Positive Non-small Cell Lung Carcinoma Research Articles

Efficacy of Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor in Treating Choroidal Metastasis from Non-small Cell Lung Cancer: A 10-Year Review.

To describe the clinical characteristics of choroidal metastasis (CM) in non-small cell lung carcinoma (NSCLC) patients and report treatment outcomes following targeted therapy versus conventional radiotherapy and/or chemotherapy. A retrospective review was conducted in two hospitals in Hong Kong between 2013-2023. Outcomes included tumor response and visual function following treatment. A total of 25 eyes of 21 Chinese patients with CM secondary to metastatic NSCLC were identified. The majority of patients were non-smokers. At the time of diagnosis, 88% of eyes were visually symptomatic with a mean visual acuity of 20/100. There were 52% of tumors locating within the macula. TKI monotherapy, TKI with radiotherapy and radiotherapy and/or chemotherapy groups were observed to demonstrate similar tumor response from 1 month, and comparable ocular progression-free indices. Untreated patients had deteriorated vision without any tumor response. Tyrosine kinase inhibitor (TKI) monotherapy group achieved the most rapid and differential vision gain than other groups. TKI achieved durable disease control in epidermal growth factor receptor mutation positive NSCLC patients with CM, while improving visual function. TKI can be considered as an alternative to conventional orbital radiotherapy or chemotherapy for these patients in view of the rapid visual recovery.

A review on macrocyclic kinase inhibitors in clinical trials

Macrocyclic kinase inhibitors have high binding affinity and selectivity towards a variety of kinases including mammalian target of rapamycin complex 1/2, janus kinases/ Fms like tyrosine kinase, cyclin-dependent kinases and anaplastic lymphoma kinase1. Recently, few macrocyclic kinase inhibitors have entered clinical trial for treatment different types of cancers including leukemia, non-small cell lung cancer, myelofibrosis, breast cancer, glioblastoma and lymphoma. Of them, ridaforomilus has completed Phase III clinical trial and is waiting to be approved for treatment of breast cancer and advanced leukemia. Pacritinib is also currently being tested in phase III clinical trial for treatment of myelofibrosis and, loratinib is being evaluated for advanced ALK gene positive nonsmall cell lung carcinoma. The broad-spectrum cyclin-dependent kinases inhibitor, TGO2, has also entered phase II clinical trial for treatment of glioblastoma and advanced leukemia.

Therapeutic drug monitoring guided dosing versus standard dosing of alectinib in advanced ALK positive non-small cell lung cancer patients: Study protocol for an international, multicenter phase IV randomized controlled trial (ADAPT ALEC).

Alectinib is first-line therapy in patients with stage IV non-small cell lung carcinoma (NSCLC) and an anaplastic lymphoma kinase (ALK) fusion. A shorter median progression-free survival (mPFS) was observed when alectinib minimum plasma concentrations during steady state (Cmin,SS) were below 435 ng/mL. This may suggest that patients should have an alectinib Cmin,SS ≥ 435 ng/mL for a more favorable outcome. This potential target could be attained by using therapeutic drug monitoring (TDM), i.e. adjusting the dose based on measured plasma trough concentrations. Hypothetically, this will increase mPFS, but this has not yet been evaluated in a randomized controlled trial (RCT). Therefore, the ADAPT ALEC trial is designed, with the primary objective to prolong mPFS in NSCLC patients treated with alectinib by using TDM. ADAPT ALEC is a multicenter, phase IV RCT, in which patients aged ≥ 18 years with advanced ALK positive (+) NSCLC eligible for alectinib in daily care are enrolled. Participants will be randomized (1:1 ratio) into intervention arm A (TDM) or B (control), stratified by brain metastases and prior ALK treatments. Starting dose in both arms is the approved flat fixed dose of alectinib 600 mg taken twice daily with food. In case of alectinib Cmin,SS < 435 ng/mL, arm A will receive increased doses of alectinib till Cmin,SS ≥ 435 ng/mL when considered tolerable. The primary outcome is mPFS, where progressive disease is defined according to RECIST v1.1 or all-cause death and assessed by CT-scans and MRI brain. Secondary endpoints are feasibility and tolerability of TDM, patient and physician adherence, overall response rate, median overall survival, intracranial PFS, quality of life, toxicity, alectinib-M4 concentrations and cost-effectiveness of TDM. Exploratory endpoints are circulating tumor DNA and body composition. The ADAPT ALEC will show whether treatment outcomes of patients with advanced ALK+ NSCLC improve when using TDM-guided dosing of alectinib instead of fixed dosing. The results will provide high quality evidence for deciding whether TDM should be implemented as standard of care and this will have important consequences for the prescribing of alectinib. ClinicalTrials.gov, identifier NCT05525338.

P2.07-02 RET Fusion Testing with FISH and Real-Time PCR: a Comparison with RNA-Based Next-Generation Sequencing in RET Positive NSCLC

Although next-generation sequencing (NGS) is now the gold-standard for lung cancer biomarker testing, single-gene assays are still used for different reasons. Therefore, we were intrigued by the perfomance of commonly used fluorescence in situ hybridization (FISH) and real-time PCR assays in a cohort of RET fusion-positive non-small cell lung carcinoma (NSCLC) patients.

SY21-3 RET-altered cancers: toward tumor agnostic therapy

RRecently, highly potent and RET-specific inhibitors were translated to the clinic. In Japan, A RET-specific inhibitor, selpercatinib, has been implemented for RET-fusion positive non-small cell lung carcinoma in December, 2021. The RET gene is an oncogene that encodes a receptor-type tyrosine kinase, and actually functions as a driver gene for various cancer types other than lung cancer. In fact, clinical trials of RET inhibitors have confirmed their therapeutic effects in various cancer types, including pancreatic, breast, and ovarian cancers, and it is expected that they will be implemented as a tumor-agnostic treatment.

Clinical Significance of Dual-Block Elastic Stain Evaluating Visceral Pleural Invasion in Peripheral Non-Small Cell Lung Cancer.

Visceral pleural invasion (VPI) is a critical component in the staging of peripheral non-small cell lung carcinoma (NSCLC). We aim to investigate whether dual-block elastic stain increases visceral pleural invasion positivity compared with single-block elastic stain. We further analyze the potential predictors of visceral pleural invasion. 8419 peripheral NSCLC patients (including 6008 patients with tumor size≤3 cm in stage I) were divided into a cohort using one paraffin block (single-block group, n = 5184) and a cohort using dual paraffin blocks (dual-block group, n = 3235) for elastic stain. The VPI-positive rate demonstrated by the dual-block elastic stains group was significantly higher than that of the single-block elastic stain group (17.7% (573/3235) versus 9.1% (474/5184), respectively, P < .001). The presence of visceral pleural invasion in T1 (≤3 cm) patients detected by single- and dual-block elastic stain was 6.3% (235/3730) and 12.0% (273/2278), respectively (P < .001). 5.7% of T1 patients (stage IA) were additionally upstaged to T2a (stage IB) by dual-block elastic stain. However, the incidence of visceral pleural invasion in pT2a patients showed no significant difference between the single-block group and the dual-block group (16.8% vs. 17.1%, P = .916). Lymphovascular invasion, lymph node metastasis, dedifferentiated carcinomas, the presence of spread through airspaces (STAS) and a poorly differentiated adenocarcinomatous growth pattern could be significant predictors of visceral pleural invasion (P < .001). Our results indicate that using dual-block elastic stain identifies more visceral pleural invasion positive T1 NSCLC patients who are upstaged to T2a, and who could benefit from optimal management post-operatively. The application of dual-block elastic stain is an efficient and practical method to detect visceral pleural invasion status.

Synthesis of 111In-p-SCN-Bn-DTPA-nimotuzumab and its preclinical evaluation in EGFR positive NSCLC animal model

Abstract The aim of this study was to investigate labeling of nimotuzumab (h-R3) with 111In using p-SCN-Bn-DTPA as bifunctional chelate, evaluate its targeting potential against SK-LU-1, H226, H650, H661, and HCC4006 non-small cell lung carcinoma (NSCLC) cell lines and correlate epidermal growth factor receptor (EGFR) expression level with internalization kinetics, biodistribution and imaging accuracy using Balb/c mice and New Zealand White rabbit (NZWR) animal model. The amount of p-SCN-Bn-DTPA attached to h-R3 was assessed by measuring relative absorbance at 652 nm with ultraviolet (UV) spectrophotometer. High-performance liquid chromatography (HPLC) was used to determine percent radiochemical purity (%RCP) and in vitro stability using excess amount of diethylenetriamine pentaacetate (DTPA). The in vitro stability in rat serum was estimated using iTLC-SG. EGFR expression level in each tumor was assessed by chemiluminescence. In vivo uptake in different organs of Balb/c mice and non-invasive imaging potential using NZWR bearing HCC4006 tumor, was evaluated with gamma camera. UV spectroscopy has confirmed the attachment of five p-SCN-Bn-DTPA (chelate) with one antibody. The HPLC indicated 98.85 ± 0.14% (n = 3) %RCP with high yield (&gt;96%), specific activity 3.5 ± 0.0.25 mCi per mg and 94.25 ± 0.34% in vitro stability at 37 °C in mice serum. In excess DTPA no considerable transchelation was experiential from the 111In labeled p-SCN-Bn-DTPA-h-R3 to the challenger. The EGFR expression in HCC4006 was higher amongst all with band density of 23.53 relative to 1.00 of H226. Initially internalization was lower which went up 1.05 × 104 molecules per HCC4006 cell in 48 h. The optimal concentration of h-R3 for maximum uptake was 15 μg per animal. Higher uptake in target organ was observed in animal infected with HCC4006 cells. However, in excess pure h-R3 the uptake was significantly reduced indicating tumor specificity. HCC4006 target site was undistinguishable relative to background activity in the initial phase of imaging due to poor uptake. However, within 60 h the HCC4006 tumor was quite apparent. This experiment suggests that at optimal dosage of 111In labeled h-R3 can be used for localization and identification of EGFR positive NSCLC using gamma camera.

MA14.02 RET Fusion Testing in Advanced Non-Small Cell Lung Carcinoma Patients: the RETING Study

The approval of selective RET inhibitors for patients with advanced RET fusion positive non-small cell lung carcinoma (NSCLC) means that the importance of accurately identifying those patients has never been greater. Due to the co-existance of single-gene testing and next-generation sequencing (NGS), we hypothesized that a comparison of RET fusions testing assays is urgently needed. Along those lines, the performance of RET immunohistochemistry (IHC) and RET fluorescence in situ hybridization (FISH) has recently been challenged. Physicians across 17 hospitals contributed to identify patients with RET fusion positive NSCLC as part of routine clinical care. In addition, RET fusion negative samples from NSCLCs diagnosed at the referral institution were also included. All tumors underwent targeted RNA-based NGS (Oncomine Comprehensive Assay v3), break-apart RET FISH (Vysis) with an automated scanning system (BioView), and RET IHC (clone EPR2871, Abcam). The material available for all tumors had been formalin-fixed and paraffin-embedded. Only cases with enough tissue available (i.e. a minimum of 50 tumor cells, as per FISH test requirements) and sufficient sequencing coverage after NGS were included. FISH and IHC results were interpreted by using previously described criteria. The NGS result was used as the gold-standard. Analyses by the three assays was succesful in all 80 tumors. Thirty-seven RET-positive samples and 43 RET-negative NSCLCs were identified. Signet ring cells and psammomatous calcifications were frequently observed in RET-positive samples (in 30% and 27% of tumors, respectively). The most common partners were KIF5B (29/37, 78%), followed by CCDC6 (6/37, 16%). Other partners included NCOA4 (1/37, 3%) and AKAP13 (1/37, 3%). Thirty-five out of the 37 (95%) NGS-positive samples were FISH-positive (86% with a split pattern and the remaining 14% with a single 3’ signal pattern). Six of those FISH-positive cases (6/35, 17%) displayed very challenging split signals (≤2 signal diameter). The two FISH-negative samples were KIF5B-RET variants that showed a narrow split (≤1 signal diameter) in 36% and 38% of tumor cells, respectively. All 43 NGS-negative tumors were also FISH-negative. Regarding RET IHC, all NGS-positive cases showed cytoplasmic staining with a mean H-score of 210 (median 210, range 5-300). Within the NGS-negative cohort, cytoplasmic staining was observed in 8 cases (19%), with a mean H-score of 9 (median 0, range 0-150). Interestingly, the two RET FISH-negative but NGS-positive samples showed relevant IHC positivity (H-score of 165 and 255, respectively). The most frequent partner in this large multicenter series of RET fusion positive NSCLCs was KIF5B. The concordance between RNA-based NGS and FISH was high, with 0% failure rate. However, the interpretation of RET FISH is very challenging due to the frequent occurrence of narrow split signals (22% in this series). RET IHC should be investigated further as an alternative method to resolve an equivocal FISH or NGS result. Funding This study was mainly funded by Lilly. We also thank Instituto de Salud Carlos III (ISCIII) (Fondos FEDER and Plan Estatal I+D+I 2008–2011 [PI11/02866] and 2013–2016 [PI14-01176 y PI17-01001]) and the iLUNG Program (B2017/BMD-3884) from the Comunidad de Madrid.

Brigatinib and lorlatinib: their effect on ALK inhibitors in NSCLC focusing on resistant mutations and central nervous system metastases.

Major issues in anaplastic lymphoma kinase-positive non-small cell lung carcinoma are acquired resistance against anaplastic lymphoma kinase inhibitors and control of central nervous system metastasis. The development of these inhibitors has changed therapeutic strategy in patients with advanced anaplastic lymphoma kinase-positive non-small cell lung carcinoma. Brigatinib and lorlatinib were designed to penetrate the blood-brain barrier and to inhibit resistant mutations against anaplastic lymphoma kinase inhibitors. We review the clinical data supporting treatment of advanced anaplastic lymphoma kinase-positive non-small cell lung carcinoma with brigatinib and lorlatinib. Brigatinib has shown promising antitumour activity, including substantial activity against central nervous system metastases, in crizotinib-treated (ALTA trial) patients and crizotinib-naïve (ALTA-1L trial) patients with anaplastic lymphoma kinase-positive non-small cell lung carcinoma. In addition, brigatinib improved progression-free survival compared with crizotinib in anaplastic lymphoma kinase inhibitor-naïve patients with anaplastic lymphoma kinase-positive non-small cell lung carcinoma. Lorlatinib has demonstrated clinical antitumour activity against both intracranial and extracranial lesions in patients with anaplastic lymphoma kinase- or c-ros oncogene 1 (ROS1)-positive non-small cell lung carcinoma. Ongoing trials and further studies of these agents' biological and clinical properties would provide insight into the optimal therapeutic strategy for administering them to achieve the best survival benefit.

Novel Crizotinib-GnRH Conjugates Revealed the Significance of Lysosomal Trapping in GnRH-Based Drug Delivery Systems.

Several promising anti-cancer drug–GnRH (gonadotropin-releasing hormone) conjugates have been developed in the last two decades, although none of them have been approved for clinical use yet. Crizotinib is an effective multi-target kinase inhibitor, approved against anaplastic lymphoma kinase (ALK)- or ROS proto-oncogene 1 (ROS-1)-positive non-small cell lung carcinoma (NSCLC); however, its application is accompanied by serious side effects. In order to deliver crizotinib selectively into the tumor cells, we synthesized novel crizotinib analogues and conjugated them to a [d-Lys6]–GnRH-I targeting peptide. Our most prominent crizotinib–GnRH conjugates, the amide-bond-containing [d-Lys6(crizotinib*)]–GnRH-I and the ester-bond-containing [d-Lys6(MJ55*)]–GnRH-I, were able to bind to GnRH-receptor (GnRHR) and exert a potent c-Met kinase inhibitory effect. The efficacy of compounds was tested on the MET-amplified and GnRHR-expressing EBC-1 NSCLC cells. In vitro pharmacological profiling led to the conclusion that that crizotinib–GnRH conjugates are transported directly into lysosomes, where the membrane permeability of crizotinib is diminished. As a consequence of GnRHR-mediated endocytosis, GnRH-conjugated crizotinib bypasses its molecular targets—the ATP-binding site of RTKs— and is sequestered in the lysosomes. These results explained the lower efficacy of crizotinib–GnRH conjugates in EBC-1 cells, and led to the conclusion that drug escape from the lysosomes is a major challenge in the development of clinically relevant anti-cancer drug–GnRH conjugates.

Abstract 1398: Modeling of drug-protein interactions to support clinical decision making for therapy-resistant EGFR or ALK-positive non-small cell lung carcinoma

Abstract Background Activating mutations in epidermal growth factor receptor (EGFR) and anaplastic lymphoma kinase (ALK) rearrangements are drugable targets in respectively 10% and 4% of advanced non-small cell lung cancer (NSCLC) patients. Different EGFR and ALK inhibitors are available for target-dependent and sometimes independent drug resistance. Mutations in the EGFR or ALK kinase domain are major mechanisms of drug-induced resistance. Prediction of protein-drug interactions using protein modeling of these mutated domains in EGFR and ALK could provide valuable information for the selection of drugs for subsequent treatment. Methods Biopsies from advanced NSCLC patients with an activating EGFR mutation or ALK rearrangement that were taken at therapy resistance were analyzed by targeted panel sequencing. In case of unknown or accumulating mutations in the EGFR and ALK kinase domains, docking poses and binding affinities of drug-protein interactions were determined. The outcome of both calculations were used to create a likelihood ranking of currently available ALK and EGFR inhibitors to bind to specific mutated targets. Follow-up data was available for a limited number of patients who were treated based on this prediction. Results Prediction Protein-drug interactions were modeled for 59 EGFR mutations and 24 ALK mutations that were detected at therapy resistance. Prediction of therapeutic activities for gefitinib, afatinib, dacomitinib, for EGFR mutations, and alectinib, ceritinib, entrectinib and lorlatinib for ALK mutations will be shown. Case example Female (35y), never-smoker, cT1bN2M1c NSCLC with metastases in lymph nodes, liver, bones and asymptomatic brain metastases, ALK translocation-positive without known ALK gatekeeper mutations. Treatment with crizotinib was started, but unacceptable liver enzyme toxicity developed after 6 weeks. Switching therapy to alectinib resulted in a complete metabolic response with absence of brain metastases by MRI. After 9 months the patient progressed with liver, brain and leptomeningeal metastases. Treatment with ceritinib had no effect. A liver biopsy revealed the ALK p.(L1196M) gatekeeper mutation. The case was discussed in the molecular tumor board, because this gatekeeper variant was reported to be sensitive to ceritinib. In contrast, ALK-drug interaction modeling indicated a better binding profile of lorlatinib than ceritinib, not only based in binding affinity but also in the ability of lorlatinib to reproduce the bioactive conformation found in literature. The patient was treated with lorlatinib and recovered in 5 days with a persistent partial response at 2 month follow up and significant clinical benefit. Conclusion Modeling of the protein-drug interactions supports clinical decision making for treatment of NSCLC patients in case of therapy resistance to EGFR and ALK inhibitors. Citation Format: Juliana F. Vilacha Madeira R Santos, Birgitta I. Hiddinga, Leon C. van Kempen, Arja ter Elst, Lucie B. Hijmering, Thijo J. Hiltermann, Ed Schuuring, Matthew R. Groves, Harry J. Groen, Anthonie J. van der Wekken. Modeling of drug-protein interactions to support clinical decision making for therapy-resistant EGFR or ALK-positive non-small cell lung carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1398.

Erlotinib monotherapy in the treatment of advanced non-small cell lung carcinoma: A single center experience with 187 patients from 2005-2018.

e20718 Background: Inhibition of epidermal growth factor receptors (EGFR) bearing somatic mutations in the tyrosine kinase domain is an important molecular target in the treatment of advanced non-small cell lung carcinoma (NSCLC). As such, first generation tyrosine kinase inhibitors such as erlotinib have been adopted as standard of care in the treatment of EGFR mutation positive advanced NSCLC. Herein we describe our experience with erlotinib monotherapy since its approval for clinical use in the United States in late 2004. Methods: A retrospective analysis of 187 patients with a diagnosis of NSCLC who underwent erlotinib monotherapy from 2005-2018 was performed. Clinical variables including patient age, disease stage, EGFR genomic status, adverse events and survival were analyzed. Likelihood Ratio Chi-Square test was applied and statistical significance was set to p &lt; 0.05. Results: Among all patients who received erlotinib monotherapy (n = 187), treatment duration longer than 3 months was associated with EGFR mutant status (p &lt; 0.001), increased rates of repeat lung biopsies (p &lt; 0.002) as well as pulmonary (p = 0.008), gastrointestinal (p = 0.005) and cutaneous adverse events (p = 0.005). Subgroup analysis based on EGFR genomic status (n = 71) revealed that EGFR mutation positive patients remained on erlotinib for at least three months at a higher rate than wildtypes (p &lt; 0.001), but also sustained higher rates of lung re-biopsy (p = 0.0121) and concomitant gastrointestinal (diarrhea) and cutaneous (acneiform, maculopapular rashes) adverse events (grades 1-3) (p = 0.003). EGFR mutant status was associated with increased rates of survival of 12 months or greater since the initiation of erlotinib (p &lt; 0.001). Conclusions: Erlotinib monotherapy duration greater than 3 months and EGFR mutant status are associated with significantly higher rates of repeat lung biopsies and gastrointestinal and cutaneous adverse events. EGFR mutant status is further associated with survival of greater than 1 year.

Predictive Biomarkers for Checkpoint Inhibitor-Based Immunotherapy: The Galectin-3 Signature in NSCLCs

Checkpoint inhibitor-based immunotherapy is opening a promising scenario in oncology, with objective responses registered in multiple cancer types. However, reliable predictive markers of tumor responsiveness are still lacking. These markers need to be urgently identified for a better selection of patients that can be candidates for immunotherapy. In this pilot study, a cohort of 34 consecutive patients bearing programmed death-ligand 1 (PD-L1)-positive non-small cell lung carcinoma (NSCLC), treated with pembrolizumab, was considered. The retrospective immuno-phenotypic analysis performed on the original tumor biopsies allowed for the identification of a specific “galectin signature”, which strongly correlated with tumor responsiveness to anti PD-1 immunotherapy. We observed that the large majority of patients (about 90%) with high galectin-3 tumor expression (score 3+) showed an early and dramatic progression of the disease after three cycles of treatments. In contrast, all patients with negative or low/intermediate expression of galectin-3 in tumor cells showed an early and durable objective response to pembrolizumab, indicating galectin-3 as an interesting predictive marker of tumor responsiveness. The galectin-3 signature, at least in NSCLCs, promises a better selection of patient candidates for immunotherapy, reducing unnecessary treatment exposures and social costs. A large multicenter study is ongoing to validate this finding.

Dichotomous ALK-IHC Is a Better Predictor for ALK Inhibition Outcome than Traditional ALK-FISH in Advanced Non-Small Cell Lung Cancer.

Purpose: ALK rearrangement detection using FISH is the standard test to identify patients with non-small cell lung carcinoma (NSCLC) eligible for treatment with ALK inhibitors. Recently, ALK protein expression in resectable NSCLC showed predictive value. We evaluated tumor response rate and survival after crizotinib treatment of patients with advanced NSCLC with ALK activation using both dichotomous immunohistochemical (IHC) staining and FISH.Experimental Design: Patients with stage IV NSCLC treated with crizotinib were selected. Tumor response was assessed. ALK rearrangements were detected by FISH (Vysis ALK-break-apart FISH-Probe KIT) and IHC [Ventana ALK (D5F3) CDx assay]. Cohorts of patients with ALK-FISH-positive advanced NSCLC from four other hospitals were used for validation.Results: Twenty-nine consecutive patients with ALK-positive advanced NSCLC diagnosed by FISH and/or IHC on small biopsies or fine-needle aspirations (FNA) were treated with ALK inhibitors. All ALK-IHC-positive patients responded to crizotinib except three with primary resistance. No tumor response was observed in 13 ALK-FISH-positive but ALK-IHC-negative patients. This was confirmed in an external cohort of 16 patients. Receiver operator characteristic (ROC) curves for ALK-IHC and ALK-FISH compared with treatment outcome showed that dichotomous ALK-IHC outperforms ALK-FISH [tumor response area under the curve: (AUC), 0.86 vs. 0.64, P = 0.03; progression-free survival (PFS): AUC 0.86 vs. 0.36, P = 0.005; overall survival (OS): AUC, 0.78 vs. 0.41, P = 0.01, respectively].Conclusions: Dichotomous ALK-IHC is superior to ALK-FISH on small biopsies and FNA to predict tumor response and survival to crizotinib for patients with advanced NSCLC. Our data strongly suggest adapting the guidelines and using dichotomous ALK-IHC as standard companion diagnostic test to select patients with NSCLC who benefit from ALK-targeting therapy. Clin Cancer Res; 23(15); 4251-8. ©2017 AACR.

Abstract 5267: Gene fusion database to create custom panels: Enabling detection of fusion transcripts and gene expression assays

Abstract Gene fusions play an important role in tumorigenesis and are increasingly recognized as important entities for the diagnosis and treatment of hematological malignancies and solid tumors. Fusion events generate a hybrid mRNA transcript comprising sequence from multiple otherwise distinct genes. Oncogenic fusion events often involve tyrosine kinases or transcription factors, leading to aberrant growth signaling, making these events potentially attractive drug targets. For instance, targeted therapies such as known tyrosine kinase inhibitors are currently approved to treat ALK fusion positive Non-Small Cell Lung Carcinoma (NSCLC) patients. Detection of known gene fusion events is an important part of genomic characterization which can inform patient diagnosis. Current methods for fusion detection include chromosome banding analysis (CBA), fluorescence in situ hybridization (FISH), and reverse transcription polymerase chain reaction (RT-PCR). New developments in next-generation sequencing (NGS) enable the efficient and simultaneous assessment of multiple gene fusion targets with high sensitivity. To enable researchers to design their own custom panels and assess a set of gene fusions of interest, we developed a comprehensive RNA gene fusion database. Oncology researchers now have the capability to create custom panels from this comprehensive database which includes over 1,000 well annotated and optimized gene fusion assays and over 20,000 gene expressions assays. To build this comprehensive gene fusion database, we identified breakpoint information for 1,178 well annotated fusions described in publications and in the COSMIC and NCBI databases. We prepared a target RNA sequence for each breakpoint using transcript sequences from the Ensembl database. We used a proprietary primer designer to generate candidates for each fusion target amenable to the AmpliSeq™ product line requirements. Quality control was performed throughout the design process to identify the best primer set for each target, to avoid primers overlapping common germline SNPs, potential primer/primer or primer/amplicon interactions, or off-target or wild-type amplifications. With this comprehensive database we provide a complete range of solutions available on ampliseq.com. Making use of the AmpliSeq™ technology, researchers now have the capability to create their own custom fusion panel and place the order within an hour. These custom panels are used with AmpliSeq™ Library reagents and Ion Torrent™ sequencing platforms for targeting next-generation sequencing. The analysis solution is provided through the Ion Reporter™ (IR) software package. Custom fusion panel workflows in IR are used to analyze sequencing data coming from the custom panels, which includes visualization of fusion transcripts and gene expression levels in a heat map feature. Citation Format: Efren Ballesteros-Villagrana, Jeoffrey Schageman, Kelli Bramlett, Paul Williams, Scott Myrand, Guoying Liu, Fiona Hyland, Seth Sadis. Gene fusion database to create custom panels: Enabling detection of fusion transcripts and gene expression assays. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5267.

Stimulation of Suicidal Erythrocyte Death by Ceritinib-Treatment of Human Erythrocytes

Background/Aims: The anaplastic lymphoma kinase (ALK) inhibitor ceritinib is utilized for the treatment of ALK positive non-small cell lung carcinoma. Side effects of the drug include decrease of blood hemoglobin concentration. Possible causes of anemia include stimulation of suicidal erythrocyte death or eryptosis, which is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Signaling of eryptosis includes increase of cytosolic Ca²⁺ activity ([Ca²⁺]_i), oxidative stress, ceramide, staurosporine sensitive protein kinase C, SB203580 sensitive p38 kinase, D4476 sensitive casein kinase 1, and zVAD sensitive caspases. The present study explored, whether ceritinib induces eryptosis and, if so, to shed light on the cellular mechanisms involved. Methods: Phosphatidylserine exposure at the cell surface was estimated from annexin-V-binding, cell volume from forward scatter, [Ca²⁺]_i from Fluo3-fluorescence, ROS formation from DCFDA dependent fluorescence, and ceramide abundance utilizing specific antibodies. Results: A 48 hours exposure of human erythrocytes to ceritinib (1 µg/ml) significantly increased the percentage of annexin-V-binding cells, significantly decreased forward scatter, significantly increased Fluo3-fluorescence, but did not significantly modify DCFDA fluorescence or ceramide abundance. The effect of ceritinib on annexin-V-binding was significantly blunted but not abolished by removal of extracellular Ca²⁺, by the kinase inhibitors staurosporine (1 µM), SB203580 (2 µM) and D4476 (10 µM), as well as by caspase inhibitor zVAD (10 µM). Conclusions: Ceritinib triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect at least in part due to Ca²⁺ entry, as well as activation of kinases and Caspases.

Anti-ALK Antibodies in Patients with ALK-Positive Malignancies Not Expressing NPM-ALK

Patients with Nucleophosmin (NPM)- Anaplastic Lymphoma Kinase (ALK) fusion positive Anaplastic Large Cell Lymphoma produce autoantibodies against ALK indicative of an immune response against epitopes of the chimeric fusion protein. We asked whether ALK-expression in other malignancies induces specific antibodies. Antibodies against ALK were detected in sera of one of 50 analysed ALK-expressing neuroblastoma patients, 13 of 21 ALK positive non-small cell lung carcinoma (NSCLC) patients, 13 of 22 ALK translocation-positive, but NPM-ALK-negative lymphoma patients and one of one ALK-positive rhabdomyosarcoma patient, but not in 20 healthy adults. These data suggest that boosting a pre-existent anti-ALK immune response may be more feasible for patients with ALK-positive NSCLC, lymphomas and rhabdomyosarcomas than for tumours expressing wild-type ALK.

Abstract 667: IMGN853, a folate receptor (FR) α-targeting antibody-drug conjugate (ADC), is highly effective against xenograft models with clinically relevant levels of receptor expression

Abstract IMGN853 comprises the anti-FR-α antibody M9346A conjugated to the cytotoxic maytansinoid molecule DM4 via a stable disulfide-containing linker, sulfo-SPDB. IMGN853 is currently in phase I clinical testing; promising clinical results were reported at ASCO 2013. To analyze determinants of the sensitivity of FR-α positive cells to IMGN853, we examined antigen density on the cell surface, internalization and processing of IMGN853, and sensitivity of the cells to IMGN853 and to non-conjugated maytansinoid, S-methyl-DM4, in vitro. Antigen density, internalization and processing were analyzed with 3[H] M9346A. Preliminary experiments demonstrated that 3[H] M9346A is an appropriate surrogate reagent to analyze binding and processing of IMGN853. Sensitivity of the cells to IMGN853 and to the unconjugated maytansinoid was analyzed by a cytotoxicity assay. A panel of seven FR-α positive cell lines was used for the study. Three of the cell lines were sensitive to IMGN853 in an antigen-dependent manner (the cytotoxic effect was blocked in the presence of a saturating excess of the non-conjugated antibody), while four lines were insensitive (the same low cytotoxic activity of IMGN853 was observed with and without the non-conjugated antibody). The proportion of cell surface-bound IMGN853 that was internalized and processed by the sensitive and insensitive cell lines was comparable. The cell lines differed in their FR-α expression, and there was a strong positive correlation between the sensitivity of cells towards IMGN853 and antigen expression on their surface. The cell lines were similar in their sensitivity to S-methyl-DM4. Taken together, the data indicate that the FR-α density on the surface of a cell is a key determinant of its sensitivity to IMGN853. Having observed a correlation between antigen density and sensitivity of the cells to IMGN853, we set out to test IMGN853 activity in xenograft models expressing FR-α at levels comparable to those on patient tumor cells. First, we developed an IHC assay with the use of anti-FR-α antibody BN3.2 (Novocastra Laboratories, UK) that detected various levels of receptor expression in tumor samples. Next, we compared FR-α expression in ovarian carcinoma (OvCa), non-small cell lung carcinoma (NSCLC) samples and in human xenograft models, and examined five models that represented antigen densities of the majority of FR-α positive OvCa and NSCLC patients. We evaluated the anti-tumor activity of IMGN853 in SCID mice bearing these xenograft models after single intravenous injections of 5, 2.5 or 1.2 mg/kg. IMGN853 was highly active with a minimally effective dose from 1.2 to 2.5 mg/kg depending on the model. There was no activity of the conjugate against a FR-α negative xenograft model. Thus, IMGN853 is highly effective against xenograft models with clinically relevant levels of FR-α expression. Citation Format: Olga Ab, Laura M. Bartle, Xiuxia Sun, Rui Wu, Holly A. Johnson, Kathleen R. Whiteman, Alyssa LaBelle, Victor S. Goldmacher. IMGN853, a folate receptor (FR) α-targeting antibody-drug conjugate (ADC), is highly effective against xenograft models with clinically relevant levels of receptor expression. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 667. doi:10.1158/1538-7445.AM2014-667

Abstract 4919: Multiplexed EGFR signaling pathway analysis in FFPE tissue using quantitative mass spectrometry

Abstract The epidermal growth factor receptor (EGFR) is a drug target for both small molecule and antibody therapeutics and has been approved in non small-cell lung carcinoma (NSCLC) and colorectal carcinoma (CRC) among other indications. These drugs block receptor signaling though blockade of the tyrosine kinase domain, or through inhibition of ligand binding. Current genomic tests measure receptor amplification, RNA levels, the mutation status of receptor or pathway molecules (EGFR or kRAS mutations) but no current assay can directly assess the activation state of the EGFR or its downstream signaling pathway components. Indeed, the EGFR mutation positive NSCLC tumors (thought to be constitutively active) show a high response rate to TKI therapy, but the many non responders (50% or more) demonstrate the limitation of genomic analysis. Since activation of EGFR is necessary for the response to these targeted agents, it is critical to measure what levels of receptor activation and downstream signaling determines tumor responsiveness to EGFR targeted therapies in these patients. For this reason, we have developed a panel of new diagnostic assays which measure the activation of the EGFR and key downstream signaling proteins through quantitation of the phosphorylation state of these proteins. These assays are based on the Liquid Tissue®-SRM technology platform. This approach enables relative and absolute quantification of proteins and their phosphorylation status directly in formalin fixed paraffin embedded (FFPE) tissue. We preclinically validated the multiplexed Liquid Tissue® phospho-SRM assay on formalin fixed EGF stimulated A431 cells. We followed up these in vitro studies with phospho-SRM analysis of FFPE NSCLC xenograft explants where extensive independent histopathologic and molecular characterization had been performed, allowing us to benchmark our phospho-SRM analysis with standard diagnostic analyses. We have now extended these quantitation studies by measuring the expression of EGFR and phospho-EGFR in FFPE tissues obtained from relevant human clinical trial cohorts – Gefitinib treated NSCLC and Cetuximab treated CRC. It is hoped that we will be able to correlate EGFR expression, activation and signaling in these tumors with responsiveness to EGFR targeted therapy, and to validate this assay for use as a companion diagnostic to guide therapy in both NSCLC and CRC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4919. doi:10.1158/1538-7445.AM2011-4919

The role of FDG-PET before and 3 weeks after neoadjuant chemoradiotherapy (NACRT) in predicting N2 clearing and survival after surgical resection in patients with biopsy-proven N2 positive non-small cell lung carcinoma (NSCLC)

7503 Background: Downstaging via NACRT is a significant prognostic factor for better survival in patients (pts) with NSCLC. Thus, identification of N2 clearing after NACRT is a critical factor for patient selection for thoracotomy. We evaluated the usefulness of FDG- PET in predicting N2 clearing after NACRT and survival after surgical resection in pts with N2 positive NSCLC. Methods: From November 2002 to October 2006, 45 pts with biopsy-proven N2 positive NSCLC were enrolled for NACRT. Thirty-four pts who had FDG-PET data before and after NACRT and underwent surgical resection were analyzed. They received 6 weekly chemotherapy with paclitaxel (50mg/m2)/cisplatin (20mg/m2) and concurrent radiotherapy (1.8Gy/day x 5/week, 45 Gy) during 5 weeks. FDG-PET scan was repeated before and 3 weeks after NACRT. Maximum standardized uptake values (SUVmax) of primary tumor and N2 nodes were measured, and percent changes in SUVmax were calculated. After surgery, the pathology of N2 nodes was assessed. Results: After NACRT, overall mean SUVmax of primary mass and N2 nodes decreased from 7.02±3.68, 6.78±4.18 to 1.80±1.27, 1.52±1.74 (74%, 78% reduction, respectively) and 17/34 pts had metabolic N2 clearing (including 8 false negative) and 17/34 pts had remained positive N2 (including 10 false positive) by FDG-PET. Mean percentage changes of SUVmax of primary mass and N2 nodes in pathologic N2 clearing group (n=19) were 75% and 76% and those with remained N2 group (n=15) were 67% and 78%, with no significant difference between two groups. Median progression-free survival (PFS) and overall survival (OS) of all 34 pts were 38 months and 45 months with median follow-up duration of 34 months (range 9–56). Median PFS in pts who achieved a reduction in SUVmax of N2 nodes of more than 60% and in pts who achieved one of less than 60% were 38 months and 14 months (log-rank test, p=0.044). OS between two groups had no significant difference. Conclusions: FDG-PET before and 3 weeks after NACRT could not predict pathologic N2 clearing. Patients who had a reduction in SUVmax of N2 nodes of more than 60% showed better PFS in patients with NSCLC treated with NACRT followed surgical resection. No significant financial relationships to disclose.