, with high overexpression of the fusion partners. RASGRF1 encodes a guanine exchange factor which activates RAS from GDP-RAS to GTP-RAS. Oncogenicity was demonstrated in NIH/3T3 models with intrinsic TMEM87A–RASGRF1 fusion. In addition, activation of MAPK was shown in PC9 models edited to express this fusion, although sensitivity to MAPK inhibition was seen without apparent sensitivity to sunitinib. Conclusions: Sunitinib exhibited limited activity in this enriched cohort of patients with advanced NSCLC. Nonetheless, we find that RNA-seq of exceptional responders represents a potentially underutilized opportunity to identify novel oncogenic targets including oncogenic activation of RASGRF1.

Purpose: Evaluating drug responses using primary patient-derived cells ex vivo represents a potentially rapid and efficient approach to screening for new treatment approaches. Here, we sought to identify neratinib combinations in HER2 mutant non–small cell lung cancer (NSCLC) patient xenograft-derived organotypic spheroids (XDOTS) using a short-term ex vivo system. Experimental Design: We generated two HER2-mutant NSCLC PDX models [DFCI359 (HER2 exon19 755_757LREdelinsRP) and DFCI315 (HER2 exon20 V777_G778insGSP)] and used the PDX tumors to generate XDOTS. Tumor spheroids were grown in a microfluidic device and treated ex vivo with neratinib-based drug combinations. Live/dead quantification was performed by dual-labeling deconvolution fluorescence microscopy. The most efficacious ex vivo combination was subsequently validated in vivo using the DFCI359 and DFCI315 PDXs and a HER2YVMA genetically engineered mouse model. Results: Both neratinib and afatinib, but not gefitinib, induced cell death in DFCI359 XDOTS. The combinations of neratinib/trastuzumab and neratinib/temsirolimus enhanced the therapeutic benefit of neratinib alone in DFCI315 and DFCI359. The combination of neratinib and trastuzumab in vivo was more effective compared with single-agent neratinib or trastuzumab and was associated with more robust inhibition of HER2 and downstream signaling. Conclusions: The XDOTS platform can be used to evaluate therapies and therapeutic combinations ex vivo using PDX tumors. This approach may accelerate the identification and clinical development of therapies for targets with no or few existing models and/or therapies.