Targetable Driver Mutations Research Articles

Nintedanib plus docetaxel after progression on first-line immunochemotherapy in patients with lung adenocarcinoma: Cohort C of the non-interventional study, VARGADO.

Immune checkpoint inhibitors (ICIs) with or without chemotherapy represent first-line standard of care for patients with advanced non-small cell lung cancer (NSCLC) without targetable driver mutations. The most appropriate second-line therapy after failing immunochemotherapy remains an open question. Nintedanib, an oral triple angiokinase inhibitor that targets the vascular endothelial growth factor receptor, fibroblast growth factor receptor, and, platelet-derived growth factor receptor, in combination with docetaxel, is approved for treatment of advanced NSCLC (adenocarcinoma histology) following progression on first-line chemotherapy. VARGADO (NCT02392455) is an ongoing, prospective, non-interventional study investigating the efficacy and safety of nintedanib plus docetaxel following first-line chemotherapy with or without ICIs in patients with locally advanced, metastatic, or locally recurrent NSCLC of adenocarcinoma histology. This analysis focuses on Cohort C, which enrolled patients who had received prior first line chemotherapy with ICIs. Patients received second-line docetaxel (75 mg/m²) by intravenous infusion on Day 1, plus oral nintedanib (200 mg twice daily) on Days 2-21 of each 21-day cycle during routine clinical care. The primary endpoint is overall survival (OS) rate 1 year after the start of treatment with nintedanib plus docetaxel. Secondary endpoints include progression-free survival (PFS), OS, and disease control rate (DCR). Safety was also assessed. Among 137 patients treated, the median age was 63 years (range, 37-84); 57 patients (41.6%) were female, most patients had Eastern Cooperative Oncology Group performance status of 0 (28.5%) or 1 (43.1%); 118 (86.1%) had stage IV NSCLC and 27 (19.7%) had brain metastases. Most (n=120, 87.6%) patients had received pembrolizumab/pemetrexed/platinum-based chemotherapy as first-line treatment. In 80 patients with available response data, the DCR was 72.5% (complete response: 1.3%; partial response: 36.3%; stable disease: 35.0%). Median progression-free survival was 4.8 months (95% confidence interval: 3.7-6.6). OS data were immature. Grade ≥3 treatment-emergent adverse events (TEAEs), serious TEAEs, and TEAEs leading to treatment discontinuation were reported in 62 (45.3%), 50 (36.5%), and 40 patients (29.2%), respectively. This analysis indicates that nintedanib plus docetaxel represents an effective second-line treatment option in patients with advanced adenocarcinoma NSCLC following progression on first-line immunochemotherapy. The safety profile was manageable with no unexpected signals.

MM-356 Deep Coverage Targeted Next Generation Sequencing Approach to Determine Somatic Mutational Spectrum of Recurrently Mutated Genes in Newly Diagnosed Multiple Myeloma in a Large Indian Cohort

To determine the mutational spectrum of recurrently mutated genes in a large cohort of Indian patients presenting with multiple myeloma (MM) in our center by performing deep coverage-targeted NGS. Samples of 151 multiple myeloma patients (149 NDMM and 6 follow-up) were sequenced. Patients were diagnosed with MM per IMWG criteria 2014. The study was initiated following approval from the institute's ethical committee, and written informed consent was obtained from all patients. BMA was subjected to plasma cell isolation using CD138+ MACS. DNA underwent automated extraction using the Maxwell System. Mutations were tested on a commercially available 47-gene custom panel (Illumina) specifically designed for MM. Libraries were prepared using a TruSeq Custom Amplicon Low Input Kit (Illumina). DNA (25 ng) was hybridized with oligos specific for targeted regions of interest. Libraries were pooled. Paired-end sequencing was carried out on Illumina's MiSeq sequencer using micro flow cell with V3 chemistry and MS600 V3 series reagent cartridge. Sequenced reads were mapped to GRCh37/Hg19 reference and data obtained as FASTQ files and processed with MiSeq Reporter. As output, .vcf files were generated. Genomic variants were annotated by ANNOVAR. Sequencing coverage of 4771 X (Range 2063X-10980X) revealed different types of variants (exonic, intronic, splice region, 3' and 5' UTR, and upstream gene). Pathogenicity was checked by COSMIC. At least one pathogenic mutation was found in 77% (116/151) of patients (median 1 mutation per patient, range 0-7) and 51% (24/47) of genes. Forty-five percent (68/151) of patients had mutations in more than one gene. The most frequently mutated genes were KRAS, NRAS, BRAF, TP53, DIS3, FAT4, FAT3, and DNAH5. Genomic aberrations found were compared with the frequency of mutations as per the MMRF and TCGA datasets and related literature; good concordance was found (especially in MAPK pathway genes). BRAF and RAS mutations were mutually exclusive. Few mutations were validated by Sanger sequencing. The high frequency of mutations in MAPK pathway genes (KRAS, NRAS, BRAF) suggested the importance of this pathway in myelomagenesis and therapeutics. Follow-up sample sequencing may help deduce clonal evolution during disease course. Identification of clinically relevant target driver mutations may aid in prognosis and therapy personification.

Novel Calcium-Binding Ablating Mutations Induce Constitutive RET Activity and Drive Tumorigenesis.

Comprehensive proteogenomic and in silico analyses of a vast number of VUSs identify a novel set of oncogenic and druggable mutations in the well-characterized RET oncogene.

EP08.02-106 KEAP1/NFE2L2 Transcriptomic Signature Predicts Survival in Advanced Stage NSCLC Patients Without Actionable Driver Mutations

We developed a KEAP1/NFE2L2 pathway signature that robustly predicts functional mutations of KEAP1- and NFE2L2-mutated NSCLC. To determine the prognostic value of this signature, we identified a cohort with KEAP1/NFE2L2 mutations and compared it with a control cohort lacking established targetable driver mutations.

Genetic Disorders with Predisposition to Paediatric Haematopoietic Malignancies-A Review.

Simple SummaryA significant progress in understanding the genetic basis of childhood cancers has been made over the past years. Genome sequencing has identified significant differences between paediatric and adult cancers. A higher prevalence of germline alterations in cancer predisposition genes is observed, in comparison to somatic mutations in paediatric cancers. Interestingly, pathogenic germline variants and somatic mutations can affect the same genes. The early recognition of genetic predispositions for childhood cancers may provide an opportunity of therapy adjustment and specific screening for different syndrome-related malignancies. Research on the biological function of gene variants with increased cancer predispositions is critical to the needs of adequate patient management. Genetic counselling with targeted therapy have become basis of integrated cancer care in paediatric oncology.The view of paediatric cancer as a genetic disease arises as genetic research develops. Germline mutations in cancer predisposition genes have been identified in about 10% of children. Paediatric cancers are characterized by heterogeneity in the types of genetic alterations that drive tumourigenesis. Interactions between germline and somatic mutations are a key determinant of cancer development. In 40% of patients, the family history does not predict the presence of inherited cancer predisposition syndromes and many cases go undetected. Paediatricians should be aware of specific symptoms, which highlight the need of evaluation for cancer syndromes. The quickest possible identification of such syndromes is of key importance, due to the possibility of early detection of neoplasms, followed by presymptomatic genetic testing of relatives, implementation of appropriate clinical procedures (e.g., avoiding radiotherapy), prophylactic surgical resection of organs at risk, or searching for donors of hematopoietic stem cells. Targetable driver mutations and corresponding signalling pathways provide a novel precision medicine strategy.Therefore, there is a need for multi-disciplinary cooperation between a paediatrician, an oncologist, a geneticist, and a psychologist during the surveillance of families with an increased cancer risk. This review aimed to emphasize the role of cancer-predisposition gene diagnostics in the genetic surveillance and medical care in paediatric oncology.

New Genetic Technologies in Diagnosis and Treatment of Cancer of Unknown Primary.

Cancer of unknown primary (CUP) represents a rare oncological and heterogeneous disease in which one or more metastases are present, but the location of the primary site is unknown. Pathological diagnosis, using immunohistochemistry, of such metastatic materials is challenging and frequently does not allow for determining the tissue of origin (ToO). The selection of systemic therapy in patients with CUP is usually based on empiric grounds, and the prognosis is generally unfavourable. New molecular techniques could identify the tissue of origin and be used to select systemic agnostic therapies in various malignancies with specific molecular abnormalities. Targetable driver mutations or gene rearrangements in cancer cells may be identified using various molecular assays, of which particularly valuable are next-generation sequencing techniques. These assays may identify tumour sources and allow personalized treatments. However, current guidelines for CUP management do not recommend routine testing of gene expression and epigenetic factors. This is mainly due to the insufficient evidence supporting the improvement of CUP's prognosis by virtue of this approach. This review summarizes the advantages and disadvantages of new genetic techniques in CUP diagnostics and proposes updating the recommendations for CUP management.

Abstract 2433: Targeting tGLI1 pharmacologically as a new therapeutic strategy for breast cancer brain metastases

Abstract Breast cancer is the second leading cause of brain metastases in women; patients with breast cancer brain metastasis (BCBM) survive only 6-18 months after diagnosis. Novel agents that act on targetable driver mutations (EGFR, PI3K, etc.) and penetrate the blood-brain barrier (BBB) have shown promise as systemic options for patients with distant lung and melanoma metastases. Nevertheless, brain metastases represent an area of unmet clinical need for which new pathways, novel mechanisms, and innovative therapies are needed. Cancer stem cells are thought to be a driving force behind not only distant metastasis, but also late-stage recurrence. The hedgehog pathway is an important mediator of breast cancer stem cells (BCSC); however, FDA-approved therapies targeting this pathway have demonstrated limited efficacy in breast cancer clinical trials. Despite advances made in understanding BCSC, it remains challenging to effectively target BCSC underscoring the need to identify and inhibit novel mediators of BCSC to treat BCBM. Our laboratory recently reported that truncated glioma-associated oncogene homolog 1 (tGLI1) promotes preferential breast cancer metastasis to the brain by activating BCSC and astrocytes in the tumor microenvironment (Oncogene 39:64-78, 2020). tGLI1 is an alternatively spliced GLI1 variant that functions as a tumor-specific gain-of-function transcription factor and terminal effector of the hedgehog pathway. tGLI1 knockdown abrogated BCBM, providing the rationale to therapeutically target tGLI1. In this study, we conducted cell-based chemical screens followed by validations and found that ketoconazole (KCZ), an FDA-approved antifungal, selectively targets tGLI1-expressing cancer cells, leading to suppression of BCSC in vitro and BCBM in vivo. Modification of KCZ moieties produced derivatives that retained tGLI1-selectivity against both BCSC in vitro and BCBM in vivo with increased BBB penetrance. Mechanistic studies suggest that KCZ-dependent cell kill is mediated through reducing tGLI1’s ability to bind and transactivate its target gene promoters, reducing expression of target genes Nanog, OCT4, and VEGFA. Based on these data, we opened an early Phase I clinical trial to determine if KCZ penetrates the BBB and modulates tGLI1 signaling in BCBM (NCT03796273). Preliminary analysis confirmed KCZ accumulation in collected brain biospecimens. We further investigated structure-activity relationship by synthesizing and testing 22 novel compounds, and identified two tGLI1-selective compounds that no longer inhibit CYP3A4. Since KCZ-mediated CYP3A4 antagonism can result in adrenal insufficiency and drug-drug interactions, these two novel tGLI1 inhibitors could be promising agents superior to KCZ. Collectively, these data establish tGLI1 as an actionable target for BCBM and validate KCZ and its novel derivatives as promising treatment options for patients with BCBM. Citation Format: Daniel Doheny, Sara Manore, Grace L. Wong, Dongqin Zhu, Sherona Sirkisoon, Marlyn Anguelov, Noah R. Augayo, Angelina T. Regua, Anderson Cox, Cristina Furdui, Terrence Smalley, Alexandra Thomas, Adrianna Masters, Roy E. Strowd, Hui-Wen Lo. Targeting tGLI1 pharmacologically as a new therapeutic strategy for breast cancer brain metastases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2433.

Abstract 3095: Breaking the crosstalk of the cellular tumorigenic network in NSCLC by a highly effective drug combination

Abstract Background: Non-small cell lung cancer (NSCLC) is commonly diagnosed at advanced stages with limited treatment options particularly in tumors without targetable driver mutations. Methods: Considering previously published criteria for selecting drug combinations for overcoming drug resistances1, we have established a low dose combination therapy with cabozantinib, afatinib, etoricoxib and plerixafor. Utilizing NSCLC patient-derived xenograft (PDX) models we aimed to use this drug regimen to disrupt paracrine substitutional signaling within the tumorigenic network in order to circumvent pre-existing and adaptive drug resistances. In contrast to daily dosing for respective monotherapies the low dose treatment was paused every five days for two days. Result: All PDX tumors treated, including highly therapy-resistant adeno- and squamous cell carcinomas without identifiable driver mutations, were completely suppressed by this drug regimen leading to an Objective Response Rate of 81% and a Clinical Benefit Rate of 100%. After treatment discontinuation growth suppression remained until the end of the follow-up period with only slight increase in tumor growth. The application and the safety profile of this low dose therapy regimen was well manageable and without pronounced side effects in this pre-clinical setting. Discussion: For the first time we show that simultaneous inhibition of pathways forming the cellular tumorigenic network in NSCLC tumors is highly effective and overcomes drug resistances for monotherapies. All of the drugs used in this combination regimen are approved for different indications and therefore readily available. Conclusion: Our results emphatically encourage a setup for a clinical study in advanced stage NSCLC patients without targetable driver mutations combining the drugs cabozantinib, afatinib, plerixafor and etoricoxib in a low dose treatment regimen as novel therapy strategy. Indication of Source: 1. Langhammer S and Scheerer J. Oncotarget 8, 43555-43570 (2017). Citation Format: Dennis Gürgen, Theresia Conrad, Michael Becker, Susanne Sebens, Christoph Röcken, Jens Hoffmann, Stefan Langhammer. Breaking the crosstalk of the cellular tumorigenic network in NSCLC by a highly effective drug combination [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3095.

Abstract 563: Circulating tumor-specific T cells preferentially recognize patient-specific mutational neoantigens and infrequently recognize shared cancer driver mutations

Abstract Background: Due to a lack of prior technologies able to generate a landscape analysis of mutational neoantigen-HLA class I complex T cell recognition, it is unclear how the frequency of T cell receptors (TCR) against patient-specific mutations compares to shared oncogenic mutations. Methods: We used the imPACT Isolation Technology®, which allows the selective capture of cancer-specific CD8 T cells from the blood of cancer patients at frequencies as low as 1 in 300K CD8 T cells. This TCR isolation platform is based on whole exome sequencing of a tumor and paired normal tissue control for the identification of non-synonymous mutations, including mutations in cancer driver genes (e.g., p53, KRAS, PI3KCA or HRAS). Bioinformatic algorithms were used to predict potential HLA-binding neoantigens (neoAg) in context of the patient’s HLA class I haplotype. A library of multimerized and barcoded predicted neoAg peptide-HLA molecules were generated to interrogate patient CD8 T cells. The paired αβ-TCR sequences were derived from the single cell sorted T cells. TCRs from antigen experienced CD8 T cell were functionally validated by generating T cells expressing neoTCRs, using non-viral precision gene editing to insert the transgenic TCR chains into the endogenous TRAC locus. We previously showed that all (40/40) TCRs isolated using this approach, reacted to patient-matched cancer cell lines (Puig Saus et al. AACR 2020). Results: Neoantigen-specific T cells isolated from the peripheral blood of 243 patients with melanoma, bladder, endometrial, ovarian, colorectal, head and neck, urothelial and breast cancers were analyzed For each patient, an average of 352 neoAg-HLA capture complexes were predicted. Across all patients 58,058 neoAg-HLA complexes were manufactured, spanning 8,804 unique mutations. Among these, only about 2% represented known oncogene driver mutations. The predicted peptide-HLA binding affinity for cancer driver mutations was significantly lower (P<0.001) compared to patient-specific neoAg. Remarkably, functional characterization of 206 TCRs showed that most TCRs (93.7%) recognized patient-specific neoAg. Despite the low percentage of neoAg-HLA capture complexes covering oncogene driver mutations (~ 2%), 6.3% of TCRs (13) targeted known cancer driver proteins. Primary human T cells were then engineered to express each TCR. TCR T cells became functionally activated only when exposed to the cognate antigen, irrespective of whether they target neoAg or cancer driver mutations, confirming the high degree of specificity of the isolated TCRs. Conclusion: A landscape analysis of neoAg-specific TCRs recognizing mutations in cancer has revealed that most T cells recognizing neoantigen-HLA complexes are specific for private mutations in each cancer, though low frequencies of TCR targeting driver mutations can still be identified. Citation Format: Barbara Sennino, Jyoti Mathur, Eva Huang, Andrew Conroy, Marc Ting, Benjamin Yuen, Zheng Pan, Eric Stawiski, Stefanie Mandl. Circulating tumor-specific T cells preferentially recognize patient-specific mutational neoantigens and infrequently recognize shared cancer driver mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 563.

Abstract 2438: Determining the role of non-mutated C-Raf kinase in metastatic disease

Abstract Many aggressive epithelial cancers do not carry targetable driver mutations (Mendiratta et al. 2021, NCI-MATCH Clinical trial NCT02465060). Commonly altered signaling pathways highlight the propensity for tumors to depend on such pathways for growth and survival where genomic alterations may not be required for pathway activation. The Raf family kinases are crucial mediators of the Ras/Raf/MEK/ERK (MAPK) cascade, which has been shown to be upregulated in a subset of metastatic cancers and are mutated in many epithelial cancers. A recent study suggests non-mutated C-Raf can drive metastasis to the lungs, bone, and various other tissues (Faltermeir et al. 2016). Though the mechanism of C-Raf driven metastasis is likely through MAPK activation, C-Raf has been demonstrated to possess MAPK independent roles that may also contribute to this phenotype. In this study, we explored various C-Raf functions as drivers of metastasis in an intracardiac mouse model system. Using a series of Raf knock-out cell lines and mutants targeting functional residues in C-Raf, we demonstrated that C-Raf dimerization is necessary to drive metastasis. We further showed that the metastasis-promoting activity of C-Raf is dependent on co-expression of its family member, B-Raf, for an accelerated metastatic phenotype. However, overexpression of a kinase-dead C-Raf mutant was still able to produce metastases albeit with lower efficiency, suggesting C-Raf possesses non-kinase dependent functions that also contributed to metastasis. Together, these results point to the importance of Raf non-canonical roles in oncogenic processes that may be unappreciated in metastatic disease. Citation Format: Lisa H. Ta, Janai R. Carr-Ascher, Weixian Deng, Brandon L. Tsai, Wendy Tran, Donny Gun, Donghui Cheng, Jihui Sha, Yeonjoo Hwang, John W. Phillips, Matthew B. Obusan, Nathanael J. Bangayan, Miyako Noguchi, Zhiyuan Mao, Chia-Chun Chen, Liang Wang, Grigor Varuzhanyan, John D. Gordon, James W. Wohlschlegel, Owen N. Witte. Determining the role of non-mutated C-Raf kinase in metastatic disease [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2438.

Comprehensive genomic profiling of MET rare tumors in China.

e15058 Background: The mesenchymal epithelial transition factor receptor (MET) is a potential therapeutic target in a number of cancers, commonly activated by amplification, mutation and fusion. Methods: Rare tumor was defined as solid tumors with an incidence lower than 2.5/100,000 per year. We retrospectively analyzed the next-generation sequencing and clinicopathological data of 3453 Chinese rare tumors patients in the Geneplus database, including 1021 genes, MSI loci, tumor mutational burden (TMB), and programmed cell death ligand-1 (PD-L1) expression analysis information. Results: MET-positive was identified in 115 patients (3.3%), including 109 cases of amplification (3.2%), 8 cases of exon 14 skipping (0.2%) and 1 cases of fusion. The prevalence of MET-positive varied widely across tumors systems and subtypes. Urinary, neural, skin and respiratory systems were the top 4 systems with 8.3% (1/12), 7.9% (58/732), 4.2% (1/24) and 2.9% (6/207) MET-positive rate, respectively. Considering the tumor subtypes, glioblastoma, sarcomatoid carcinoma, neuroendocrine tumor and glioma were higher, reaching 15.7% (16/102), 13.2% (7/53), 9.8% (4/41), and 7.4% (36/487) respectively (Table). Totally, 73.0% of patients had at least one common targetable gene mutation, including ATM，BRAF，BRCA，CDKN2A，EGFR substitution, ERBB2, FGFR1,2,3, KIT, NF1, PIK3CA, PTEN and RET. The proportions of TMB-H (≥9 mut/Mb) and MSI-H were very low, with only 8.9% and 2.2%, while PD-L1 positive rate reached to 60.9%. Conclusions: The treatment options for rare tumors are still limited. The approval of MET receptor tyrosine kinase has brought hope to patients with Non-Small Cell Lung Cancer. The MET-positive rate in rare tumors is higher than 2.7% in lung adenocarcinoma, so MET receptor tyrosine kinase have great application potential in rare tumors. However, since most patients co-occurred with other targetable driver mutations or PD-L1 positive, how to choose the best treatment strategy should be solved. The deployment of the best treatment strategy is a problem that should be solved in the follow-up study.[Table: see text]

Outcomes in non-small cell lung cancer (NSCLC) patients treated with second-line therapy after progression or recurrence after initial first line immune checkpoint-based therapy.

e21027 Background: The use of immune checkpoint inhibitors (ICI) based therapies, including in combination with platinum-based or as monotherapy, is currently the standard of care in stage IV NSCLC patients as a first line therapy. However, there is limited data on the options and outcomes of NSCLC patients if they progress after first-line therapy. This study aims to investigate the clinical outcomes to second lines of treatment for these patients. Methods: This is a retrospective study conducted at Houston Methodist Hospital involving adult patients (> 18 years old) with metastatic stage IV NSCLC who received ICI based therapy (combination or monotherapy) for the treatment of NSCLC and subsequently progressed or recurred and received a second line of therapy between 11/1/2016 and 12/31/2019. We excluded patients with targetable driver mutations (except KRAS) and patients with a concurrent cancer diagnosis. We collected information on the second line of therapy received and evaluated outcomes including progression-free survival (PFS), overall survival (OS) and overall response rate (ORR). PFS was defined from time of second-line therapy initiation to time of documented recurrence/progression. ORR was defined as patients who achieve partial response (PR) or complete response (CR). The Kaplan Meier method was used for survival analysis. Results: Among patients who received ICI-based first line therapy, 32 patients were identified to meet the inclusion criteria. Twenty-seven (84%) patients had adenocarcinoma and 17 patients had negative PD-L1 stain. Twenty-seven (84%) patients received a combination of carboplatin, pembrolizumab, and pemetrexed, whereas five (16%) patients received ICI monotherapy (3 pembrolizumab, 1 nivolumab and 1 durvalumab). Thirteen patients had an initial response to first-line therapy (11 PR, 2 CR). After progression/recurrence, various therapies were administered as second-line with the most common being the use of ICI + chemotherapy combination (6 pts, 19%) or ramucirumab + taxotere combination (6 pts, 19%). The remaining patients received other types of chemotherapy, targeted therapy, ICI, or were enrolled in a clinical trial. Nine patients (28%) had a response after 2nd line therapy [4 CR and 5 PR], 16 (50%) had progressive disease, and 5 (16%) had mixed response/stable disease. The cohort median PFS after 2nd line of therapy was 16.9 weeks (95% CI: 14.4-19.3). Conclusions: These findings reflect the variability in practices in stage IV NSCLC patients who progress after receiving ICI-based first line therapy. Outcomes of patients who progress after their first line remain suboptimal with short-lived responses. Further studies are needed to investigate the efficacy and safety of different second line treatment options.

Real-world biomarker testing trends in metastatic non-small cell lung cancer (NSCLC).

e18728 Background: NSCLC is now recognized as not one disease, but many—differentiated by an increasing number of molecular targets. About 50% of patients with advanced nonsquamous NSCLC have a targetable driver mutation. We retrospectively assessed biomarker testing trends in a large community oncology practice to identify evolving real-world testing rates and patterns in the areas of methodology, specimen types, and vendors. Methods: Natural-language processing queries were used to extract data from biomarker testing reports of patients diagnosed with stage IV NSCLC in 2015-2019. Tests were included if done within 1 year of diagnosis and were classified as NGS panels (20+ genes) or targeted single-gene tests. Results: Records of 587 patients were included. Over the 5-year study window, testing rates increased for all 6 mutations analyzed (Table). In 2015, only ALK, ROS1, and EGFR were profiled in our data (n = 20 patients), all using targeted single-gene tests. The use of multigene panels increased steadily over time, constituting the majority by 2019. MET, RET, and NTRK were tested almost exclusively using NGS panels (data beginning in 2016). Tissue-only specimens were used for 93.8% of patients diagnosed in 2015, while tissue plus blood were used in 6.3%. Blood-only samples began appearing in our data in 2016, making up 7% of patients. By 2019, the use of blood tests had increased substantially, to 47% of patients, although tissue-only remained the most common type (54%). While blood testing has become more popular, it is often still performed with tissue tests. The primary testing methods used in 2015-2017 were FISH for ALK and ROS1; NGS for MET, NTRK, and RET; and Sanger or NGS for EGFR. By 2018, NGS had become the predominant test method across all 6 biomarkers. During 2015-2016, several vendors were used for testing, with a very segmented market for ALK and EGFR tests. By 2019, the market had aligned to a few key vendors for the majority of tests. In 2016, the top 5 vendors did 76% of 180 total tests, which increased to 93% of 1880 tests in 2019. Almost all blood testing in our data was performed by a single vendor. Conclusions: Testing rates for ALK, ROS1, and EGFR were more consistent than those for MET, RET, and NTRK throughout the study window, possibly because of earlier availability of tests and treatments for these biomarkers. NGS was more available by the time MET, RET, and NTRK became established as actionable biomarkers, explaining the earlier use of panels. A limitation is that nearly 75% of the data are from the final 2 years. These results, however, represent notable testing trends from a large practice and illustrate the expanding application of molecular testing in community oncology.[Table: see text]

Effect of performance status (ECOG PS) on treatment outcome with second-line (2L) nintedanib (NIN) + docetaxel (DOC) for patients (pts) with lung adenocarcinoma after first-line (1L) immune checkpoint inhibitor (ICI) combination therapy.

9092 Background: ICIs with or without chemotherapy represent 1L standard of care for patients with advanced non-small cell lung cancer (NSCLC) without targetable driver mutations. Given the paucity of prospective randomized trials assessing second-line treatment options post-immunochemotherapy, non-interventional data may help support clinical decision making. Patients with ECOG PS > 1 are generally underrepresented in clinical trials relative to routine clinical practice. We therefore examined treatment outcomes with 2L NIN + DOC after 1L ICI combination therapy with respect to ECOG PS at baseline. Methods: In this analysis of Cohort C of the ongoing, non-interventional VARGADO study (NCT02392455), eligible pts had locally advanced, metastatic or locally recurrent adenocarcinoma NSCLC and received 2L NIN+DOC in routine clinical practice after failure on 1L ICI in combination with chemotherapy. The primary endpoint is 1-year survival rate (not yet mature). Tumor response was according to investigator review. Results: In 164 pts enrolled in Cohort C, median age was 63 years (range: 37 – 84), 100 pts (61.0%) were men, and 123 pts (75.0%) were ECOG PS 0–1. 146 pts (89.0%) had received prior 1L pembrolizumab-based combination therapy. Objective response rate (ORR) with 2L NIN+DOC was 35.4% (40/113 pts) in the overall population and 41.2%, 30.8% and 28.6% for pts with ECOG PS 0, 1 and > 1, respectively. Disease control rate (DCR) was 67.3% (76/113 pts) in the overall population and 76.5%, 65.4% and 50.0% for pts with ECOG PS 0, 1 and > 1, respectively. Median PFS was 4.7 months (95% CI: 3.4 – 5.3) in the overall population and 5.1 months (95% CI: 2.8 – 8.3), 3.7 months (95% CI: 2.5 – 5.3) and 2.1 months (95% CI: 1.3 – 6.5) for pts with ECOG PS 0, 1 and > 1, respectively. The median OS was 8.1 months (95% CI: 6.3 – 11.2) in the overall population and 9.1 months (95% CI: 5.4 – 14.5), 10.5 months (95% CI: 6.3 – 14.9) and 4.0 months (95% CI: 2.5 – 6.3) for pts with ECOG PS 0, 1 and > 1, respectively. Conclusions: Our results support 2L NIN+DOC as an effective treatment option in pts with advanced adenocarcinoma NSCLC following 1L ICI in combination with chemotherapy. ECOG PS > 1 was associated with lower ORR, DCR and shorter PFS and OS. These findings warrant further evaluation of the effect of ECOG PS on 2L treatment outcomes in patients after 1L ICI combination therapy. Clinical trial information: NCT02392455.

Immunophenotypic correlates and response to first-line pembrolizumab among elderly patients with PD-L1-high (≥ 50%) non–small cell lung cancer.

9054 Background: Older age is associated with increased levels of systemic inflammation and altered immunosurveillance in cancer. Whether aging correlates with a distinct immunophenotype or impacts clinical outcomes to first-line pembrolizumab in patients with advanced non-small-cell lung cancer (NSCLC) and a PD-L1 tumor proportion score (TPS) of ≥50% is unclear. Methods: We performed a retrospective analysis of patients with NSCLC. Multiplexed immunofluorescence (mIF) for CD8+, PD-1+, PD-1+CD8+ and FOXP3+ was performed to explore tumor immunophenotype. Clinical outcomes were analyzed on a separate cohort of patients with PD-L1-high (TPS of ≥50%) NSCLC (negative for sensitizing genomic alterations in EGFR and ALK) who received treatment with first-line pembrolizumab. Variables demonstrating a univariate signal of association of p < 0.1 were included in the multivariate model. The results were compared in patients < 80 vs ≥80 years old. Results: Among 541 patients with NSCLCs profiled by mIF, the median age was 67 (28-90). When comparing patients < 80y (n = 497) to ≥80y (n = 44), there was no difference in median CD8+ T cells/mm2 (171 vs 148; p = 0.69), PD-1+ immune cells/mm2 (81.1 vs 87.2; p = 0.95), or PD-1+CD8+ T cells/mm2 (18.0 vs 13.1; p = 0.56). NSCLCs from patients ≥80y had a higher median of intratumoral-associated FOXP3+ T cells/mm2 (63.6 vs 91.1; p = 0.03). In a cohort of 271 patients with PD-L1 ≥50% who received first-line pembrolizumab, baseline clinicopathological characteristics were balanced in the < 80y (n = 225) vs ≥80y (n = 46) groups in terms of sex, tobacco use, Eastern Cooperative Oncology Group-Performance Status (ECOG-PS), histology, presence of potentially targetable driver mutations (KRAS, MET, BRAF, HER2, RET), and PD-L1 TPS distribution (50-89% vs ≥90%). Compared to patients < 80y, patients ≥80y had no difference in objective response rate (ORR 39.1% vs 28.2%; p = 0.22) or median progression-free survival (mPFS 6.0 vs 3.0 months; p = 0.16). However, patients ≥80y had a shorter median overall survival (mOS 25.7 vs 7.6 months; p = 0.02), and this result remained significant after adjusting for ECOG-PS. Among those who experienced disease progression on pembrolizumab, patients ≥80y were significantly less likely to receive any second-line systemic therapy compared to patients < 80y (55.6% vs 30.8%; p = 0.008). Conclusions: In patients with NSCLC and PD-L1 ≥50%, the ORR and mPFS to first-line pembrolizumab were similar between patients < vs ≥80 years old. OS was shorter among patients ≥80y, potentially reflecting lower use of second-line therapy in elderly patients after progression on pembrolizumab. The immunophenotypic correlates of NSCLC in older patients need further investigation.

A phase I/II multisite study of rucaparib and pembrolizumab maintenance therapy in stage IV non-squamous non–small cell lung cancer after initial therapy with carboplatin, pemetrexed, and pembrolizumab.

TPS9138 Background: Carboplatin, pemetrexed, and pembrolizumab (CPP) followed by pemetrexed and pembrolizumab maintenance is a standard of care (SOC) for non-squamous metastatic non-small cell lung cancer (NSCLC), based on results of the KEYNOTE-189 study. The progression free survival (PFS) on this study was 8.8 months. There is increasing data to support that certain sporadic tumors harbor somatic mutations in genes of the homologous recombinant repair (HRR) pathway resulting in a “BRCAness” phenotype rendering sensitivity to PARP inhibitors (PARPi). TCGA data suggest up to 38% of NSCLC have mutations in HRR pathway genes. Furthermore, treatment with PARPi is shown to upregulate PD-L1 expression, which is associated with pembrolizumab response in NSCLC. We therefore hypothesized that maintenance therapy with a PARPi and pembrolizumab will improve PFS compared to SOC pemetrexed and pembrolizumab. Methods: This is a single arm, multi-site, investigator-initiated phase I/II study that is enrolling treatment-naïve patients with stage IV nonsquamous metastatic NSCLC without any targetable driver mutations eligible for CPP. Patients without progression after 4 cycles of CPP then proceed to maintenance with rucaparib 600mg PO BID and pembrolizumab 200mg IV every 21 days. The primary objective is efficacy assessed by PFS and second objectives include overall survival, safety, tolerability, and objective response rate (ORR). Correlatives in this study include PD-L1 expression, tumor mutational burden (TMB), and measurements of HRR deficiency as assessed by homologous repair deficiency (HRD) and loss of heterozygosity (LOH) using established methods. Key exclusion criteria include untreated brain metastases and prior exposure to a PARPi or PD-1/PD-L1 inhibitor. The phase I cohort which consisted of 6 patients treated with the approved full doses of rucaparib and pembrolizumab was completed without dose limiting toxicity (DLT). Using a Bayesian decision-theoretic two-stage design, the first stage will enroll 38 patients. If predefined PFS parameters are met, an additional 17 patients, for a total of 55 patients, will be enrolled. To date, 21 patients have been enrolled. Clinical trial information: NCT03559049.

OncoPan®: An NGS-Based Screening Methodology to Identify Molecular Markers for Therapy and Risk Assessment in Pancreatic Ductal Adenocarcinoma.

Pancreatic cancer has a high morbidity and mortality with the majority being PC ductal adenocarcinomas (PDAC). Whole genome sequencing provides a wide description of genomic events involved in pancreatic carcinogenesis and identifies putative biomarkers for new therapeutic approaches. However, currently, there are no approved treatments targeting driver mutations in PDAC that could produce clinical benefit for PDAC patients. A proportion of 5–10% of PDAC have a hereditary origin involving germline variants of homologous recombination genes, such as Mismatch Repair (MMR), STK11 and CDKN2A genes. Very recently, BRCA genes have been demonstrated as a useful biomarker for PARP-inhibitor (PARPi) treatments. In this study, a series of 21 FFPE PDACs were analyzed using OncoPan®, a strategic next-generation sequencing (NGS) panel of 37 genes, useful for identification of therapeutic targets and inherited cancer syndromes. Interestingly, this approach, successful also on minute pancreatic specimens, identified biomarkers for personalized therapy in five PDAC patients, including two cases with HER2 amplification and three cases with mutations in HR genes (BRCA1, BRCA2 and FANCM) and potentially eligible to PARPi therapy. Molecular analysis on normal tissue identified one PDAC patient as a carrier of a germline BRCA1 pathogenetic variant and, noteworthy, this patient was a member of a family affected by inherited breast and ovarian cancer conditions. This study demonstrates that the OncoPan® NGS-based panel constitutes an efficient methodology for the molecular profiling of PDAC, suitable for identifying molecular markers both for therapy and risk assessment. Our data demonstrate the feasibility and utility of these NGS analysis in the routine setting of PDAC molecular characterization.

Reduced FEV1 as Prognostic Factors in Patients With Advanced NSCLC Receiving Immune Checkpoint Inhibitors.

BackgroundThe aim of study is to investigate the influence of pulmonary function on the prognosis in patients with advanced non-small cell lung cancer (NSCLC) receiving immune checkpoint inhibitors (ICI).Patients and MethodsData were collected retrospectively from 151 patients with stage IV NSCLC who received ICI and completed spirometry before ICI therapy in Taipei Veterans General Hospital between January 2016 and December 2020. The co-primary end points were overall survival (OS) and progression-free survival (PFS) between groups divided by 80% predicted FEV1 since ICI therapy started; the secondary outcomes were objective response rate.ResultsAmong 151 patients enrolled to this study, 67.5% of patients were men, 75.5% were adenocarcinoma, 24.5% had known targetable driver mutation, 33.8% received first-line ICI, and 62.8% received ICI monotherapy. The objective response rate was 24.5% and disease control rate was 54.3%. In multivariable analysis, patient with reduced FEV1 had inferior PFS (FEV1 < 80% vs. FEV1 ≥ 80%, adjusted HR = 1.80, P = 0.006) and OS (FEV1 < 80% vs. FEV1 ≥ 80%, adjusted HR = 2.50, P < 0.001). Median PFS and OS in the preserved FEV1 group (≥80% predicted FEV1) compared to the reduced FEV1 group (<80% predicted FEV1) were 5.4 vs. 2.9 months (HR = 1.76, P = 0.003) and 34.9 vs. 11.1 months (HR = 2.44, P < 0.001), respectively. The other independent prognostic factors of OS include stage IVA disease (adjusted HR = 0.57, P = 0.037), initial liver metastasis (adjusted HR = 2.00, P = 0.049), ICI monotherapy (adjusted HR = 1.73, P = 0.042) and ICI related pneumonitis (adjusted HR = 3 .44, P = 0.025).ConclusionsReduced FEV1 is strongly associated with inferior clinical outcomes in patients with advanced NSCLC treated with ICI.

The impact of driver mutation on the treatment outcome of early-stage lung cancer patients receiving neoadjuvant immunotherapy and chemotherapy

Neoadjuvant immunotherapy and chemotherapy have improved the major pathological response (MPR) in patients with early-stage operable non-small cell lung cancer (NSCLC). This study aimed to assess whether the presence of targetable driver mutations affects the efficacy of the combination of immunotherapy and chemotherapy. We enrolled patients with early-stage operable NSCLC who received preoperative neoadjuvant therapy between January 1, 2017, and December 30, 2020. Neoadjuvant therapy was delivered with platinum-doublet chemotherapy; moreover, pembrolizumab was added at the attending physician’s discretion based on patient’s request. Pathological responses were assessed; moreover, disease-free survival was estimated. Next-generation sequencing was performed in case sufficient preoperative biopsy specimens were obtained. We included 23 patients; among them, 11 received a combination of neoadjuvant immunotherapy and chemotherapy while 12 received neoadjuvant chemotherapy alone. The MPR and pathological complete response rates were 54.5% and 27.3%, respectively, in patients who received a combination of neoadjuvant immunotherapy and chemotherapy. These rates were significantly higher than those in patients who only received neoadjuvant chemotherapy. Three patients in the combination group experienced disease recurrence during the follow-up period even though two of them showed an MPR. These three patients had targetable driver mutations, including an EGFR exon 20 insertion, EGFR exon 21 L858R substitution, and MET exon 14 skipping. Only one patient who remained disease-free had a targetable driver mutation. Among patients with early-stage operable NSCLC requiring neoadjuvant therapy, comprehensive genomic profiling is crucial before the administration of the combination of neoadjuvant immunotherapy and chemotherapy.

Splice-switching of the insulin receptor pre-mRNA alleviates tumorigenic hallmarks in rhabdomyosarcoma

Rhabdomyosarcoma (RMS) is an aggressive pediatric tumor with a poor prognosis for metastasis and recurrent disease. Large-scale sequencing endeavors demonstrate that Rhabdomyosarcomas have a dearth of precisely targetable driver mutations. However, IGF-2 signaling is known to be grossly altered in RMS. The insulin receptor (IR) exists in two alternatively spliced isoforms, IR-A and IR-B. The IGF-2 signaling molecule binds both its innate IGF-1 receptor as well as the insulin receptor variant A (IR-A) with high affinity. Mitogenic and proliferative signaling via the canonical IGF-2 pathway is, therefore, augmented by IR-A. This study shows that RMS patients express increased IR-A levels compared to control tissues that predominantly express the IR-B isoform. We also found that Hif-1α is significantly increased in RMS tumors, portraying their hypoxic phenotype. Concordantly, the alternative splicing of IR adapts to produce more IR-A in response to hypoxic stress. Upon examining the pre-mRNA structure of the gene, we identified a potential hypoxia-responsive element, which is also the binding site for the RNA-binding protein CUG-BP1 (CELF1). We designed Splice Switching Oligonucleotides (SSO) against this binding site to decrease IR-A levels in RMS cell lines and, consequently, rescue the IR-B expression levels. SSO treatment resulted in a significant reduction in cell proliferation, migration, and angiogenesis. Our data shows promising insight into how impeding the IGF-2 pathway by reducing IR-A expression mitigates tumor growth. It is evident that Rhabdomyosarcomas use IR alternative splicing as yet another survival strategy that can be exploited as a therapeutic intervention in conjunction with already established anti-IGF-1 receptor therapies.