Anaplastic Lymphoma Kinase Fusion Variants Research Articles

Survival outcomes in ALK-positive NSCLC patients (p) treated 1st line brigatinib and impact of the ALK fusion variant.

e21015 Background: There is limited data on brigatinib (a next-generation anaplastic lymphoma kinase (ALK) inhibitor) efficacy according to the ALK fusion variant. Here we investigate the prevalence of ALK fusion variants and evaluate treatment outcomes in ALK-positive NSCLC treated with first-line brigatinib. Methods: This is an open-label, non-randomized, phase II, exploratory, multi-center clinical trial. Stage IV, ALK-positive NSCLC p were treated with brigatinib 90mg for the first 7 days (D 1-7 at cycle 1) and then 180 mg daily thereafter for QW4 cycles of duration (28 days ±3days). Brigatinib was administered until loss of clinical benefit, unacceptable toxicity, p or physician decision to discontinue, or death. Response to treatment was evaluated as per RECIST v1.1. ALK rearrangement was identified locally by immunohistochemistry and confirmed by fluorescence in situ hybridization. In addition, baseline tissue samples were analyzed by NGS using the Oncomine™ Focus Assay in a centralized laboratory. Results: Between May 2020 and October 2021, 33 p were enrolled from eight different institutions. One p was considered not valid because there no tissue or plasma sample was available at baseline. Therefore, 32 p were finally considered for the study. Baseline characteristics were as follows: median age (range): 53 (35-86) years; female, n = 15 (47%); ECOG PS of 0 n = 6 (19%), 1 n = 23 (72%), 2 n = 3 (9%); never smoker, n = 14 (44%). All tumors were adenocarcinoma. The average number of metastases per p was four. In total, seven (22%) p had brain metastases. A sample was valid for NGS analysis in 29 cases. ALK fusion partners were EML4 in 28 cases (97%) and HIP1 in one case (3%). The ALK fusion variant 1 was the most frequently detected (n = 13, 45%), followed by variant 3 (n = 9; 31%), and variant 2 (n = 4; 14%). The EML4(6)-ALK(19) variant was detected in 2 cases, and variant 8 was detected in one case. The median follow-up time was 18 months. During the study, 13 deaths were recorded, and progressive disease (PD) was observed in 15 p. With a data maturity of 87%, median progression-free survival (PFS) was 16.7 months (95%CI 6.2-NR), and median overall survival (OS) was NR (95%CI 12.4-NR). There were no significant differences in survival outcomes according to the type of variant. The same was true when grouping long (variants 1,2 and 8) and short (variant 3, EML4(6) - ALK(19)) variants. The p whose tumor harbored the HIP1(21)-ALK(20) variant was alive and with and in partial response at the time of data cutoff. On the other hand, a patient whose tumor harbored the short variant EML4(6) - ALK(19) along with the KRAS G12C mutation was diagnosed with disease progression after three months of treatment initiation and deceased after eight months from the start of treatment. Conclusions: In this interim analysis we were unable to demonstrate a significant difference in survival outcomes according to variant type. Clinical trial information: NCT04223596 .

ALK fusion NSCLC oncogenes promote survival and inhibit NK cell responses via SERPINB4 expression

Anaplastic lymphoma kinase (ALK) fusion variants in Non-Small Cell Lung Cancer (NSCLC) consist of numerous dimerizing fusion partners. Retrospective investigations suggest that treatment benefit in response to ALK tyrosine kinase inhibitors (TKIs) differs dependent on the fusion variant present in the patient tumor. Therefore, understanding the oncogenic signaling networks driven by different ALK fusion variants is important. To do this, we developed controlled inducible cell models expressing either Echinoderm Microtubule Associated Protein Like 4 (EML4)-ALK-V1, EML4-ALK-V3, Kinesin Family Member 5B (KIF5B)-ALK, or TRK-fused gene (TFG)-ALK and investigated their transcriptomic and proteomic responses to ALK activity modulation together with patient-derived ALK-positive NSCLC cell lines. This allowed identification of both common and isoform-specific responses downstream of these four ALK fusions. An inflammatory signature that included upregulation of the Serpin B4 serine protease inhibitor was observed in both ALK fusion inducible and patient-derived cells. We show that Signal transducer and activator of transcription 3 (STAT3), Nuclear Factor Kappa B (NF-κB) and Activator protein 1 (AP1) are major transcriptional regulators of SERPINB4 downstream of ALK fusions. Upregulation of SERPINB4 promotes survival and inhibits natural killer cell-mediated cytotoxicity, which has potential for therapeutic impact targeting the immune response together with ALK TKIs in NSCLC.

Detecting anaplastic lymphoma kinase (ALK) gene rearrangements with next-generation sequencing remains a reliable approach in patients with non-small-cell lung cancer.

Next-generation sequencing (NGS) is rapidly becoming routine in clinical oncology practice to identify therapeutic biomarkers, including gene rearrangements in anaplastic lymphoma kinase (ALK). Our study investigated the concordance of ALK positivity evaluated by DNA-based NGS with orthogonal ALK testing methods such as fluorescence in situ hybridization (FISH), immunohistochemistry (IHC), and RNA-based NGS (RNA-NGS). Thirty-eight patients with lung adenocarcinoma who were detected with ALK rearrangements using DNA-NGS and also had adequate tissue samples submitted for FISH, IHC, and RNA-NGS, were included in this study. Of the 38 patients, RNA samples from 3 patients failed quality control for RNA-NGS. The concordance of ALK positivity was calculated relative to DNA-NGS results. The concordance rates were 97.1% (34/35) for RNA-NGS, 94.7% (36/38) for IHC, and 97.4% (37/38) for FISH. DNA-NGS detected single ALK rearrangements in 14 (35.0%) patients and complex ALK rearrangements in 26 (65.0%). RNA-NGS detected only single transcripts of the primary ALK fusions. A novel LANCL1-ALK (L7:A20) detected using DNA-NGS was detected as EML4-ALK (E13:A20) transcripts using RNA-NGS. Interestingly, patients with single ALK rearrangements were more likely to be detected with atypical isolated red signals (p < 0.001), while patients with complex ALK rearrangements were more likely to be detected with atypical split red and green signals less than 2 signal diameters apart (p < 0.001). Our study highlights the reliability of NGS in the accurate detection of specific ALK fusion variants and concomitant mutations that are crucial for individualized treatment decisions in patients with lung cancer.

ALK fusion variant 3a/b, concomitant mutations, and high PD-L1 expression were associated with unfavorable clinical response to second-generation ALK TKIs in patients with advanced ALK-rearranged non-small cell lung cancer (GASTO 1061)

ObjectivesSecond-generation anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitors (TKIs) have significantly improved clinical outcomes in patients with advanced ALK-positive non-small cell lung cancer (NSCLC), but clinical responses vary widely. In this study, the impacts of ALK fusion variants, concomitant mutations, and PD-L1 expression on the clinical response were evaluated in patients receiving second-generation ALK TKIs. Materials and methodsWe retrospectively enrolled 193 patients with ALK-rearranged advanced NSCLC who received second-generation ALK TKIs at Sun-yat Sen University Cancer Center from January 2015 to December 2020. The ALK fusion variants and concomitant mutations were identified with next-generation sequencing, while PD-L1 expression was assessed by immunohistochemistry. ResultsThe median progression-free survival (PFS) was significantly shorter for variant 3a/b than for other variants (9.93 months vs 16.97 months, HR 1.941, P = 0.0014). Baseline concomitant mutations were significantly associated with shorter PFS while on ALK TKIs (median PFS, 10.87 months vs 22.47 months, HR 1.984, P = 0.002). A subset of 68 patients was analyzed for PD-L1 expression: TPS 0% in 32.4% (22/68) of the patients, 1–49% in 30.9% (21/68) of the patients, and ≥ 50% in 36.7% (25/68) of the patients. Expression of PD-L1 was significantly associated with variant 3a/b and concomitant mutations. Median PFS was shorter in patients with high PD-L1 expression (median PFS in patients with PD-L1 TPS of 0% vs 1–49% vs ≥ 50% were 27.43 months vs 30.63 months vs 9.50 months, respectively, P = 0.001). In multivariate analysis, PD-L1 expression (TPS ≥ 50%), concomitant mutations, and variant 3a/b remained negative prognostic factors for the clinical efficacy of second-generation ALK TKIs in ALK-rearranged non-small cell lung cancer. ConclusionALK fusion variant 3a/b, concomitant mutations, and high PD-L1 expression were associated with unfavorable clinical response to second-generation TKIs in ALK-rearranged NSCLC.

Concomitant mutation status of ALK-rearranged non-small cell lung cancers and its prognostic impact on patients treated with crizotinib.

BackgroundIn non-small cell lung cancer (NSCLC), anaplastic lymphoma kinase (ALK) rearrangement characterizes a subgroup of patients who show sensitivity to ALK tyrosine kinase inhibitors (TKIs). However, the prognoses of these patients are heterogeneous. A better understanding of the genomic alterations occurring in these tumors could explain the prognostic heterogeneity observed in these patients.MethodsWe retrospectively analyzed 96 patients with NSCLC with ALK detected by immunohistochemical staining (VENTANA anti-ALK(D5F3) Rabbit Monoclonal Primary Antibody). Cancer tissues were subjected to next-generation sequencing using a panel of 520 cancer-related genes. The genomic landscape, distribution of ALK fusion variants, and clinicopathological characteristics of the patients were evaluated. The correlations of genomic alterations with clinical outcomes were also assessed.ResultsAmong the 96 patients with immunohistochemically identified ALK fusions, 80 (83%) were confirmed by next-generation sequencing. TP53 mutation was the most commonly co-occurring mutation with ALK rearrangement. Concomitant driver mutations [2 Kirsten rat sarcoma viral oncogene homolog (KRAS) G12, 1 epidermal growth factor receptor (EGFR) 19del, and 1 MET exon 14 skipping] were also observed in 4 adenocarcinomas. Echinoderm microtubule associated protein-like 4 (EML4)-ALK fusions were identified in 95% of ALK-rearranged patients, with 16.2% of them also harboring additional non–EML4-ALK fusions. Nineteen non-EML4 translocation partners were also discovered, including 10 novel ones. Survival analyses revealed that patients concurrently harboring PIK3R2 alterations showed a trend toward shorter progression-free survival (6 vs. 13 months, P=0.064) and significantly shorter overall survival (11 vs. 32 months, P=0.004) than did PIK3R2-wild-type patients. Patients with concomitant alterations in PI3K the signaling pathway also had a shorter median overall survival than those without such alterations (23 vs. 32 months, P=0.014), whereas progression-free survival did not differ significantly.ConclusionsThe spectrum of ALK-fusion variants and the landscape of concomitant genomic alterations were delineated in 96 NSCLC patients. Our study also demonstrated the prognostic value of concomitant alterations in crizotinib-treated patients, which could facilitate improved stratification of ALK-rearranged NSCLC patients in the selection of candidates who could optimally benefit from therapy.

Distribution of EML4-ALK fusion variants and clinical outcomes in patients with resected non-small cell lung cancer

ObjectivesThe molecular profiles and prognosis of anaplastic lymphoma kinase (ALK) fusion and resectable non-small cell lung cancer (NSCLC) remain unclear. This study aimed to explore the distribution of ALK fusion variants and prognostic factors in patients with surgically resected NSCLC. Material and methodsAmong the 93 ALK positive surgical patients screened by immunohistochemistry (IHC) or real-time polymerase chain reaction (RT-PCR), 63 patients were confirmed as ALK rearrangement by next-generation sequencing (NGS), including 55 cases of stage I-III and 8 cases of stage IV. Medical records were retrospectively reviewed, the distribution of ALK fusion variants and prognostic factors were analyzed. ResultsAll of the 55 early stage patients were histological adenocarcinoma. No other fusion types were found except for echinoderm microtubule-associated protein-like 4- anaplastic lymphoma kinase (EML4-ALK). EML4-ALK variant 1 (E13:A20; 25/55, 45.5 %) was the predominant variant type, followed by EML4-ALK variant 3 (E6:A20; 19/55, 34.5 %) and variant 2 (E20:A20; 8/55, 14.5 %). Concomitant mutations occurred in 22 patients (22/55, 40.0 %), which involved in 32 co-mutations from 12 kinds of mutated genes. TP53 mutations were most common in coexisting mutations (13/32, 40.6 %). TP53 mutations were less frequently occurred in variant 1 group (3/25, 12.0 %) than in non-variant 1 group (10/30, 33.3 %, P = 0.064). The median disease-free survival (DFS) of the 55 patients was 22.1 months, and the median overall survival (OS) was not mature at the time of analysis. Multivariable analysis showed that stage T3 and EML4-ALK variant 3 were independent prognostic factors for shorter DFS. Neither TP53 mutations nor any coexisting mutations were related to prognosis. ConclusionsThis study illustrated the patterns of EML4-ALK fusion variants and gene profiles in patients with resected NSCLC. Advanced T stage and EML4-ALK variant 3 were associated with worse prognosis. The role of TP53 mutations in prognosis is worthy of further study.

Association of Programmed Death-Ligand 1 Expression with Fusion Variants and Clinical Outcomes in Patients with Anaplastic Lymphoma Kinase-Positive Lung Adenocarcinoma Receiving Crizotinib.

Programmed death-ligand 1 (PD-L1) expression is associated with clinical outcomes of epidermal growth factor receptor (EGFR) mutant lung adenocarcinoma (ADC) treated with tyrosine kinase inhibitors (TKIs). However, whether PD-L1 expression plays a role in anaplastic lymphoma kinase (ALK)-positive lung ADC is unknown. We aimed to evaluate the impact of PD-L1 in patients with ALK-positive lung ADC receiving crizotinib. PD-L1 expression was identified by immunohistochemistry (IHC). Reverse transcriptase-polymerase chain reaction was used for ALK variant detection, and immunofluorescence-based multiplex staining was applied for exploring immune cells in tumor microenvironments. A total of 78 patients with ALK-positive advanced ADC were enrolled in our study, of whom 52 received crizotinib. Compared with EGFR/ALK wild-type tumors, PD-L1 expression was lower in ALK-positive ADC. ALK fusion variants were identified in 32 patients, and those with variant 3 and 5 (short variants) had higher PD-L1 expression than those with other variants. The crizotinib objective response rate (ORR) and progression-free survival (PFS) was better in tumors with negative PD-L1 expression (ORR/PFS in PD-L1 0% vs. 1%-49% vs. 50%-100%: 60.7%/11.8 months vs. 38.5%/6.5 months vs. 36.4%/4.0 months, p = .007/.022). The multivariate Cox proportional hazards model revealed that PD-L1 0% (vs. ≥1%) was an independent factor for longer PFS (adjusted hazard ratio 0.322, 95% confidence interval 0.160-0.650, p = .002). Multiplex IHC in three cases showed a varied extent of immune cell infiltrations in tumors with different PD-L1 expression. Positive PD-L1 expression was associated with unfavorable clinical outcomes in patients with ALK-positive lung ADC receiving crizotinib. Not all lung adenocarcinoma with sensitizing driver mutations experienced durable responses to small-molecule tyrosine kinase inhibitors (TKIs). Similar to the negative impact of programmed death-ligand 1 (PD-L1) in epidermal growth factor receptor mutant tumors treated with TKIs, this study demonstrated that positive PD-L1 expression was also associated with worse response rate and shorter progression-free survival of anaplastic lymphoma kinase (ALK)-positive adenocarcinoma treated with crizotinib. Among different ALK fusion partners, tumors with short variants (V3 and V5) had higher PD-L1 compared with long variants (V1, V2, and V6). Testing PD-L1 before initiating crizotinib for ALK-positive lung cancer could be a simple method to provide important prognostic information.

The Impact of Clinical Factors, ALK Fusion Variants, and BIM Polymorphism on Crizotinib-Treated Advanced EML4-ALK Rearranged Non-small Cell Lung Cancer.

Patients' clinical factors and genetics factors such as anaplastic lymphoma kinase (ALK) fusion variants and BIM (Bcl-2-like 11) polymorphism were reported to be associated with clinical outcome in crizotinib-treated advanced non-small cell lung cancer (NSCLC). However, the results were still controversial. We analyzed outcome of 54 patients with known ALK fusion variants who received crizotinib for advanced NSCLC. Thirty of them had successful BIM polymorphism analysis and 6 (20%) had a BIM deletion. Multivariate Cox regression analysis found that previous anticancer therapy [adjusted hazard ratio (aHR) 1.35, 95% confidence interval (CI), 1.04–1.76 for each additional line of therapy, p = 0.025] and Eastern Cooperative Oncology Group (ECOG) performance status ≥2 (aHR 8.35, 95% CI, 1.52–45.94, p = 0.015) were independent factors for progression-free survival (PFS). Only ECOG performance status ≥2 (aHR 7.20, 95% CI, 1.27–40.79, p = 0.026) was an independent factor for overall survival (OS). Neither ALK fusion variants nor the presence of a BIM deletion was associated with crizotinib PFS or OS. After adjusting with clinical factors, different ALK variants and BIM polymorphism might not be independent factors for crizotinib PFS or OS in advanced NSCLC with ALK rearrangement.

Long-term complete response in a patient with postoperative recurrent ALK-rearranged lung adenocarcinoma treated with crizotinib: A case report.

Anaplastic lymphoma kinase (ALK) gene rearrangements are identified in approximately 5% of patients with non-small cell lung cancer (NSCLC). Despite initial dramatic responses to ALK inhibitors, the majority of patients relapse within 1 year, owing to the development of resistance. Herein we present a case of variant type 2 ALK-rearranged lung adenocarcinoma recurrence with multiple lung metastasis that maintained complete response over 5 years with crizotinib, which is the first approved ALK inhibitor. The efficacy of crizotinib may vary among ALK fusion variants and thus, variant type may represent an important factor in guiding the treatment strategy for ALK-rearranged lung adenocarcinoma.

ALK Fusion Partners Impact Response to ALK Inhibition: Differential Effects on Sensitivity, Cellular Phenotypes, and Biochemical Properties.

Oncogenic tyrosine kinase fusions involving the anaplastic lymphoma kinase (ALK) are detected in numerous tumor types. Although more than 30 distinct 5' fusion partner genes have been reported, treatment of ALK-rearranged cancers is decided without regard to which 5' partner is present. There is little data addressing how the 5' partner affects the biology of the fusion or responsiveness to ALK tyrosine kinase inhibitors (TKI). On the basis of the hypothesis that the 5' partner influences the intrinsic properties of the fusion protein, cellular functions that impact oncogenic potential, and sensitivity to ALK TKIs, clonal 3T3 cell lines stably expressing seven different ALK fusion variants were generated. Biochemical and cellular assays were used to assess the efficacy of various ALK TKIs in clinical use, transformative phenotypes, and biochemical properties of each fusion. All seven ALK fusions induced focus formation and colonies in soft agar, albeit to varying degrees. IC50s were calculated for different ALK TKIs (crizotinib, ensartinib, alectinib, lorlatinib) and consistent differences (5-10 fold) in drug sensitivity were noted across the seven ALK fusions tested. Finally, biochemical analyses revealed negative correlations between kinase activity and protein stability. These results demonstrate that the 5' fusion partner plays an important biological role that affects sensitivity to ALK TKIs.Implications: This study shows that the 5' ALK fusion partner influences ALK TKI drug sensitivity. As many other kinase fusions are found in numerous cancers, often with overlapping fusion partners, these studies have ramifications for other kinase-driven malignancies. Mol Cancer Res; 16(11); 1724-36. ©2018 AACR.

526P - Correlation between the response to critzotinib and concomitant genetic alterations in advanced lung cancer patients with ALK rearrangement

Background: The fusion gene echinoderm microtubule-associated protein-like 4 (EML4) - anaplastic lymphoma kinase (ALK) was found to be a driver mutation in lung cancer in 2007. Crizotinib can significantly prolong a patient’s survival and improve the quality of life in patients harboring ALK fusion gene. Methods: The specimens enrolled in the study underwent next generation sequencing. The types of ALK fusion gene and contaminant mutations were identified and their correlation with crizotinib efficacy was explored. Results: 197 patients with advanced ALK-positive lung cancer received crizotinib treatment at Peking Union Medical College Hospital and Peking University Cancer Hospital from January 2013 to December 2017. After excluding cases with incomplete medical records and inadequate tissue samples, 16 cases were enrolled in the study and the specimen underwent next-generation sequencing. The main ALK fusion type was V1 fusion (37.5%); followed by V3 fusion (31.25%). No difference was found in clinical characteristics and crizotinib efficacy between patients with different types of ALK fusion genes. Patients with shorter progression free survival (PFS) have a larger number of genetic mutations (p = 0.048). According to the mutation types, three mutational signatures were identified, namely, signature A, B and C. Patients with mutation signature A have a prolonged PFS (p = 0.026). Conclusions: No association between crizotinib efficacy and types of ALK fusion variant was found. However, crizotinib efficacy was associated with mutation signature and mutation number. Legal entity responsible for the study: Peking Union Medical College Hospital. Funding: Has not received any funding. Disclosure: All authors have declared no conflicts of interest.

Identification of a highly lethal V3+ TP53+ subset in ALK+ lung adenocarcinoma.

Tyrosine kinase inhibitors (TKI) have improved prognosis in metastatic anaplastic lymphoma kinase (ALK)-driven lung adenocarcinoma, but patient outcomes vary widely. We retrospectively analyzed the clinical course of all cases with assessable baseline TP53 status and/or ALK fusion variant treated at our institutions (n = 102). TP53 mutations were present in 17/87 (20%) and the echinoderm microtubule-associated protein-like 4 (EML4)-ALK variant 3 (V3) in 41/92 (45%) patients. The number of metastatic sites at diagnosis was affected more by the presence of V3 than by TP53 mutations, and highest with both factors (mean 5.3, p < 0.001). Under treatment with ALK TKI, progression-free survival (PFS) was shorter with either TP53 mutations or V3, while double positive cases appeared to have an even higher risk (hazard ratio [HR] = 2.9, p = 0.015). The negative effect of V3 on PFS of TKI-treated patients was strong already in the first line (HR = 2.5, p = 0.037) and decreased subsequently, whereas a trend for PFS impairment under first-line TKI by TP53 mutations became stronger and statistically significant only when considering all treatment lines together. Overall survival was impaired more by TP53 mutations (HR = 4.9, p = 0.003) than by V3 (HR = 2.4, p = 0.018), while patients with TP53 mutated V3-driven tumors carried the highest risk of death (HR = 9.1, p = 0.02). Thus, TP53 mutations and V3 are independently associated with enhanced metastatic spread, shorter TKI responses and inferior overall survival in ALK+ lung adenocarcinoma. Both markers could assist selection of cases for more aggressive management and guide development of novel therapeutic strategies. In combination, they define a patient subset with very poor outcome.

Cost Saving Opportunities in NSCLC Therapy by Optimized Diagnostics.

With an incidence of 68 new cases per 100,000 people per year, an estimated total number of up to 350,000 new non-small-cell lung cancer (NSCLC) cases are diagnosed each year in the European Union. Up to 10% of NSCLC patients are eligible for therapy with novel ALK (anaplastic lymphoma kinase) inhibitors, as they have been diagnosed with a mutation in the gene coding for ALK. The ALK inhibitor therapy costs add up to approx. 9000 € per patient per month, with treatment durations of up to one year. Recent studies have shown that up to 10% of ALK cases are misdiagnosed by nearly 40% of pathologic investigations. The current state-of-the-art ALK diagnostic procedure comprises a Fluorescent in situ Hybridization (FISH) assay accompanied by ALK inhibitor therapy (Crizotinib). The therapy success ranges between a full therapy failure and the complete remission of the tumor (i.e., healing), but the biomedical and systemic reasons for this range remain unknown so far. It appears that the variety of different ALK mutations and variants contributes to the discrepancy in therapy results. Although the major known fusion partner for ALK in NSCLC is the Echinoderm microtubule-associated protein-like 4 (EML4), of which a minimum of 15 variants have been described, an additional 20 further ALK fusion variants with other genes are known, of which three have already been found in NSCLC. We hypothesize that the wide variety of known (and unknown) ALK mutations is associated with a variable therapy success, thus rendering current companion diagnostic procedures (FISH) and therapy (Crizotinib) only partly applicable in ALK-related NSCLC treatment. In cell culture, differing sensitivity to Crizotinib has been shown for some fusion variants, but it is as yet unknown which of them are really biologically active in cancer patients, and how the respective variants affect the response to Crizotinib treatment. Moreover, it has been demonstrated that translocated ALK genes can also be observed in healthy tissues and are not compulsorily associated with tumors. Therefore, it is important to keep in mind that even for the known variants of ALK fusion genes, the biological function is not known for all variants, and that no information is available on the homogeneity of ALK fusion variants within a single tumor. These facts, in concert with data for ALK mutation prevalence and therapy outcomes of a German cohort of NSCLC patients, support the hypothesis that, by using novel companion diagnostic tools in combination with therapy outcome predictions, massive cost savings could be possible in European Health Care systems without a loss of patient care.

Abstract 3164: A comprehensive analysis of autopsied specimens and patient-derived cell lines in ALK-positive lung cancers with rapid acquired resistance to alectinib

Abstract Background Alectinib, a highly selective anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitor (TKI), demonstrated a stunning disease control rate (95.7%) and unprecedented median progression-free survival (not reached: 95% CI: 20.3-) (Lancet Oncology 2013, ASCO 2016, Abstract #9008). However, acquired resistance to alectinib is inevitable; some ALK-positive lung tumors rapidly acquire alectinib resistance. Therefore, we sought to investigate the mechanism of the rapid resistance to alectinib using clinical samples and patient-derived ALK-positive cell lines. Materials and Methods Autopsied samples (primary lung tumors and metastatic tumors from the esophagus, liver, and bilateral kidney) were obtained from a 51-year-old male with advanced ALK-positive lung cancer. The lung tumors acquired resistance to first-line treatment of alectinib in three months, and subsequent treatment with ceritinib or crizotinib showed only a moderate and temporary effect. We established novel ALK-positive cell lines; ABC-14 was established from a pleural effusion after alectinib treatment, and ABC-17 was established from patient-derived xenograft (PDX) tumors of autopsied liver metastases. Western blot analysis, immunostaining, a receptor tyrosine kinase assay, fluorescence in situ hybridization (FISH), and next-generation sequencing were performed. Results ALK overexpression was confirmed in all autopsied samples by immunostaining. PCR revealed that ABC-14 harbored the EML-4/ALK fusion variant 3. An in vitro cell proliferation assay showed that ABC-14 exhibited resistance to alectinib (IC50 &amp;gt; 1 µM), but was sensitive to crizotinib (IC50 = 98 nM). The receptor tyrosine kinase assay revealed the activation of MET and EGFR in ABC-14. Quantitative RT-PCR and FISH confirmed MET gene amplification. Quantitative RT-PCR also indicated the overexpression of an EGFR ligand, EGF, TGF-α but no EGFR mRNA overexpression. The combination of crizotinib (dual ALK/MET-TKI) and an EGFR-TKI, gefitinib, showed an additive inhibitory effect on cell growth compared with each drug alone in vitro. ABC-17 showed resistance to both alectinib and crizotinib; consistently showed no activation of MET and no MET gene amplification. Interestingly, ABC-17 showed metastatic ability in the lung in NOG mouse PDX models. Conclusion The mechanism of rapid resistance to alectinib may be complicated and heterogeneous. Crizotinib combined with gefitinib, which inhibits the ALK, EGFR, and MET pathways, may represent one potent strategy against alectinib resistance. Further next-generation sequencing of clinically relevant samples should provide deeper insights into its resistance. (This work was supported by KAKEN 16K19454 and KAKEN 15H04830.) Citation Format: Go Makimoto, Kadoaki Ohashi, Kazuya Nishii, Shuta Tomida, Hiroe Kayatani, Tomoki Tamura, Hisao Higo, Kiichiro Ninomiya, Takashi Ninomiya, Toshio Kubo, Eiki Ichihara, Akiko Sato, Katsuyuki Hotta, Masahiro Tabata, Katsuyuki Kiura. A comprehensive analysis of autopsied specimens and patient-derived cell lines in ALK-positive lung cancers with rapid acquired resistance to alectinib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3164. doi:10.1158/1538-7445.AM2017-3164

Abstract 4648: Development of a rapid, precise, and sensitive molecular assay for ALK fusion detection

Abstract Introduction In non-small cell lung cancer (NSCLC), chromosomal translocation events that result in overactivation of the anaplastic lymphoma kinase (ALK) have been identified and shown to play a key role in tumorigenesis. Inhibitors of the ALK kinase, such as the FDA-approved crizotinib, are effective in treating ALK-fusion-positive non-small cell lung cancer. Molecular studies have identified many ALK fusion variants in various cancer types, encompassing a combination of 22 different gene partners as well as various break points within the partner genes. The most common fusion events in non-small cell lung cancer are between the echinoderm microtubule associated protein like 4 (EML4) gene and the ALK gene. Fusion events between the Kinesin Family Member 5B (KIF5B) gene and ALK have also been identified. Existing assays for ALK fusion detection include the time-consuming, costly, and challenging fluorescence in situ hybridization (FISH) method, which is the current gold standard. Next generation sequencing approaches are also available, which are also costly to implement, difficult to interpret, and have lengthy workflows. Reverse-transcriptase PCR methods are ideal, as they are cost-effective, sensitive, rapid, and require little interpretation expertise. Methodology We designed a multiplexed real-time RT-PCR method to enable indiscriminate detection of ten EML4-ALK and three KIF5B-ALK fusion variants (encompassing approximately 95% of EML4-ALK and 90% of KIF5B-ALK characterized fusion variants, based on the Catalogue of Somatic Mutations in Cancer (COSMIC) database). The assay uses standard real-time PCR instrumentation and was characterized using the Bio-Rad CFX384™, Roche LightCycler® 96, and Roche LightCycler® 480 II instruments. Assay preparation and run time requires 2 hours or less. Sensitivity testing showed that for the Roche LightCycler® 480 II and Bio-Rad CFX384™, 100% detection was achieved for 50 copies of fusion template for each of the 13 targeted variants. The Roche LightCycler® 96 showed slightly poorer sensitivity, with 100% detection observed at 50 copies for 9 fusion variants and 100 copies for the remaining four variants. Inter- and intra-run precision testing demonstrated good reproducibility and repeatability, with a coefficient of variation of less than 10% for all targeted fusion variants. Using ALK-positive and ALK-negative FFPE reference materials, it was demonstrated that 50 ng of total FFPE RNA was sufficient and up to approximately 200 ng showed good performance. Conclusion In summary, we have developed a rapid, precise, and sensitive assay for the detection of thirteen EML4-ALK and KIF5B-ALK fusion events to enable molecular characterization of non-small cell lung cancer tumors. Citation Format: Mona D. Shahbazian, Yulei Shang, Maidar Jamba, Michael J. Powell. Development of a rapid, precise, and sensitive molecular assay for ALK fusion detection [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4648. doi:10.1158/1538-7445.AM2017-4648

Differential protein stability and clinical responses ofEML4-ALK fusion variants to various ALK inhibitors in advancedALK-rearranged non-small cell lung cancer

Anaplastic lymphoma kinase (ALK) inhibition using crizotinib has become the standard of care in advanced ALK-rearranged non-small cell lung cancer (NSCLC), but the treatment outcomes and duration of response vary widely. Echinoderm microtubule-associated protein-like 4 (EML4)-ALK is the most common translocation, and the fusion variants show different sensitivity to crizotinib in vitro. However, there are only limited data on the specific EML4-ALK variants and clinical responses of patients to various ALK inhibitors. By multiplex reverse-transcriptase PCR, which detects 12 variants of known EML4-ALK rearrangements, we retrospectively determined ALK fusion variants in 54 advanced ALK rearrangement-positive NSCLCs. We subdivided the patients into two groups (variants 1/2/others and variants 3a/b) by protein stability and evaluated correlations of the variant status with clinical responses to crizotinib, alectinib, or ceritinib. Moreover, we established the EML4-ALK variant-expressing system and analyzed patterns of sensitivity of the variants to ALK inhibitors. Of the 54 tumors analyzed, EML4-ALK variants 3a/b (44.4%) was the most common type, followed by variants 1 (33.3%) and 2 (11.1%). The 2-year progression-free survival (PFS) rate was 76.0% [95% confidence interval (CI) 56.8-100] in group EML4-ALK variants 1/2/others versus 26.4% (95% CI 10.5-66.6) in group variants 3a/b (P = 0.034) among crizotinib-treated patients. Meanwhile, the 2-year PFS rate was 69.0% (95% CI 49.9-95.4) in group variants 1/2/others versus 32.7% (95% CI 15.6-68.4) in group variants 3a/b (P = 0.108) among all crizotinib-, alectinib-, and ceritinib-treated patients. Variant 3a- or 5a-harboring cells were resistant to ALK inhibitors with >10-fold higher half maximal inhibitory concentration in vitro. Our findings show that group EML4-ALK variants 3a/b may be a major source of ALK inhibitor resistance in the clinic. The variant-specific genotype of the EML4-ALK fusion allows for more precise stratification of patients with advanced NSCLC.

Clinical outcomes in ALK-rearranged lung adenocarcinomas according to ALK fusion variants.

BackgroundClinical outcomes of anaplastic lymphoma kinase (ALK)-rearranged non-small cell lung cancer according to ALK fusion variants are not clear. We aimed to investigate the prevalence of ALK fusion variants and to compare clinical outcomes according to ALK fusion variants.MethodsA retrospective analysis was conducted on patients with advanced ALK-rearranged adenocarcinoma treated with chemotherapy and ALK inhibitors. ALK rearrangement was identified by fluorescence in situ hybridization and confirmed by immunohistochemistry. Peptide nucleic acid-mediated quantitative polymerase chain reaction assays, designed to detect 28 types of echinoderm microtubule-associated protein-like 4 (EML)-ALK rearrangements, were performed. Clinicopathological analysis and treatment outcomes with platinum-based chemotherapy, pemetrexed therapy, and ALK inhibitors—including crizotinib and ceritinib—were evaluated.ResultsA total of 52 patients with ALK-rearranged lung adenocarcinoma were enrolled. EML4-ALK variant 1 (v1) was the most common variant (38.5 %) followed by the non-EML4 variant (36.5 %), EML4-ALK variant 3a/b (19.2 %), and EML4-ALK variant 2 (5.8 %). No clinicopathological distinction was found between the different ALK fusion variants. Treatment response rates for each therapeutic agent did not differ according to ALK fusion variant. However, EML4 variants, especially v1, showed significantly longer progression-free survival (PFS) on pemetrexed treatment than did non-EML4 variants (median 31.1 months versus 5.7 months, P = 0.003). PFS with platinum-based chemotherapy and ALK inhibitors did not differ according to ALK fusion variant. Multivariate survival analysis using Cox’s regression model revealed v1 as the only predictive factor for prolonged PFS on pemetrexed.ConclusionsAmong ALK fusion variants, v1 is the most common subtype. It showed superior progression-free survival on pemetrexed than did non-EML4 variants. No survival difference was demonstrated between variants treated with crizotinib or ceritinib.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-016-1061-z) contains supplementary material, which is available to authorized users.

Efficacy of crizotinib in ALK fusion variants

In 1995, rearrangement of the anaplastic lymphoma kinase ( ALK ) gene was first reported as a fusion gene in anaplastic large cell non-Hodgkin lymphoma, and in 2005 it was again on focus when Mano et al. reported that echinoderm microtubule-associated protein-like4 (EML4)-ALK fusion gene is a powerful oncogene in lung cancer (1,2).

Abstract 1144: Detection of a novel ALK fusion variant in lung adenocarcinoma using a comprehensive genomic analysis

Abstract Background: Anaplastic lymphoma kinase (ALK) is a tyrosine kinase that is constitutively activated in a subset of non-small cell lung cancer (NSCLC), mainly as a result of chromosomal translocation. In ALK-rearranged NSCLC, the growth of tumors is strongly addicted to ALK activity, and suppression of ALK is a potent therapeutic strategy. To date, 6 known fusion partners of ALK onco-kinase have been reported in NSCLC, although the sensitivity to ALK inhibitor according to fusion variant except for EML4-ALK is still unclear. In this study, we reviewed clinical trial samples analyzed by comprehensive genomic profiling approach to detect unknown actionable ALK rearrangements. Materials and methods: We reviewed gene alteration and expression profile of clinical samples in preliminary molecular studies from the ongoing BATTLE-2, phase II clinical trial of targeted drugs in chemo-refractory NSCLC. Results: In 187 NSCLC samples, 3 samples harbored ALK fusion gene: 2 EML4-ALK and 1 novel ALK fusion variant PRKAR1A-ALK in a lung adenocarcinoma of male former smoker patient. The ALK gene expression levels of ALK-rearranged samples were significantly higher compared to other samples. Interestingly, the patient harboring PRKAR1A-ALK was treated with ALK inhibitor crizotinib and showed long time stable disease. Conclusions: Our findings suggest that PRKAR1A-ALK fusion could be a novel oncogenic driver in lung adenocarcinoma and an indicator of ALK inhibitor response. (NHI-NCI CA155196; 2P50CA070907-16A1) Citation Format: Kazuhiko Shien, Dennis Ruder, Ecaterina E. Ileana, Vassiliki A. Papadimitrakopoulou, Garrett M. Frampton, Carmen Behrens, Neda Kalhor, J. Jack Lee, Ximing Tang, Roy S. Herbst, Ignacio I. Wistuba, Julie G. Izzo. Detection of a novel ALK fusion variant in lung adenocarcinoma using a comprehensive genomic analysis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1144.

Differential Crizotinib Response Duration Among ALK Fusion Variants in ALK-Positive Non-Small-Cell Lung Cancer.

Anaplastic lymphoma kinase (ALK) rearrangement-positive non-small-cell lung cancers can be effectively treated with an ALK tyrosine kinase inhibitor (TKI) such as crizotinib, but the response magnitude and duration are heterogeneous. Several ALK variants have been identified, but few studies have focused on the effects of different ALK variants on the efficacy of crizotinib. Among 55 patients treated with crizotinib as the initial ALK-TKI between January 2007 and December 2014, we identified 35 patients with tumor specimens that could be evaluated for ALK variants by reverse transcription polymerase chain reaction. We retrospectively evaluated the efficacy of crizotinib on the basis of the objective response rate and progression-free survival (PFS) according to the ALK variants. The most frequent ALK variant was variant 1 in 19 patients (54%), followed by variant 2 in five patients (14%), variant 3a/3b in four patients (12%), and other variants in seven patients (20%). Objective response rate was 69% in all patients, whereas it was 74% and 63% in the variant 1 and non-variant 1 groups, respectively. The median PFS time was significantly longer in patients with variant 1 than in those with non-variant 1 (median PFS, 11.0 months [95% CI, 6.5 to 43.0 months] v 4.2 months [95% CI, 1.6 to 10.2 months], respectively; P < .05). Multivariable analysis identified two significant factors associated with PFS duration, ALK variant 1 (hazard ratio, 0.350; 95% CI, 0.128 to 0.929; P < .05) and advanced stage (hazard ratio, 4.646; 95% CI, 1.381 to 21.750; P < .05). Our results indicate the better efficacy of crizotinib in patients with ALK variant 1 versus non-variant 1. The ALK variant status might affect the efficacy of ALK-TKIs.