ROS1 Fusion Research Articles

Detecting ALK, ROS1, and RET fusions and the METΔex14 splicing variant in liquid biopsies of non-small-cell lung cancer patients using RNA-based techniques.

ALK, ROS1, and RET fusions and MET∆ex14 variant associate with response to targeted therapies in non-small-cell lung cancer (NSCLC). Technologies for fusion testing in tissue must be adapted to liquid biopsies, which are often the only material available. In this study, circulating-free RNA (cfRNA) and extracellular vesicle RNA (EV-RNA) were purified from liquid biopsies. Fusion and MET∆ex14 transcripts were analyzed by nCounter (Nanostring) and digital PCR (dPCR) using the QuantStudio® System (Applied Biosystems). We found that nCounter detected ALK, ROS1, RET, or MET∆ex14 aberrant transcripts in 28/40 cfRNA samples from positive patients and 0/16 of control individuals (70% sensitivity). Regarding dPCR, aberrant transcripts were detected in the cfRNA of 25/40 positive patients. Concordance between the two techniques was 58%. Inferior results were obtained when analyzing EV-RNA, where nCounter often failed due to a low amount of input RNA. Finally, results of dPCR testing in serial liquid biopsies of five patients correlated with response to targeted therapy. We conclude that nCounter can be used for multiplex detection of fusion and MET∆ex14 transcripts in liquid biopsies, showing a performance comparable with next-generation sequencing platforms. dPCR could be employed for disease follow-up in patients with a known alteration. cfRNA should be preferred over EV-RNA for these analyses.

Mechanisms of resistance to tyrosine kinase inhibitor treatments in patients with ROS1 fusion-positive non-small cell lung cancer.

9062 Background: ROS1 rearrangement was found in about 1% non-small cell lung cancer (NSCLC), and the fusion proteins created during this process will result in the successive activation of the ROS1 kinase domain. NSCLC patients with ROS1 gene fusions were considered a unique subtype highly sensitive to related tyrosine kinase inhibitor (TKI) treatments. However, the acquired TKI resistance was the major hurdle preventing patients from getting prolonged treatment benefits. Methods: 107 patients diagnosed with advanced or metastatic NSCLC harboring ROS1 fusion were retrospectively recruited. All patients were treated with crizotinib as first-line treatment, and 21 patients received lorlatinib after crizotinib progression. Samples were collected at baseline, after crizotinib progression, and after lorlatinib progression, which all underwent targeted DNA sequencing. TKIs binding to mutated ROS1 fusion proteins was simulated using molecular dynamics simulations. Results: The most witnessed fusion partner of ROS1 was CD74 (58%), followed by SDC4 (14%), EZR (11%), and SLC34A2 (9%). The median progression-free survival was 12.9 months for crizotinib and 6.4 months for lorlatinib. Patients with CD74-ROS1 and SLC34A2-ROS1 had significantly longer PFS than those with other ROS1 fusion types when treated with crizotinib. Patients with baseline TP53 mutations showed worse PFS compared to TP53 wild-type (WT) patients (P < 0.001, HR: 0.19, 95%CI: 0.09-0.39) under crizotinib treatment. An accumulation of both on-target (baseline vs. post-crizotinib vs. post-lorlatinib: 0% vs. 43% vs. 62%) and off-target resistant mutations (baseline vs. post-crizotinib vs. post-lorlatinib: 22% vs. 26% vs. 43%) after multiple TKI treatments was observed. A total of ten on-target resistance mutations were detected after TKI therapies, with 4 first-reported mutations (ROS1 L2010M, G1957A, D1988N, L1982V). According to the molecular dynamics simulation results, ROS1 L2010M mutations (happening in the ROS1 kinase ATP binding cassette) maybe resistant to lorlatinib, entrectinib, cabozantinib, and crizotinib. ROS1 G1957A may lead to resistance to cabozantinib. Moreover, all four novel on-target mutations may lead to resistance to crizotinib. Conclusions: In summary, CD74- and SLC34A2-ROS1 patients showed better crizotinib efficacy with different resistance mutation patterns. Patients of these subtypes are potential beneficiaries of molecular testing after crizotinib progression, directing future treatment strategies. Multiple TKI treatments may lead to the accumulation of both on-target and off-target resistance mutations. In addition, 4 novel ROS1 on-target mutations identified underwent molecular dynamics simulations, unveiling potential resistance to different TKIs, which can provide vital information for future ROS1 fusion-positive NSCLC patient treatment selections.

High-confidence AI-based biomarker profiling for H&E slides to optimize pathology workflow in lung cancer.

e21207 Background: In recent years AI methodologies have been increasingly adopted in the oncology space as part of the clinical decision process. Our previous research has shown accurate detection of actionable biomarkers based on AI algorithms applied to Hematoxylin & Eosin (H&E) scanned slides. While investigating a wide actionable biomarker panel, this profiling process can also flag patients with no actionable biomarkers. In Non-Small Cell Lung Cancer (NSCLC), EGFR, ALK, and ROS1 are the most commonly tested actionable alterations and the primary exclusion criteria for immunotherapy. Here we present a novel AI-based solution that can accurately and almost instantaneously detects patients without the main actionable biomarkers from the H&E image alone, thus helping prioritize the pathology lab workflow and navigate patients to a faster and more optimal diagnosis route. Methods: A retrospective cohort of 435 NSCLC patients was collected from the Sheba Medical Center pathology department. All cases (from primary and metastatic sites) with documented results for EGFR mutations, ALK, and ROS1-fusions were included. Molecular profiling was based on Oncomine Comprehensive, Dx target, or Solid tumor DNA assays, with 4 cases also supported by the Idylla EGFR mutation test. Deidentified digital whole slide images (WSIs) were run using Imagene’s AI classifier and compared to the officially reported results. The classifier was generated using self-supervised algorithm, combined with multiple instance learning algorithms applied using untagged and tagged FFPE H&E scan (40x or 20x magnification) from Imagene’s internal database (including TCGA research network slides). A categorical prediction (positive/negative) inferred on the validation set by the combined AI classifier model. Results: The combined AI model for detecting EGFR, ALK and ROS1 actionable alteration resulted in 127 patients (29.4% of the cohort) reported with no EGFR actionable mutation, ALK, or ROS1 fusions. Only two patients (1.57% of the negatively detected patients) were found to be false negative ( EGFR: p.G719D and p.L858R), demonstrating 98.43% accuracy. Conclusions: Image-based molecular detection can serve as an accurate and fast detection method to be used in the clinical setting for monitoring and prioritizing current pathology workflow. Specifically, it can detect a major fraction of the non-targetable patients and optimize their diagnostic flow.

Response to neoadjuvant immune checkpoint inhibitor (ICI)-based therapy in oncogene-driven resectable non-small cell lung cancer (NSCLC).

8523 Background: Neoadjuvant (NEO) chemotherapy (chemo) plus nivolumab (nivo) has received FDA approval for treatment of resectable NSCLC, demonstrating superior event-free survival compared to chemo alone. However, outcomes of NEO ICI-based therapies in patients (pts) with oncogene-driven (OD)-NSCLC remains an unanswered question. Methods: We conducted a retrospective secondary analysis of pts enrolled in the clinical trial NCT02259621 with clinical stage I-IIIA NSCLC, treated with NEO ICI-based therapies: nivo alone, nivo+ipilimumab (ipi), or nivo+chemo followed by surgery. Pts underwent baseline genomic profiling using NGS. Pts with KRAS, EGFR, MET, BRAF, ALK, HER2, ROS1, RET or NTRK alterations were identified as OD-NSCLC. STK11 and KEAP1 mutations (muts) were also recorded. For pts who underwent definitive resection (DR) and baseline NGS testing, association of OD-status and clinical outcomes, including major pathologic response (MPR) and recurrence-free survival (RFS) are reported. Proportions are reported with exact 95% binomial confidence intervals (CI). Binomial probabilities are compared with Chi-square or Fisher’s exact tests. RFS and median follow-up (f/u) are reported using the Kaplan-Meier (KM) and reverse KM methods, respectively. Results: 61 pts received NEO ICI-based therapy: 60% treated with nivo alone, 15% with ipi+nvo and 25% with chemo+nivo. 92% of pts underwent DR. Pathologic complete response (pCR) and MPR rates were, 12.5% and 37.5%, respectively. Baseline NGS testing was available for 47 pts, of whom, 49% had OD-NSCLC, with the majority (78%) harboring a KRAS mut. Additional OD alterations included non-classical EGFR (9%), METex14 skipping (9%) and ROS1 fusion (4%), of whom, 3/5 pts had a MPR after NEO-ICI. STK11 muts were noted in 16% pts, and co-altered with KRAS in 9%. Median f/u for OD-NSCLC was 42.58 months. Pts with OD-NSCLC had comparable RFS after NEO-ICI compared to those with non-OD-NSCLC (HR 0.64, CI 0.19-2.13, p=0.5), including those with KRAS+ disease (HR 0.73, CI 0.2-2.73, p=0.6). Pts with co-mut KRAS+ STK11+ trended toward shorter RFS compared to KRAS+ STK11- (HR 6.36, CI 0.56-72.82, p=0.1). STK11+ also trended toward shorter RFS without reaching statistical significance (HR 2.51, CI 0.67-9.35, p=0.17). No significant associations between OD-status and MPR rates were seen, however KRAS+ (4/18) vs. Kras- (13/29) trended toward lower MPR rate (p=0.1). Four of 5 pts with disease progression preventing DR harbored an STK11 or KEAP1 mut. Conclusions: Findings from this cohort treated with various NEO ICI-based therapies, suggests comparable RFS, regardless of OD status- with the majority harboring KRAS muts. Lower MPR rates were observed for pts with KRAS+ tumors. STK11 mut and KRAS+ STK11+ co-mut status both trended toward shorter RFS, though definitive conclusions are limited by cohort size and treatment heterogeneity.

Preliminary efficacy and safety of camrelizumab plus metronomic oral vinorelbine as first-line therapy for people aged 70 and above with advanced non-small cell lung cancer (NSCLC) from a phase II trial.

e14677 Background: Traditional platinum-based combination chemotherapy used as the standard first‐line treatment for advanced NSCLC poses potential safety risk for patients (pts) aged 70 years and above. Moreover, data on immune checkpoint inhibitors treating older pts with lung cancer in the current pivotal trials is limited. Metronomic chemotherapy (MCT) is the frequent administration of chemotherapy drugs at lower doses each time. Compared with traditional chemotherapy, MCT shows better safety profile and antitumor angiogenesis and immunomodulatory effects. Camrelizumab, an anti-PD-1 inhibitor, demonstrated encouraging efficacy in NSCLC (CameL study and CameL-sq study). Here, we designed a phase II trial to evaluate the efficacy and safety of camrelizumab plus metronomic oral vinorelbine (mOV) as first-line therapy for Chinese older pts with advanced NSCLC. Methods: Key inclusion factors were pts aged 70 years and above, histologically or cytologically confirmed unresectable stage III and IV NSCLC without positive EGFR mutation, ALK fusions or ROS1 fusions, ECOG performance status 0-2, no previous systematic treatment. Pts received camrelizumab (200mg, D1, q3w) combined with mOV (30mg, tiw1, day 1-3-5 per week) for 6 cycles, followed by camrelizumab monotherapy maintenance until disease progression or intolerable side effects. The primary endpoint was progression free survival (PFS). The secondary endpoints included overall survival (OS), objective response rate (ORR), disease control rate (DCR), duration of response (DoR) and safety. Results: From March 2021 to November 2022, 12 pts were evaluable for inclusion in the study. The median age was 77 (range: 70-85) with 8 (67%) males. Median follow-up was 7.2 months (range 2.8 months-22.1 months). The median PFS was 9.3 months (95%CI: 3.9 months-14.7 months). The one-year OS rate was 66.3%. The ORR and DCR were 50% (95%CI: 25.4%-74.6%) and 83.3% (95%CI: 50.9%-90.1%), respectively. Pts with positive or negative PD-L1 responded similarly to the treatment (respectively at 5/8 and 1/2 on ORR). The median OS and DoR have not been reached. Adverse events (AEs) with any grade were observed in 10 (83.3%) pts, of which grade III-IV AEs were observed in 2 (16.7%) pts with neutropenia and interstitial pneumonia, respectively. Immune-related adverse events (irAEs) occurred in 3 (25%) pts, of which grade III-IV irAEs occurred in one (8.3%) patient with interstitial pneumonia. No grade 5 adverse event happened. Conclusions: The regimen of camrelizumab plus mOV for the first-line therapy for older NSCLC pts showed initial promising efficacy and safety profile, which suggests it is worth further exploration. Clinical trial information: ChiCTR2100049487 .

The Additional Exclusions of ROS1 Fusions (In Addition to EGFR Mutation and ALK Fusions) in the Cemiplimab NSCLC FDA Indication (EMPOWER-Lung 1 and -Lung 3). Catching Up with Current Scientific View of Immunotherapy in Never-Smoker Predominant Actionable Driver Mutation Positive NSCLC?

Cemiplimab is one of seven immune checkpoint inhibitors (ICIs) approved for the first-line (1L) treatment of advanced NSCLC in the US based on EMPOWER-Lung 1 and -Lung 3 trials. In addition to exclusion of NSCLC patients harboring EGFR mutations and ALK fusion from 1L treatment with ICIs, exclusion of ROS1 fusion is an additional unique exclusion the use of criterion for cemiplimab in the US FDA indication based on the design of the EMPOWER lung trials. We review the effectiveness of ICIs in never-smoker predominant NSCLC with driver mutations (EGFR, ALK, ROS1, RET, HER2) and question whether exclusion of ROS1 fusion would put cemiplimab at a competitive disadvantage given the requirement for insurance to prove ROS1 fusion negativity. We further discuss whether the US FDA as a regulatory authority has the right and responsibility to harmonize the use of ICIs in these actionable driver mutations to standardize community practice for the benefit of patients and to advance the development of next-generation treatment for these driver mutations.

A comparative analysis of next-generation sequencing based on plasma cfDNA and tumor tissue DNA in patients with advanced lung cancer.

e21021 Background: Although liquid biopsy based on plasma cell-free DNA (cfDNA) provides a non-invasive detection method for cancer patients, tissue biopsy is still the gold standard for molecular subtyping. The concordance of these two techniques has been evaluated in certain cohorts. The comparison of plasma cfDNA and tissue DNA NGS ina large Chinese cancer cohort is yet to be analyzed. Methods: We retrospectively reviewed HaploX's genomic sequencing data from July 2021 to December 2022 to assess the concordance between plasma cfDNA and cancer tissue DNA. A total of 589 advanced lung cancer patients with matched tissue and plasma sample were included in the study. Tissue and peripheral blood was collected at the same time during surgical operation or biopsy. Tissue DNA and plasma cfDNA were isolated and prepared for the NGS with commercially available kits. The positive percent agreement (PPA) between plasma cfDNA sequencing and tissue sequencing, the negative percent agreement (NPA), positive predictive value (PPV), negative predictive value (NPV), and overall percent agreement (OPA) were calculated and compared. Results: The positive percent agreement (PPA) of plasma cfDNA sequencing compared with tissue sequencing were 78% (EGFR, 82%; KRAS, 71%; HER2 mutation, 76%; BRAF V600E mutation, 75%; ALK fusion, 74%; RET fusion, 60%; ROS1 fusion,33.3%; MET exon14 skipping, 50%). EGFR mutations included EGFR p.L858R,19del, p.C797S, p.L861Q, p.T790M, KRAS mutations included p.G12X and p.Q61X.， HER2 mutations included gain-of-function mutations；NPA was 92.4%，PPV was 93.8%，NPV was 74.4%，OPA was 84%. Conclusions: The positive mutation detection consistency of plasma cfDNA sequencing and tissue sequencing in advanced lung cancer patients is 78%, indicating that liquid biopsy based on plasma cfDNA is reliable in most advanced lung cancer patients, but there are some limitations in gene fusion and MET exon 14 jumping detection. Liquid biopsy based on plasma cfDNA can meet most clinical genomic testing needs in general, especially when tissue is not available, but physicians need to be more cautious with negative results. Keywords: Liquid biopsy, Next-generation sequencing (NGS), Concordance Analysis, Lung Cancer.

Liquid and tissue profiling of targetable co-mutations with KRAS in non-small cell lung cancer.

e21201 Background: The primary objective of this study was to understand the co-occurrence of KRAS with other actionable drivers in Indian lung cancers patients, and to assess the utility of liquid biopsy in this cohort. Methods: We evaluated single nucleotide alterations, copy number alterations, and fusions in 507 tumor tissue and 1271 blood specimens with targeted NGS panels in NSCLC patients. Results: In tissue-based analysis (n = 507), KRAS mutations were detected in 68 (13.4%) samples. Of these, 63 (92.6%) samples did not show the presence of any targetable co-mutations. Targetable co-mutations were detected in 5 (7.4%), with concomitant EGFR mutations in all 5 samples(100%) and BRAF mutations in 2 samples (33.3%) In the liquid biopsy (n = 1271) analysis, KRAS mutations were observed in 94 (7.4%) samples. The liquid biopsy analysis showed higher presence of targetable co-mutations in 11.7% (n = 11). EGFR was the most common co-occurring targetable gene observed in 9 samples (81.8%). Mutations in BRAF were detected in 3 (27.3%) samples. ERBB2 Exon 20 insertion was detected in 1 sample (9.1%). No ALK, RET or ROS1 fusions were seen in our KRAS positive cohort. In the tissue cohort, one newly diagnosed adenocarcinoma case showed primary co-mutations in EGFR (Exon 19del VAF 76.8%) and KRAS (G13D VAF 10.5%), impacting utility of EGFR TKI as the first line therapy. Another EGFR Exon 19 del patient developed KRAS G12D mutation as an acquired resistance mutation post Osimertinib therapy. In the liquid biopsy cohort (n = 11), the KRAS positive cohort had four newly diagnosed patients with 3 having EGFR and 1 with BRAF co-mutation. Among the patients with prior therapy, one patient at presentations had EGFR Exon 19 deletion and developed EGFR T790M at progression on Afatinib. The baseline tissue did not have coexisting KRAS mutation. Fresh tissue biopsy was performed at the progression on Osimertinib. The noteworthy finding was the KRAS G12C (VAF 0.36%) presence in liquid biopsy but absences in the simultaneous fresh tissue sample. The EGFR Exon 19 was present at lesser frequency than KRAS in the liquid (VAF 0.23%). In the patients from liquid biopsy cohort with Osimertinib therapy (n = 4), all presented with KRAS as acquired resistance mutation. One among these also showed concomitant EGFR C797S mutation. Conclusions: KRAS mutation virtually makes it unlikely for other targetable mutations to coexist. It makes it thus compelling to further study the role of liquid biopsy with KRAS first and decide if chemotherapy with IO can be started considering the standard of care is to wait for the entire targeted panel before initiating IO. Equally its important to include KRAS in the lung panel and although small the concomitant KRAS may compromise the efficacy to TKI. Liquid biopsy is a promising alternative for detection of actionable alterations, including KRAS and can be harnessed for longitudinal profiling, monitoring tumor evolution and treatment outcome.

Prevalence of actionable oncogenic alterations (AGA) among patients with advanced (adv) non-squamous (NSQ) non-small cell lung cancer (NSCLC) by age, sex, and race in the United States (US).

e21206 Background: Identification of AGA in NSCLC has improved the understanding of lung cancer pathogenesis and introduced targeted therapies directed against specific genomic alterations. Given the limited data on the prevalence of AGA by demographics, we evaluated prevalence of AGA by age at diagnosis (dx), sex, and race. Methods: A descriptive study used the US nationwide de-identified Flatiron Health-Foundation Medicine NSCLC clinico-genomic database. Eligible patients were adults with a dx of adv NSQ NSCLC from Jan-2016 to Sep-2020 who initiated a first line therapy. The prevalence of AGA for each age, sex, or race stratum was calculated. Results: The cohort included 3938 patients; 67.9% White, 52.9% female, and median age at dx was 68 y. Table 1 presents the prevalence of AGA. The prevalence of EGFR and BRAF V600E mutations increased with age at dx. ALK rearrangement and ROS1 fusion were more prevalent among patients < 50 y vs ≥50 y while MET exon 14 skipping mutation was more prevalent among patients ≥75 y vs < 75 y. EGFR and KRAS G12C mutations were more prevalent in female vs male. Compared to White or Black, Asians had a higher prevalence of EGFR mutations and lower prevalence of KRAS G12C. Conclusions: The prevalence of certain AGA among patients with adv NSCLC vary by demographics, which may contribute to disproportionate disease burden in certain strata. These results highlight the need to enhance diversity of clinical trial populations and ensure the disease burden across demographics in the general population are represented in clinical trials. [Table: see text]

First results from the RETgistry: A global consortium for the study of resistance to RET inhibition in RET-altered solid tumors.

9065 Background: Rearranged during transfection ( RET) gene alterations are the oncogenic driver in diverse tumor types, including RET fusions identified in 1-2% of non-small cell lung cancers (NSCLC). While RET-selective tyrosine kinase inhibitors (TKIs) selpercatinib and pralsetinib are effective, acquired drug resistance remains a challenge. Here, we report the initial results from the RETgistry, an international consortium aimed at elucidating mechanisms of resistance to RET TKIs across RET-altered solid tumors. Methods: This was a retrospective analysis performed across 16 institutions. Patients (pts) were eligible if they had advanced solid tumor harboring an oncogenic RET alteration, received ≥1 RET TKI with disease progression, and underwent resistant tumor or liquid biopsy for next-generation sequencing (NGS). Results: A total of 103 time-distinct biopsies (62 tissue, 30 plasma, 11 paired tissue/plasma) were included in analysis, obtained from 88 pts with progression on a RET-selective TKI [selpercatinib (n = 70), pralsetinib (n = 14), selpercatinib and pralsetinib sequentially (n = 4)]. Pts had the following tumor types: 72 NSCLC (69% KIF5B-RET, 21% CCDC6-RET, 10% other RET fusion), 13 medullary thyroid cancer (TC) (54% RET M918T, 46% other RET mutation), and 2 papillary and 1 anaplastic TC (all, RET fusion). Median age was 58 (range, 21-86); 48% were male with 89% never/light smokers. Median duration of RET TKI preceding biopsies [first-line, n = 32 (31%); second-line, n = 42 (41%); third-/greater-line, n = 29 (28%)] was 16.5 months (mos) (95% CI, 14.0-19.6); median PFS was 14.1 mos (95% CI, 9.3-17.0). Resistant biopsies were obtained at median 15.0 mos from TKI initiation (range, 1.8-58.8). Acquired RET mutations were detected in 14 (14%), most common being G810 substitutions in 12 (12%). Potential off-target resistance gene alterations identified in 43 cases (42%) included MET amplification (14%), BRAF V600E or fusion (2%), KRAS gain or mutation (5%), ERBB2 amplification (2%), EGFR amplification (3%), ROS1 fusion (1%), and activating PIK3CA mutation or PTEN loss (4%). No resistant lung cancer biopsy demonstrated small cell transformation. The duration of TKI therapy (HR 0.64, p = 0.11) or PFS (HR 0.75, p = 0.33) did not differ according to the presence of on-target vs off-target resistance. NGS analyses of pre-RET-selective TKI tumors (n = 92) revealed frequent co-occurring alterations in TP53 (29%) and CDKN2A/B (12%). Conclusions: On-target resistance to RET inhibition due to acquired RET mutations was less common than off-target resistance, identified in 14%. The majority of RET TKI resistance is mediated by off-target mechanisms such as bypass receptor tyrosine kinase activation. Further studies are warranted to enable the development of therapeutic strategies to address resistance in pts with RET-altered tumors. The RETgistry accrual and data analyses continue.

Relationship between PD-L1 expression and molecular aberrances in lung adenocarcinoma with solid components.

Previous studies have evaluated the expression of programmed cell death ligand 1 (PD-L1) in terms of genetic mutation in lung adenocarcinoma (LUAD). However, there are no corresponding large-sample studies in Chinese patients with LUAD with solid components (LUAD-SC). Furthermore, it remains unknown whether the relationship that exists between PD-L1 expression levels and clinicopathological and molecular profiles in small biopsy specimens is consistent with that in surgically-resected specimens. The present study explored the clinicopathological features and genetic correlation of PD-L1 expression in LUAD-SC. We collected 1,186 LUAD-SC specimens from Fudan University, Zhongshan Hospital. The tumors were divided into PD-L1 negative, low, and high groups according to the tumor proportion score (TPS)-assessed expression of PD-L1. The mutational information of all specimens was assessed. Each group's clinicopathological features were also assessed. The relationship between PD-L1 expression levels and clinicopathological features, the overlap with driver genes and the prognostic value were analyzed. In 1,090 resected specimens, a high PD-L1 expression level was more prevalent in the group with predominant SCs, which was remarkably correlated with lymphovascular invasion and a more advanced clinical stage. In addition, the PD-L1 expression level was significantly related to EGFR, KRAS, and BRAF mutations and ROS1 fusions. Meanwhile, in 96 biopsy specimens, the solid-dominant type and EGFR showed a significant difference in PD-L1 expression. Furthermore, compared with their resected counterparts, the biopsy specimens were significantly associated with solid predominant, advanced tumor-node-metastasis (TNM) stage, and high PD-L1 expression. Finally, high PD-L1 expression can be considered a poor prognostic factor for overall survival (OS). LUAD-SC with high PD-L1 expression levels is linked to unique clinicopathologic characteristics as well as driver mutations. It is important to evaluate the percentage of solid components in both punctured and excised specimens, which may help identify cases of high PD-L1 expression.

Biomarker testing and treatment patterns in US patients (pts) with advanced/metastatic non-small cell lung cancer (NSCLC) harboring MET amplification.

e21057 Background: Limited published evidence exists on pts with advanced stage NSCLC harboring MET amplification ( METamp), a rare oncogenic driver. We report here real-world biomarker testing and treatment patterns for these patients. Methods: This was a non-interventional, descriptive cohort study using ConcertAI electronic medical record data derived from US community oncology centers and linked with ASCO/CancerLinQ data. Eligible pts had stage IIIB–IV NSCLC and METamp. Study period: Jan 1, 2004 – Oct 31, 2021. Results: Of 8,454 lung cancer pts with minimum 1 molecular test including MET, 164 had METamp (1.9%). Pts were mainly identified from 2015 onwards and primarily in oncology in the Southern and Midwest regions (86.6%). Majority of pts were tested by tissue biopsy (67.1%), 20.7% by liquid biopsy and 12.2% not specified. At least 64.5% of pts were highly MET amplified (either gene copy number ≥8 or by liquid biopsy ++, +++). Other mutations screened for were ALK, ROS1 and EGFR (99.4%, 98.2% and 97.6%), with oncogenic ALK, ROS1 fusions and activating EGFR mutations in 0.6% and 13.1%, respectively. BRAF and KRAS were less frequently screened for (90.2% and 87.2%), with potential oncogenic alterations in 2.0% and 12.6%. Of 127 pts tested for PD-L1 (by immunohistochemistry), 63.0% were positive, and 72.5% had tumor proportion score ≥50. Eighteen pts had METamp detected after receiving an EGFR tyrosine kinase inhibitor (TKI), indicating METamp as a secondary oncogenic driver. RB1 loss and MYC amplification, as potential mechanisms of MET inhibition resistance, were not frequently screened for (30.5% and 46.3%). RB1 loss was identified in 2.0% and MYC amplification in 15.8%. Next generation sequencing was the most frequent diagnostic used overall. A total of 144 pts (87.8% of 164 pts) received first-line (1L) anticancer therapy; platinum-based chemotherapy 57/144 (39.6%), immune-checkpoint inhibitors 50/144 (34.7%) or EGFR TKI monotherapy 24/144 (16.7%), other 2/144 (1.4%) and MET TKIs (mainly crizotinib) were used in 11/144 pts (7.6%) in 1L, 14/78 (17.9) in second line and 7/42 (16.7%) in third line. Conclusions: In this cohort of pts identified in a US community oncology setting, biomarker screening even for rarer genetic alterations occurred. Concomitant genetic alterations to METamp were infrequent except for alterations of EGFR and KRAS. The frequency of pts with high level of METamp in the cohort may explain the lower frequency of other biomarkers, as high level of amplification indicates a cleaner MET profile. Overall MET inhibitor treatment was not common. Screening for novel and rare biomarkers such as METamp as a primary or secondary oncogenic driver is becoming increasingly important to enable efficacious targeted treatment.

MYC amplification segment-size mutual exclusivity analysis with other oncogenic events in a pan-cancer sequencing cohort.

3073 Background: MYC gene alterations resulting in increased copy number are recurrent oncogenic alterations across human malignancies. Presently no clear data exists to aid in interpreting MYC amplification characteristics for identifying MYC-dependent tumors. Methods: Analysis was conducted on a center-wide cohort of 68,920 tumor-normal matched samples from pediatric and adult patients sequenced with an FDA-authorized targeted sequencing panel (MSK-IMPACT). To ascertain tumor content, ploidy and allelic specific gene copy number, tumor-normal samples were analyzed with FACETS. Samples with tumor purity <20% were excluded. MYC amplification was defined as total copy number (TCN) minus ploidy ≥ 4. All samples were queried for presence of concurrent oncogenic or likely-oncogenic alterations based on OncoKb annotation. Co-occurrence (Co) and mutual-exclusivity (Me) analysis between MYC amplification according to segment size and other oncogenic alterations was performed with DISCOVER (false discovery rate threshold of 0.01) for each tumor type. Results: After removing low-quality samples (n=19,010/68,920), 49,910 samples were included in the analysis. 3,219 (6.4%) samples harbored MYC amplification, with segment size ranging from less than 100Kb to more than 100Mb, with 38% samples displaying focal amplifications shorter than 10 Mb and 62% broad amplifications longer than 10 Mb. TCN was inversely correlated with segment size (R= -0.34, p=2.2e-16). Triple-negative breast cancer (13.3%), hepatocellular carcinoma (8.7%), neuroendocrine tumors of the rectum (8.3%), high-grade serous ovarian cancer (7.6%) and large cell neuroendocrine carcinomas of the lung (6.7%) had the highest prevalence of MYC amplification without concurrent potentially driver oncogene mutations, amplifications or fusions. CoMe analysis demonstrated mutual exclusivity of MYC gene amplification regardless of segment size with most major tumor-type specific oncogenic events. All MYC amplifications were mutually exclusive with MET splice variants and mutations (q< 0.001) and KRAS mutations (q< 0.001) in lung adenocarcinoma (LUAD), FGFR3 mutations (q< 0.001) and fusions (q< 0.001) in bladder cancer, PTEN mutations (q< 0.001) in hormone receptor-positive breast cancer, KRAS mutations (q< 0.001) in colorectal cancer and CDK4 amplifications (q< 0.001) in sarcomas. Broad MYC amplifications were mutually exclusive with RET, ROS1 and ALK fusions in LUAD, while focal ones co-occurred with RET (q=0.007) and ALK fusions (q< 0.001) or secondary mutations (q< 0.001). Conclusions: This dataset defines a subgroup of MYC amplified cancers without co-occurring drivers and further subdivided by segment size, providing information that may help better identify patients for trials of novel MYC-directed therapies.

Anaplastic lymphoma kinase (ALK) and ROS 1– positive advanced NSCLC: A real-world institutional experience.

e21074 Background: A novel fusion gene of EML4-ALK and ROS 1 has been identified in a subset of non-small-cell lung cancers (NSCLCs). Patients with the ALK-EML4 and ROS1 fusion gene demonstrate unique clinicopathological characteristics and treatment with targeted therapy led to improved survivals. Various clinical trials had shown the efficacy of ALK inhibitors, and they had become the standard of care. The real-world data of ALK inhibitors from a developing country like India is sparse and the present study was designed to analyze the incidence and outcomes of ALK positive NSCLC. Methods: Data of advanced NSCLC patients diagnosed between 2016 and 2022 with ALK and ROS1 positivity was analyzed. Detection of ALK was done by IHC or fluorescent in situ hybridization (FISH) and ROS1 was done by FISH. Results: A total of 776 advanced NSCLC patients between 2016 and 2022 were tested for ALK of which 63(8.1%) were positive. The median age at presentation was 50 years (range, 19-80 years). Of 63 patients, 34(54%) and 29(46%) were males and females. Cough (83%) was the most common symptom at presentation followed by breathlessness (73%), anorexia (59%) weight loss (59%) and chest pain (43%). The median duration of symptoms was 3 months (range, 1-18). 44(86%) patients were never smokers and 9(14%) patients had history of smoking or tobacco usage. The most common site of metastases was opposite lung (59%) followed by bone (50%), pleural effusion (41%) and brain metastasis were seen in 13(21%) patients. Adenocarcinoma was the most common histology (95%). Detection of ALK positivity was done by IHC and FISH in 45(72%) and 18(28%) patients. Of 63 patients, 52 patients received at least 4 months of treatment and were assessed for survival. First line therapy was crizotinib, chemotherapy followed by maintenance crizotinib, ceritinib, Alectinib in 40(77%), 4(7.7%), 6(11.5%) and 2(3.8%) patients respectively. At a median follow up of 14 months, the one-year progression free and overall survival was 71% and 75% respectively. The 2 year and 3-year OS were 54% and 30% respectively. Of 322 patients tested for ROS1, 9(2.8%) were positive for ROS1 translocation and received first line therapy with crizotinib. The one-year PFS and OS of ROS 1 positive patients were 75% and 89% respectively. Conclusions: ALK and ROS1 positivity was seen in 8.1% and 2.8% of advanced NSCLC patients. Majority of patients were never smokers. Opposite lung and bone were the most common site of metastases. Approximately more than half of the patients had two-year survival rate and survivals are almost similar to clinical trials. Crizotinib is well tolerated in majority of patients.

“Evaluation of ROS1 expression and rearrangements in a large cohort of early-stage lung cancer”

BackgroundROS1 fusion is an infrequent, but attractive target for therapy in patients with metastatic non- small-cell lung cancer. In studies on mainly late-stage disease, the prevalence of ROS1 fusions is about 1–3%. In early-stage lung cancer ROS1 might also provide a fruitful target for neoadjuvant or adjuvant therapy. In the present study, we investigated the prevalence of ROS1 fusion in a Norwegian cohort of early-stage lung cancer. We also explored whether positive ROS1 immunohistochemical (IHC) stain was associated with certain mutations, clinical characteristics and outcomes.MethodsThe study was performed using biobank material from 921 lung cancer patients including 542 patients with adenocarcinoma surgically resected during 2006–2018. Initially, we screened the samples with two different IHC clones (D4D6 and SP384) targeting ROS1. All samples that showed more than weak or focal staining, as well as a subgroup of negative samples, were analyzed with ROS1 fluorescence in situ hybridization (FISH) and next-generation sequencing (NGS) with a comprehensive NGS DNA and RNA panel. Positive ROS1-fusion was defined as those samples positive in at least two of the three methods (IHC, FISH, NGS).ResultsFifty cases were IHC positive. Of these, three samples were both NGS and FISH-positive and considered positive for ROS1 fusion. Two more samples were FISH positive only, and whilst IHC and NGS were negative. These were also negative with Reverse Transcription quantitative real time Polymerase Chain Reaction (RT-qPCR). The prevalence of ROS1 fusion in adenocarcinomas was 0.6%. All cases with ROS1 fusion had TP53 mutations. IHC-positivity was associated with adenocarcinoma. Among SP384-IHC positive cases we also found an association with never smoking status. There was no association between positive IHC and overall survival, time to relapse, age, stage, sex or pack-year of smoking.ConclusionsROS1 seems to be less frequent in early-stage disease than in advanced stages. IHC is a sensitive, but less specific method and the results need to be confirmed with another method like FISH or NGS.

ERBB family fusions are recurrent and actionable oncogenic targets across cancer types.

Gene fusions involving receptor tyrosine kinases (RTKs) define an important class of genomic alterations with many successful targeted therapies now approved for ALK, ROS1, RET and NTRK gene fusions. Fusions involving the ERBB family of RTKs have been sporadically reported, but their frequency has not yet been comprehensively analyzed and functional characterization is lacking on many types of ERBB fusions. We analyzed tumor samples submitted to Caris Life Sciences (n=64,354), as well as the TCGA (n=10,967), MSK IMPACT (n=10,945) and AACR GENIE (n=96,324) databases for evidence of EGFR, ERBB2 and ERBB4 gene fusions. We also expressed several novel fusions in cancer cell lines and analyzed their response to EGFR and HER2 tyrosine kinase inhibitors (TKIs). In total, we identified 1,251 ERBB family fusions, representing an incidence of approximately 0.7% across all cancer types. EGFR, ERBB2, and ERBB4 fusions were most frequently found in glioblastoma, breast cancer and ovarian cancer, respectively. We modeled two novel types of EGFR and ERBB2 fusions, one with a tethered kinase domain and the other with a tethered adapter protein. Specifically, we expressed EGFR-ERBB4, EGFR-SHC1, ERBB2-GRB7 and ERBB2-SHC1, in cancer cell lines and demonstrated that they are oncogenic, regulate downstream signaling and are sensitive to small molecule inhibition with EGFR and HER2 TKIs. We found that ERBB fusions are recurrent mutations that occur across multiple cancer types. We also establish that adapter-tethered and kinase-tethered fusions are oncogenic and can be inhibited with EGFR or HER2 inhibitors. We further propose a nomenclature system to categorize these fusions into several functional classes.

Spectrum of kinase gene rearrangements in a large series of paediatric inflammatory myofibroblastic tumours.

Inflammatory myofibroblastic tumours (IMTs), being an exceptionally rare category of paediatric neoplasms, often contain druggable gene rearrangements involving tyrosine kinases. This study presents a large consecutive series of IMTs which were analysed for the presence of translocations by the PCR test for 5'/3'-end ALK, ROS1, RET, NTRK1, NTRK2 and NTRK3 unbalanced expression, variant-specific PCR for 47 common gene fusions and NGS TruSight RNA fusion panel. Kinase gene rearrangements were detected in 71 of 82 (87%) IMTs (ALK: n = 47; ROS1: n = 20; NTRK3: n = 3; PDGFRb: n = 1). The test for unbalanced expression had 100% reliability in identifying tumours with ALK fusions, but failed to reveal ROS1 rearrangements in eight of 20 (40%) ROS1-driven IMTs; however, ROS1 alterations were detectable by variant-specific PCR in 19 of 20 (95%) cases. ALK rearrangements were particularly common in patients below 1 year of age (10 of 11 (91%) versus 37 of 71 (52%), P = 0.039). ROS1 fusions occurred more often in lung IMTs than in tumours of other organs (14 of 35 (40%) versus six of 47 (13%), P = 0.007). Among 11 IMTs with no kinase gene rearrangement identified, one tumour demonstrated ALK activationvia gene amplification and overexpression, and another neoplasm carried COL1A1::USP6 translocation. PCR-based pipeline provides a highly efficient and non-expensive alternative for molecular testing of IMTs. IMTs with no detectable rearrangements need further studies.

Abstract 1403: Comprehensive characterization of 1038 Chinese non-small cell lung cancer patients of different PD-L1 expression levels

Abstract Background: Programmed death ligand 1 (PD-L1) is currently one of the most well-characterized markers for predicting response to immunotherapy in non-small cell lung cancer (NSCLC). Multiple clinicohistologic and genetic characteristics have been proposed as correlates of PD-L1 expression. However, few studies have examined the associations between PD-L1 expression and immune infiltration or chromosomal arm-level alterations. In this comprehensive characterization, we identified clinical features, oncogenic drivers and other genomic abnormalities, impaired biological pathways, arm-level amplifications and losses, and infiltration of immune cell populations that were associated with PD-L1 expression. Methods: We retrospectively included 1038 NSCLC patients who had both PD-L1 Tumor Proportion Score (TPS) by the 22C3 assay and genomic profiles acquired with targeted sequencing using 8-, 168-, and 520-gene panels (Burning Rock Biotech, Guangzhou, China). Multiplex immunohistochemistry (mIHC) was used to analyze immune infiltration. Association analysis was performed to identify patient characteristics associated with PD-L1 expression. Results: The negative (TPS <1%), low (1-49%), and high (≥50%) PD-L1 expression classes accounted for 25.2% (n = 262), 43.2% (n = 449), and 31.5% (n = 327) patients, respectively. PD-L1 expression was positively associated with advanced tumor stage, the male sex, squamous carcinoma, ALK fusions, KRAS G12/13X mutations, BRAF V600E, ROS1 fusion, MET exon 14 skipping, altered TP53, LPR1B, FAT1, PTPRD, KDR, and CD274, and altered pathways such as MAPK, RAS, DNA damage response, p53 signaling, and checkpoint factors. The negative correlates included driver-positive status, EGFR L858R, EGFR exon 19 deletions, ERBB2 exon 20 insertions, altered RMB10, STK11, RICTOR, GNAS, and PIK3C2G, and aberrant receptor tyrosine kinase, ERBB2 signaling, and non-homologous end joining. In terms of arm-level aberrations, PD-L1 positivity was associated with 3p, 3q, 7p or 7q amplification and 18p, 20p, or 20q deletion. We also found that 9p, 9q, 10p, 12p, 12q, and 19p amplifications were most frequent in high PD-L1-high patients, who were also more likely to harbor 18p, 29p, or 20q deletion when compared with compared with PD-L1-negative patients. mIHC analysis revealed that PD-L1 expression was associated with proportions of CD3+, CD3+CD8+, CD3+PD-1+, CD8+PD-1+, and PD-1+ cells in the tumor and CD3+PD-1+, CD8+PD-1+, and PD-1+ cells in the stroma, suggesting increased tumor infiltration of cytotoxic T cells associated with PD-L1 expression. Conclusions: This study provides a comprehensive analysis of the associations between PD-L1 expression and a multitude of histopathologic, genomic, and immunologic features. Further research is warranted for validation and association of these factors with clinical outcomes. Citation Format: Weiqiang Yin, Jinghao Liang, Zhihua Guo, Zixian Xie, Haonan Zhao, Fayuan Wu. Comprehensive characterization of 1038 Chinese non-small cell lung cancer patients of different PD-L1 expression levels [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1403.

Abstract 2857: Patient-derived cells (PDCs) and organoids (PDOs) as platforms for screening novel therapeutics for NSCLC

Abstract Purpose: Patient-derived cells (PDCs) and organoids (PDOs) are constructed from patient tissue to mimic the biological features of patients. In cancer research, PDC/PDOs have been crucial as they can recapitulate tumor mutations. As heterogeneity between traditional cell lines and the human body leads to clinical trial failure, PDC/PDOs are widely used for predicting the preclinical drug efficacy. The aim of this study is to show that the PDC/PDOs can be used as effective tools for screening novel therapies on non-small cell lung cancer (NSCLC). Experimental design: PDC/PDO models from malignant effusions were established as following. We succeeded the establishment with samples which are positive for malignancy in cytology tests and tumor colony formation. Genotypes are analyzed by Sanger sequencing, Whole exome sequencing, or RNA-sequencing. Cell viability assay was performed using currently approved drugs or drugs in clinical development or their combinations. Results: A total of 46 PDCs and 150 PDOs was established from NSCLC patients, including models harboring sensitizing EGFR mutations, ALK fusions, ROS1 fusions, EGFR exon20 insertion, BRAF V600E, and those harboring various resistance mechanisms to EGFR-TKIs (T790M, C797S/C797G, MET amplification), to ALK-TKIs (G1202R), and to ROS1 TKI (G2032R). Osimertinib-resistant YU-1097 harboring EGFR resistance mutation (E19del/T790M/C797S) revealed sensitivity to BLU-945 (IC50, 108nM), a novel fourth-generation EGFR-TKI. A similar inhibition of cell viability was observed with repotrectinib (IC50, 21nM), a next-generation ROS1-TKI and lorlatinib (IC50, 9nM) in YU-1078 harboring CD74-ROS1, whereas more robust tumor regression was seen with repotrectinib in YU1078-derived xenograft model. Amivantamab, a EGFR-MET bispecific antibody, showed a robust activity in YU-1163 and YUO-036 in vitro and in vivo. YU-1077 harboring ALK G1202R solvent-front mutation showed sensitivity to NVL-655, a next-generation ALK-TKI, with a potency > 10-fold than that of lorlatinib. YUO-010 with MET amplification following osimertinib was sensitive to RGEN 5093-M114, a METxMET bispecific antibody-drug conjugate. Conclusions: PDC/PDO models can be utilized for evaluating activity of novel agents and will accelerate novel drug development in NSCLC. Citation Format: Yunjoo Joo, Sewon Park, Ju-hyeon Lee, Mi Ran Yun, Mi Ra Yu, Chun-Bong Synn, Seung Yeon Oh, Eun Ji Lee, Dong Kwon Kim, Seul Lee, Kyumin Lim, Min Hee Hong, Sun Min Lim, Chang Gon Kim, Ji Yun Lee, Jii Bum Lee, Byoung Chul Cho. Patient-derived cells (PDCs) and organoids (PDOs) as platforms for screening novel therapeutics for NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2857.

Abstract 3460: Targeting the MIF-CD74 axis to overcome resistance to tyrosine kinase inhibitors in non-small cell lung cancer

Abstract Development of tyrosine kinase inhibitors (TKIs) against oncogenic drivers has significantly improved survival of patients with oncogene-mutated non-small cell lung cancer (NSCLC). However, acquired resistance to TKIs emerges over time in essentially all patients who initially respond. Recent evidence suggests that drug-tolerant persister (DTP) cells, which survive and adapt to targeted therapies during an early phase of treatment, play an important role in the emergence of drug resistance. We previously reported that CD74 expression is upregulated in EGFR-mutated lung cancer after treatment with EGFR-TKIs and that CD74 can be one of the DTP cell markers. However, the mechanism underlying CD74 expression and the role of CD74 in DTP cells remain unclear. Here, we sought to identify the mechanism using cell culture systems and transgenic mouse models. We confirmed that CD74 upregulation at the mRNA level after TKIs treatments in various oncogene-mutated cell lines, including those with EGFR mutations, ROS1 fusions, and ALK fusions. We found that CIITA, upstream of CD74, and TNF-α expression were induced by TKIs treatments in tumor cells, leading to an increase in CD74 expression. In addition, we found that TKIs treatments enhanced the autocrine secretion of macrophage migration inhibitory factor (MIF), a ligand of CD74, from tumor cells, suggesting that autocrine stimulation of the CD74 signaling results in block of apoptosis and emergence of DTP cells. To examine whether CD74 plays an important role in the emergence of resistance to TKIs in vivo, we crossed lung-specific EGFR-L858R-T790M transgenic mice with Cd74 knockout mice. Complete deletion of CD74 overcame or delayed resistance to TKIs. Taken together, the MIF-CD74 axis can be a novel target to overcome resistance in driver-mutated NSCLC. Citation Format: Motohiro Izumi, Meghan Lee, Daisuke Shibahara, Ikei S. Kobayashi, David Plotnick, Susumu S. Kobayashi. Targeting the MIF-CD74 axis to overcome resistance to tyrosine kinase inhibitors in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3460.