Intrathoracic Lymph Node Microcalcifications are Associated With a High Prevalence of Malignancy and Anaplastic Lymphoma Kinase Rearrangement: The "Calce" Study.

Next-Generation Sequencing Identified a Novel Crizotinib-Sensitive PLB1-ALK Rearrangement in Lung Large-Cell Neuroendocrine Carcinoma

P3.02a-002 Pulmonary Sarcomatoid Carcinoma with ALK Rearrangement: Frequency, Clinical-Pathologic Characteristics, and Response to ALK Inhibitor: Topic: ALK

Background. Inflammatory myofibroblastic tumor (IMFT) is a rare mesenchymal neoplasm, mainly driven by anaplastic lymphoma kinase (ALK) rearrangement, which is present in 50% of cases and target for ALK inhibition. For the current project we used tumor material from IMFT patients treated with the ALK/ROS1/MET inhibitor crizotinib in the frame of EORTC 90101, where IMFT patients were attributed to ALK+/- sub-cohorts based on the presence (>15% of cells) or absence (<15%) of ALK rearrangement by fluorescence in situ hybridization (FISH) and/or immunohistochemistry (IHC) [Schöffski at al. Lancet Respir Med. 2018]. The aim of the project was to evaluate the frequency of ALK rearrangement, compare different methods of ALK detection, perform a detailed characterization of ALK rearrangement partners, and to identify other potential molecular drivers with potential pharmacodynamic relevance. Material and Methods. Archival material from 24 IMFT cases, both primary or metastatic samples, was analyzed by IHC using ALK antibodies: ALK1 (DAKO) and ALK-D5F3 (Cell Signalling), and pan-NTRK (Ventana, clone EPR17341). ALK and ROS1 rearrangement were studied by FISH and fusion genes were detected using the Archer CTL Fusion Panel. Results. ALK immunopositivity by ALK1 and ALK-D5F3 was observed in 14 out of 24 cases and 11/21, respectively. ALK rearrangement by FISH was found in 13/23 samples. With Archer, fusion transcripts were identified in 13/20 specimens, all but one involving ALK with 11 different fusion partners. In one case we detected an ETV6-NTRK3 fusion; NTRK positivity was confirmed by IHC. Interestingly, the NTRK fusion case responded to crizotinib, which shows cone anti-NTRK activity [Okamura et al. JCO Precis Oncol 2019]. We did not detect ROS1 rearrangement by FISH in any sample. Two samples with ALK rearrangement by Archer were negative by FISH, with 11% and 0% of cells with ALK split signal, but both showed protein expression with both antibodies used. They responded to crizotinib with either a RECIST 1.1 complete response or stable disease, for 43 and 9 months, respectively. All but one case with ALK rearrangement by Archer were positive by ALK IHC with ALK1 antibody. The only immunonegative sample had an EML4-ALK1 fusion. Two cases were positive with ALK-D5F3 antibody but no ALK positivity was detected using ALK1, and no ALK rearrangement was detected by FISH or Archer panel, suggesting false positive results. Conclusions. ALK rearrangement is the most common driver in IMFT and the fusion can involve multiple partners. ALK1 but not the ALK-D5F3 antibody shows high ALK specificity in IMFT. The Archer CTL Fusion panel is a reliable and sensitive method for detecting fusion transcripts in IMFT, also allowing to detect alternative fusion genes, which may be responsible for the sensitivity to kinase inhibitors. Citation Format: Agnieszka Wozniak, Che-Jui Lee, Tom van Wezel, Jozef Sufliarsky, Hans Gelderblom, Jean-Yves Blay, Maria Debiec-Rychter, Raf Sciot, Judith V.M.G. Bovee, Patrick Schöffski. Detection of molecular drivers in inflammatory myofibroblastic tumor: study on archival tissue from EORTC 90101 “CREATE” phase II clinical trial [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3191.

10527 Background: Epidermal growth factor receptor (EGFR) mutation or anaplastic lymphoma kinase (ALK) rearrangement was found not only predict the efficacy of targeted drugs, but also associate with the efficacy of chemotherapy drugs in non-small cell lung cancer (NSCLC) patients. We investigated the relationship of EGFR mutation status or ALK rearrangement and DNA repair or synthesis genes, such as excision repair cross-complementing 1 (ERCC1), ribonucleotide reductase subunit M1 (RRM1), thymidylate synthetase (TS) and breast cancer gene one (BRCA1) gene expression, as a potential explanation for these observations. Methods: In this surgical series, 104 resected lung adenocarcinomas from nonsmoker females were analyzed concurrently for the EGFR mutation, ALK rearrangement status and mRNA expression of ERCC1, RRM1, TS and BRCA1 genes. EGFR mutation detection was performed by the method of ADx-ARMS, ALK rearrangement was detected by PCR and the mRNA expression of different genes were tested using the method of real-time PCR. Results: 73 (70.2%) patients harbored EGFR mutations and 10 (9.6%) had ALK rearrangement. The ERCC1 mRNA level in patients with EGFR mutation was 3.44±1.94×10-3 , which is significantly lower than in the patients with ALK positive and both negative(4.60±1.95×10-3 and 4.95±2.33×10-3 respectively, P=0.010). While the TS mRNA levels were significantly lower in the patients with EGFR mutation(1.15±1.38×10-3 VS 2.69±3.97×10-3, P=0.006) or ALK positive (1.21±0.78×10-3VS 2.69±3.97×10-3, P=0.020) than in patients with both negative. Conclusions: NSCLC specimens harboring activating EGFR mutations are more likely to express low ERCC1 and TS mRNA levels, while NSCLC patients with ALK rearrangement are more likely to express low TS mRNA levels, which could be helpful to select a proper chemotherapy regimen for NSCLC patients with known EGFR mutation or ALK fusion status.

Anaplastic lymphoma kinase (ALK) rearrangement is a rare feature in advanced non-small cell lung cancer (NSCLC), occurring in around 5% of the patients (1). This oncogenic addiction is more frequent in no/light-smokers, in thyroid transcription factor-1 (TTF1)-positive adenocarcinoma, with cribriform architecture and ring cells (2). ALK rearrangement—most of the time a translocation with a partner gene—induces the formation of a fusion protein with oncogenic properties. The first targeted therapy developed for ALK -rearranged NSCLC was crizotinib, an ALK tyrosine kinase inhibitor (TKI). Crizotinib was proved to be superior to chemotherapy in first-line setting in ALK -rearranged NSCLC, with an overall response rate (ORR) of 74% and a progression-free survival (PFS) of 10.9 months (3). Crizotinib in first-line treatment is now widely used. Other ALK TKIs have been developed, as brigatinib. Results of the phase I-II trial published in 2016 showed in 79 advanced NSCLC patients treated with brigatinib an ORR at 75% [72% in previously crizotinib treated patients (n=71)] and a PFS (median) of 13.2 months (4). Safety profile showed specific lung toxicity, occurring at the beginning of the treatment (median 2 days) in 8% of the patients, with incidence increasing with the dose of brigatinib. The ALTA trial, published in May 2017 in Journal of Clinical Oncology by Kim et al . was a phase II trial that randomized patients with advanced ALK -rearranged NSCLC between 2 doses of brigatinib (90 mg daily and 90 mg daily for 7 days then 180 mg daily) (5). The primary endpoint was ORR, and no comparison between the 2 arms was pre-planned. Assuming an ORR ≥35% with a power of 90% and a bilateral alpha risk at 0.025, the study had to include 109 patients per arm. Patients should have an advanced ALK -rearranged NSCLC with progression with crizotinib and a performance status (PS) between 0 and 2. Brain metastases were allowed only if asymptomatic and with stable doses of steroid treatment. Two-hundred and twenty-two patients were randomized [112 patients arm A (90 mg) and 110 patients arm B (90 mg then 180 mg)]. Both arms were well-balanced, with a median age at 54 years, 57% of women, 31% of Asian, 60% of non-smokers, 97% of adenocarcinoma, 69% of brain metastases. ORR (investigator-assessed) was 45% in arm A and 54% in arm B, and disease-control rate was 82% (arm A) and 86% (arm B). Median time to response was 1.8 months (arm A) and 1.9 months (arm B), and duration of response was 13.8 months (arm A) and 11.8 months (arm B). Median PFS was 9.2 months (arm A) and 12.9 months (arm B). Post-hoc comparison between the 2 arms for PFS showed a clear benefit for arm B, with a hazard ratio (HR) at 0.55 [95% confidence interval (CI), 0.36–0.86]. One-year overall survival (OS) probability was 71% (arm A) and 80% (arm B). As shown in the phase I-II trial, brigatinib had intracranial efficacy. ORR for measurable brain metastases was 42% (arm A, n=26) and 67% (arm B, n=18). For active brain metastases (i.e., metastases without previous brain radiotherapy, or progression after brain radiotherapy), intracranial ORR was 42% (arm A, n=19) and 73% (arm B, n=15). Intracranial PFS (median) was 15.6 months (arm A) and 12.8 months (arm B), with a duration of intracranial response (median) not reached in both arms. In term of safety, no new signal was detected. In both arms, main toxicities (≥10% patients) were nausea (33%/40%), diarrhea (19%/38%), vomiting (24%/23%), headache (28%/27%), decrease appetite (22%/15%) and hypertension (11%/21%). Grade 3–4 toxicities were rare: hypertension (6%/6%), increased creatine phosphokinase (3%/9%) and rash (1%/3%), mainly. Pulmonary early toxicity occurred in 6% of patients (n=14), during the first 2 days of treatment (always at 90mg daily of brigatinib). Grade 3–5 pulmonary toxicities concerned 3% of patients (n=7). Half of the patients (n=7) had successfully continued brigatinib after suspension, whereas 7 patients stopped brigatinib definitely. One patient died from acute respiratory failure (<1%). Multivariate analysis showed that advanced age [odds ratio (OR) =2.10; 95% CI, 1.21–3.65] and time from last crizotinib dose to first brigatinib dose less than 7 days (OR =3.88; 95% CI, 1.10–13.68) were associated with early pulmonary toxicity. Seven percent (arm A) and 20% (arm B) patients had dose reduction, due to increase of creatine phosphokinase, pneumonitis and rash.

e21144 Background: Anaplastic lymphoma kinase ( ALK) gene rearrangements are found in approximately 5% of non-small-cell lung cancer (NSCLC). Since ALK-TKIs have been developed for patients with ALK-positive advanced NSCLC, it remained 20%-30% non-responders, thus other treatment strategies would be explored to prolong overall survival. Previous studies have shown that ALK rearrangement can upregulate PD-L1 expression via signaling pathways such as ERK/AKT, and the high expression of PD-L1 negatively affects the efficacy of crizotinib and would be more suitable for immunotherapy. However, only a few immunotherapy clinical studies recruited tiny amounts of ALK rearrangement NSCLC, making it difficult to investigate the relationship between this oncogenic gene alteration and therapeutic effects. Herein, we described the immune-related molecular characteristics of ALK-rearranged patients in a large group of Chinese lung cancer, hoping to provide more precise treatment strategies. Methods: A total of 1410 lung cancer patients harboring ALK rearrangement were included in this analysis. Using wide NGS panel testing, we elucidated ALK gene fusions, intergenic region fusions ( ALK-IR) as well as other genomic alterations and TMB levels in each patients’ tumor specimen. PD-L1 expression level was evaluated by the tumor positive score (TPS). Results: Over two hundreds of types of ALK rearrangement were identified. The whole population was divided into six subgroups according to types of ALK rearrangements: 3'- ALK-only (71.1%), 5'-non- EML4-ALK-only (1.0%), multi- ALK fusion (14.2%), single ALK-IR (3.2%), multi-3'- ALK-IR (10.1%), and multi-5'- ALK-IR (0.5%). The overall PD-L1 expression level was high in ALK rearrangement lung cancer, of which the proportion of patients with TPS ≥ 50% was 19.5%, and the TPS ≥ 50% proportion in the single ALK-IR subgroup was the highest (28.6%) among all subgroups. We then evaluated the distribution of TMB levels and found the median TMB level was higher than 30% lung cancer. Of note, the single ALK-IR subgroup also present a trend of higher TMB level among all subgroups. We also observed more DNA damage response (DDR) gene mutations (median number: 2, range: 1-22) in the single ALK-IR subgroup. Somatic copy number amplifications (SCNAs) were detected in 195 (13.8%) ALK -rearranged lung cancer patients, and the proportion of SCNAs in each subgroup was similar. The most frequently high-level amplified genes were MYC (23.9%) and EGFR (10.9%). Furthermore, the 3'- ALK-only subgroup mainly occurred low-level SCNAs (68.5%), whereas the multi- ALK fusion and multi-3'- ALK-IR subgroups were more likely to harbor high and moderate level SCNAs. Conclusions: Our findings demonstrated that lung cancer with ALK-IR might have active immune microenvironment, indicating it’s a subset of ALK-rearranged patients who would benefit from immunotherapy.

Anaplastic lymphoma kinase (ALK) rearrangement is a representative driver mutation in lung cancer. However, the biology of early-stage ALK-rearranged lung cancer remains unclear. We aimed to assess the clinicopathological features, prognostic implications, and influence of ALK rearrangement on the postoperative course in surgically resected lung cancer. We retrospectively analyzed data from the Japanese Joint Committee of Lung Cancer Registry database. Of the 12 730 patients with lung adenocarcinoma, 794 (6.2%) were tested for ALK rearrangement and were included. ALK rearrangements were detected in 76 patients (10%). The 5-year overall survival (OS) rate was significantly higher in the ALK rearrangement-positive group than in the ALK rearrangement-negative group (p = 0.030). Multivariable analysis revealed that ALK rearrangement was an independent prognostic factor for improved OS (hazard ratio, 0.521; 95% confidence interval, 0.298-0.911; p = 0.022). Regarding the postrecurrence state, there was no difference in the initial recurrence sites between both groups. Administration of ALK-tyrosine kinase inhibitors (TKIs) improved postrecurrence survival in any treatment lines. In one of the largest national surveys, ALK rearrangement was associated with improved long-term outcomes in surgically resected patients. ALK-TKIs may be an important treatment strategy for ALK rearrangement-positive lung adenocarcinoma in the postrecurrence state.

3555 Background: Anaplastic lymphoma kinase (ALK) rearrangement is a validated therapeutic driver gene in non-small cell lung cancer (NSCLC). More than 30 different fusion partner genes of ALK in NSCLC have been reportedand most of these ALK fusions respond well to ALK inhibitors crizotinib. With the development of next-generation sequencing (NGS), more novel partners for ALK rearrangement have been identified. Here, we aimed to report the landscape of ALK rearrangement in Chinese patients with solid tumors. Methods: Tissue or blood samples were subjected to NGS in a College of American Pathologists-certified and Clinical Laboratory Improvement Amendments-accredited lab for ALK arrangement. Results: In total, we profiled more than 40,000 patients, among which 72 cases with 52 ALK fusion partner, harboring 17 reported partners and 35 novel partners. The average ALK rearrangement patients' age was 53 years (range, 17-76 years). Among all the ALK fusion cases (n = 72), lung cancer were the largest proportion with 77.8% (n = 56), colorectal cancer accounted for, 5.5% (n = 4), liver cancer accounted for 4.2% (n = 3), biliary cancer, melanoma, carcinosarcoma and inflammatory myofibroblastic tumor accounted for 2.8% (n = 2) respectively, and only one case (n = 1) was malignant peritoneal mesothelioma. The most common ALK fusion partners were KIF5B (n = 6), DCTN1 (n = 5) and STRN (n = 5). In 38 cases, 35 novel ALK fusion partners were discovered. The novel CLIP4-ALK, EHBP1-ALK, PLB1-ALK occurred twice in 6 patients, which were two lung cancer patients with CLIP4-ALK fusion, two lung cancer patients with PLB1-ALK fusion, one hepatic cellular cancer patients with EHBP1-ALK, and one melanoma patients with EHBP1-ALK. There were two special lung cancer cases with two ALK fusions. One case detected the novel LRIG1-ALK fusion and novel PLB1-ALK fusion, the other case detected novel GLI3-ALK fusion and reported HIP1-ALK fusion. Conclusions: Novel ALK fusions are detected in patients with not only NSCLC but also other solid tumors. NGS fusion assay is an optional method for screening novel fusions.

BackgroundAlthough some studies have assessed the correlation between computed tomography (CT) features and anaplastic lymphoma kinase (ALK) rearrangement in patients with non-small-cell lung cancer (NSCLC), few have focused on early-stage patients. The results of some previous studies are inconsistent and contradictory. Therefore, this study aimed to analyze the clinicopathological and CT features of patients with early-stage NSCLC harboring ALK rearrangement.MethodsThis retrospective analysis included 65 patients with ALK rearrangement and 629 ALK-negative patients. All patients had surgically resected NSCLC and were diagnosed with stage IA or stage IIB NSCLC. Clinicopathological features and CT signs, including tumor size and density, consolidation tumor ratio (CTR), lesion location, round or irregular shape, lobulated or spiculated margins, air bronchograms, bubble-like lucency or cavities, and pleural retraction, were investigated according to different genotypes.ResultsThe prevalence of ALK rearrangement in patients with early-stage NSCLC was 9.3% (65/694). Patients with ALK rearrangement were significantly younger than those without ALK rearrangement (P = 0.033). The frequency of moderate cell differentiation was significantly lower in tumors with ALK rearrangement than in those without ALK rearrangement (46.2% vs. 59.8%, P = 0.034). The frequency of the mucinous subtype was significantly higher in the ALK-positive group than in the ALK-negative group (13.8% vs. 5.4%, P = 0.007). No significant differences were found in any CT signs between the ALK-positive and ALK-negative groups.ConclusionsPatients with ALK-positive lung cancer may have specific clinicopathological features, including younger age, lower frequency of moderate cell differentiation, and higher frequency of the mucinous type. CT features may not correlate with ALK rearrangement in early-stage lung cancer. Immunohistochemistry or next-generation sequencing is needed to further clarify the genomic mutation status.

Pulmonary Sarcomatoid Carcinoma with ALK Rearrangement: Frequency, Clinical-Pathologic Characteristics, and Response to ALK Inhibitor

Anaplastic lymphoma kinase (ALK) rearrangement, one of the common oncogene rearrangements in the mutational history of lung adenocarcinoma, occurs in approximately 5% of non-small cell lung cancer (NSCLC) patients who could be effectively treated with ALK tyrosine kinase inhibitors (TKIs). The earlier phase III PROFILE 1014 study has shown that crizotinib, a first-generation ALK-TKI, significantly improved progression-free survival (PFS) compared with platinum-based chemotherapy in patients with previously untreated advanced ALK-positive NSCLC. Thus, clinicians must screen potential candidates for this driver alteration to guide ALK inhibitor therapy with a molecular testing platform capable of capturing all ALK fusions. Echinoderm microtubule-associated proteins, including the EML4 gene, are the most common ALK rearrangement partner. With the widespread use of the next-generation sequencing (NGS) techniques, which could approach enable the simultaneous screening of multiple genetic alterations, increasingly ALK rearrangement partners have been documented. However, the concurrent two ALK rearrangements within the same patient have rarely previously been reported. Here, we describe a novel CCNY-ALK (C1:A20) and ATIC-ALK (A7:A20), coexisting in the same case with poorly differentiated NSCLC and providing evidence of its sensitivity to ALK inhibitors. The newly identified rearrangement partners can be added to the list of ALK rearrangements that occurred in ALK-positive NSCLC, as it could lead to prolonged disease control. Also, while different ALK rearrangement variants might bring differing clinical outcomes, we discuss the impact of the co-mutations of these two ALK rearrangements on the sensitivity to ALK inhibitors. However, the impact of co-mutations on the pathogenesis of NSCLC should be further studied to supply more theoretical insight that co-mutations present for personalized anti-cancer therapy.

PC04.03 Adjuvant Targeted Therapy for PTS with Genomic Alterations - Yes

A novel HIP1-ALK fusion variant in lung adenocarcinoma showing resistance to Crizotinib

ObjectivesThere have been few studies that have fully elucidated the relationship between genomic mutations in pulmonary adenocarcinomas and occult lymph node (LN) metastases (pN1-2) despite a preoperative clinical N0 stage (cN0). It is well known that anaplastic lymphoma kinase (ALK) rearrangements are more likely to occur in younger patients with high grade tumors. The aim of this study was to investigate the genomic status, examine the clinicopathologic features, and evaluate whether ALK mutations are associated with occult LN metastases.Materials and methodsWe retrospectively evaluated 459 Japanese patients who underwent pulmonary resection of cN0 adenocarcinomas between January 2012 and December 2015. The clinicopathologic characteristics, including age, sex, smoking index, tumor maximum diameter and consolidation/tumor ratio on computed tomography (CT), maximum standardized uptake value on positron emission tomography (PET) and gene mutations (epidermal growth factor receptor [EGFR], ALK, and kirsten ras genes (KRAS), were evaluated.ResultsALK and EGFR and KRAS mutations were all mutually exclusive. Among 324 patients found to have mutations, ALK was involved in 19 (5.9%), EGFR in 266 (82.1%), and KRAS in 39 (12.0%). The incidence of occult LN metastases did not differ significantly between those with or without mutations (p=0.27). On univariate and multivariate analyses, tumors with ALK were more likely to have occult LN metastases (p=0.03). In cN0 tumors with ALK, pN1 was diagnosed in 26.3% and pN2 in 10.5%, whereas pN1 or pN2 stage was found in <10.0% in those with EGFR or KRAS mutations or with no mutations at all. No significant difference was found in the 2-year disease-free survival among those with gene mutations (p=0.08).ConclusionThis study highlights the frequency of PET- and CT-negative occult LN metastases in resected adenocarcinomas with ALK rearrangement. Our multivariate analysis showed that ALK rearrangements were associated with a significantly higher incidence of occult LN metastasis compared with ALK-negative adenocarcinomas.

Gene analysis could not be performed in all patients, especially in advanced non-small cell lung cancer (NSCLC). We aimed to find some clinical futures and CT or FDG-PET characteristics, which could be combined to help distinguish anaplastic lymphoma kinase (ALK) rearrangement form epidermal growth factor receptor (EGFR) mutations in treatment naïve advanced lung adenocarcinoma of Chinese patients. We retrospectively reviewed clinical and radiological characteristics of 145 patients with treatment naïve advanced lung adenocarcinoma. The one-way ANOVA, the Mann-Whitney test, chi-square test and logistic regression were used for comparison between patients with ALK rearrangement and those with EGFR mutation. Among 145 patients with advanced lung adenocarcinoma, only six patients had both ALK rearrangement and EGFR mutation, the sample size was too small to analysis. Univariate analysis revealed that patients with ALK rearrangement were younger (P=0.001) and with lower serum carcinoembryonic antigen (CEA) level (P=0.008) than those with EGFR mutation. More of tumors with ALK rearrangement were well defined (P=0.023) and have bubble lucency (P=0.026) compared with those with EGFR mutation (P=0.026). Lymphadenopathy was seen more frequently in patients with ALK rearrangement (P=0.167). Twenty-six patients received FDG-PET/CT, among this population, lesion standardized uptake values (SUV) >6.95 and lymph nodes SUVmax >6.25 were more often seen in ALK rearrangement group (P=0.011, both). In multivariate analysis, patients younger than 50 years (RR =9.878, 95% CI: 2.318-42.090, P=0.002), with lower CEA level than 4.95 µg/L (RR =8.166, 95% CI: 1.085-31.983, P=0.003) and without brain metastasis (RR =7.304, 95% CI: 1.099-48.558, P=0.040) were more likely to be ALK rearrangement than EGFR mutation. Tumor diameter less than 36 mm were prone to be EGFR mutation (RR =0.078, 95% CI: 0.017-0.356, P=0.001). Treatment naïve advanced lung adenocarcinomas with ALK rearrangement were more likely to have younger age, lower serum CEA level, larger tumor volume, well defined tumor border, and non-brain metastasis than those with EGFR mutation. Bubble lucency and higher FDG uptake of lesion and lymph nodes may help distinguish ALK rearrangement from EGFR mutation in the absence of genetic analysis.