Enteral administration of alectinib for ALK-positive non-small cell lung cancer in an elderly patient: A case report.

RATIONALE: Alectinib is a tyrosine kinase inhibitor (TKI) approved for use as first-line metastatic therapy for patients with anaplastic lymphoma kinase-rearranged non-small cell lung cancer. Certain medical conditions related to the tumor lesions may not allow oral administration of TKIs. PATIENT CONCERNS: We hereby report the case of a 90-year-old patient with anaplasic lymphoma kinase-rearranged lung cancer with severely impaired general condition and swallowing disorders. DIAGNOSIS: A thoracic computerized tomography (CT)-scan confirmed the presence of a mediastinal tumor lesion explaining the swallowing disorders secondary to recurrent paralysis. INTERVENTIONS: As no oral administration was feasible, alectinib was administered by percutaneous gastrostomy. OUTCOMES: The patient had few side-effects. He presented a major clinical and radiological response. After 2 months of treatment with alectinib, his mini-mental state examination had increased from 8/30 to 23/30. He had a 60% reduction in targeted pulmonary, bone and node lesions according to the Response Evaluation Criteria in Solid Tumors version 1.1 (RECIST 1.1). After 6 months of treatment, the patient's performance status had evolved from 3 to 1. This improvement in general condition made it possible to remove the feeding tube. LESSONS: In cases of lung cancer with oncogenic addiction, enteral administration of TKIs should be considered for elderly patients with an impaired general condition.

Identification of novel ALK rearrangements in gynecologic clear cell carcinoma.

Clear cell carcinomas (CCCs) of the gynecologic tract are aggressive tumors with high resistance rate to conventional platinum-based chemotherapies. Currently, the molecular features of these tumors remain largely unknown and there is no targeted therapy available. The aim of our study was to identify anaplastic lymphoma kinase (ALK) translocations, a potential molecular target for therapy. Ninety-seven patients with gynecologic CCC (62 ovarian, 27 uterine corpus and 8 uterine cervical) were screened for ALK rearrangement and ALK copy number gain using an ALK break-apart fluorescence in situ hybridization probe. The genomic landscape of all cases with ALK rearrangements and 10 random cases with ALK copy number gain was queried using a hybrid capture-based DNA next-generation sequencing assay and an Illumina Fusion RNA assay. Findings were then correlated with ALK immunohistochemistry (clone D5F3) expression. ALK rearrangement was detected in 5% (5/97) and ALK copy number gain in 79% (77/97) of gynecologic CCCs. Next-generation sequencing in ALK-rearranged CCCs identified a novel BABAM2-ALK fusion in one case. ALK translocation partners were not identified in the remaining cases. Our findings show that ALK fusion, which is targetable in other cancers, may be a pathogenetic mechanism in a small number of gynecologic CCCs.

ALK-transformed mature T lymphocytes restore early thymus progenitor features.

Anaplastic large cell lymphoma (ALCL) is a mature T cell neoplasm that often expresses the CD4+ T cell surface marker. It usually harbors the t(2;5) (p23;q35) translocation, leading to the ectopic expression of NPM-ALK, a chimeric tyrosine kinase. We demonstrated that in vitro transduction of normal human CD4+ T lymphocytes with NPM-ALK results in their immortalization and malignant transformation. The tumor cells displayed morphological and immunophenotypical characteristics of primary patient-derived anaplastic large cell lymphomas. Cell growth, proliferation, and survival were strictly dependent on NPM-ALK activity and include activation of the key factors STAT3 and DNMT1 and expression of CD30 (the hallmark of anaplastic large-cell lymphoma). Implantation of NPM-ALK-transformed CD4+ T lymphocytes into immunodeficient mice resulted in the formation of tumors indistinguishable from patients' anaplastic large cell lymphomas. Integration of "Omic" data revealed that NPM-ALK-transformed CD4+ T lymphocytes and primary NPM-ALK+ ALCL biopsies share similarities with early T cell precursors. Of note, these NPM-ALK+ lymphoma cells overexpress stem cell regulators (OCT4, SOX2, and NANOG) and HIF2A, which is known to affect hematopoietic precursor differentiation and NPM-ALK+ cell growth. Altogether, for the first time our findings suggest that NPM-ALK could restore progenitor-like features in mature CD30+ peripheral CD4+ T cells, in keeping with a thymic progenitor-like pattern.

Lenalidomide demonstrates clinical activity in anaplastic lymphoma kinase-positive large B-cell lymphoma.

Anaplastic lymphoma kinase-positive large B-cell lymphoma (ALK+ LBCL) is known to be a rare and aggressive form of lymphoma that relapses quickly after both conventional chemotherapy and more targeted therapy. Lenalidomide is an immunomodulator that has shown safety and efficacy in multiple myeloma and is also approved for use in several types of lymphoma. In the case described here, the patient had a significant partial response to lenalidomide, which has not previously been described in this type of lymphoma. Given how aggressive and difficult to treat ALK+ LBCL is, further research is warranted to more completely elucidate the mechanism of action of lenalidomide in ALK+ LBCL and its role in treatment.

Synergistic Effect of Alectinib and Everolimus on ALK-positive Anaplastic Large Cell Lymphoma Growth Inhibition.

BACKGROUND/AIM: The aim of the study was to examine the efficacy of the combination of anaplastic lymphoma kinase (ALK) inhibitors with other inhibitors for the treatment of ALK-positive lymphomas. This approach is predicted to be an alternative way for suppressing ALK-positive anaplastic large cell lymphoma (ALCL). MATERIALS AND METHODS: We treated ALK-positive ALCL cell lines, KARPAS-299 and SU-DHL-1, with the ALK inhibitor alectinib and the mammalian target of rapamycin (mTOR) inhibitor everolimus. RESULTS: The ALK inhibitor alectinib had a selective ALK-dependent inhibitory effect on ALK-positive cancer cell proliferation. Treatment with alectinib or everolimus inhibited target molecules, and their combination augmented their inhibitory effect on the mTOR pathway. Furthermore, the combination treatment significantly inhibited cell proliferation and induced cell cycle arrest at the G0/G1 phase in ALK-positive ALCL cells. CONCLUSION: The combination of both inhibitors synergistically inhibits ALK-positive ALCL cell growth via the ALK/mTOR pathway.

Crizotinib-Associated Renal Cysts in Anaplastic Lymphoma Kinase-Positive Lung Cancer Patients: A Single-Center Experience.

Crizotinib is an anaplastic lymphoma kinase (ALK) inhibitor that was approved for ALK-harboring lung cancer. There have been reports about the development and progression of renal cysts from crizotinib. We report a series of 3 cases of crizotinib-associated renal cysts in patients admitted to our institution, with different kinds of presentation. A monitor for complex renal cysts is warranted in patients receiving crizotinib.

Patient-reported outcomes from the randomized phase III ALEX study of alectinib versus crizotinib in patients with ALK-positive non-small-cell lung cancer.

OBJECTIVES: Alectinib demonstrated superior efficacy and a safety profile that compared favorably with crizotinib in treatment-naïve ALK+ non-small-cell lung cancer (NSCLC) in the phase III ALEX study. We present patient-reported outcomes (PROs) from ALEX to assess disease burden, treatment-related symptom tolerability, and health-related quality of life (HRQoL) with alectinib versus crizotinib. MATERIALS AND METHODS: Patients were randomized to receive alectinib 600 mg or crizotinib 250 mg twice daily until disease progression, death, or withdrawal. Pre-specified PRO endpoints were: mean change from baseline in symptoms, HRQoL, and functioning; and time to deterioration (TTD) in cough, dyspnea, chest pain, arm/shoulder pain, fatigue, and a composite of three symptoms (cough, dyspnea, chest pain). PRO data were collected using EORTC QLQ-C30 and LC13 questionnaires. Raw scores were standardized to a 0-100-point range, with a ≥10-point score change defined as clinically meaningful. TTD was defined as the time from randomization until confirmed clinically meaningful deterioration (i.e., a ≥10-point score change from baseline). RESULTS: Baseline completion rates and characteristics were balanced in the PRO-evaluable population (alectinib n = 100, 66%; crizotinib n = 97, 64%). On average, alectinib-treated patients reported clinically meaningful improvements in lung cancer symptoms for longer than crizotinib-treated patients. Between-treatment differences in lung cancer symptoms tended to favor alectinib from 11.1 months (45 weeks) onwards, around the time of median PFS with crizotinib (11.1 months). TTD in lung cancer symptoms was similar between treatment arms, despite longer duration of symptom improvement with alectinib; composite symptom endpoint (hazard ratio 1.10 [95% confidence interval: 0.72-1.68]). Duration of clinically meaningful improvement in HRQoL was longer with alectinib versus crizotinib (Week 88 vs. Week 68, respectively). Better patient-reported tolerability was observed with alectinib versus crizotinib on common treatment-related symptoms. CONCLUSION: PRO data support the superior efficacy and tolerability of alectinib relative to crizotinib demonstrated in the ALEX study.

Monitoring Therapeutic Response and Resistance: Analysis of Circulating Tumor DNA in Patients With ALK+ Lung Cancer.

INTRODUCTION: Despite initial effectiveness of ALK receptor tyrosine kinase inhibitors (TKIs) in patients with ALK+ NSCLC, therapeutic resistance will ultimately develop. Serial tracking of genetic alterations detected in circulating tumor DNA (ctDNA) can be an informative strategy to identify response and resistance. This study evaluated the utility of analyzing ctDNA as a function of response to ensartinib, a potent second-generation ALK TKI. METHODS: Pre-treatment plasma was collected from 76 patients with ALK+ NSCLC who were ALK TKI-naive or had received prior ALK TKI, and analyzed for specific genetic alterations. Longitudinal plasma samples were analyzed from a subset (n = 11) of patients. Analysis of pre-treatment tumor biopsy specimens from 22 patients was compared with plasma. RESULTS: Disease-associated genetic alterations were detected in 74% (56 of 76) of patients, the most common being EML4-ALK. Concordance of ALK fusion between plasma and tissue was 91% (20 of 22 blood and tissue samples). Twenty-four ALK kinase domain mutations were detected in 15 patients, all had previously received an ALK TKI; G1269A was the most prevalent (4 of 24). Patients with a detectable EML4-ALK variant 1 (V1) fusion had improved response (9 of 17 patients; 53%) to ensartinib compared to patients with EML4-ALK V3 fusion (one of seven patients; 14%). Serial changes in ALK alterations were observed during therapy. CONCLUSIONS: Clinical utility of ctDNA was shown, both at pre-treatment by identifying a potential subgroup of ALK+ NSCLC patients who may derive more benefit from ensartinib and longitudinally by tracking resistance. Prospective application of this technology may translate to improved outcomes for NSCLC patients treated with ALK TKIs.

Intracranial Inflammatory Myofibroblastic Tumor with Negative Expression of Anaplastic Lymphoma Kinase: A Case Report and Review of the Literature.

BACKGROUND: Inflammatory myofibroblastic tumor (IMT) is an indolent tumor mainly affecting children and young adults. As a rare mesenchymal tumor with unknown etiology and pathogenesis, IMT has a predilection for the lung and abdominopelvic region. Previous literature featuring IMT in the central nervous system (IMT-CNS) is rare. The clinical symptoms and radiologic features of IMT-CNS are not specific; therefore, the diagnosis is predominately based on the histopathologic and immunohistochemical analysis of the specimen. CASE DESCRIPTION: We herein present a case of a 21-year-old woman who complained of bilateral blurred vision for 15 days. Head magnetic resonance imaging demonstrated a round-shaped and irregular lesion located in the right frontal lobe. The boundary of the lesion was clear, and the lesion was homogeneously enhanced. Peripheral edema of the lesion was observed, and the mass effect was obvious. Supratentorial craniotomy tumor resection was performed. Histopathologic and immunohistochemical analysis revealed IMT, which had negative expression of anaplastic lymphoma kinase. CONCLUSIONS: Remission of her symptoms was observed, and no recurrence was recorded during a 6-month follow-up.

ALK Expression in Angiomatoid Fibrous Histiocytoma: A Potential Diagnostic Pitfall.

We recently encountered a case of primary pulmonary angiomatoid fibrous histiocytoma (AFH), which was initially misdiagnosed as inflammatory myofibroblastic tumor (IMT) based in part on anaplastic lymphoma kinase (ALK) expression by immunohistochemistry (IHC). Prompted by this experience, we evaluated ALK expression in 11 AFH, 15 IMT, and 11 follicular dendritic cell sarcomas using 3 different antibody clones: D5F3, 5A4, and ALK1. ALK IHC positive cases were analyzed with fluorescence in situ hybridization (FISH) using dual color ALK break-apart probe kit. The majority of AFH cases studied were positive for ALK IHC with at least 1 antibody (9/11 D5F3, 6/9 5A4, 1/9 ALK1), most demonstrating moderate to strong cytoplasmic staining. AFH with positive ALK IHC showed no ALK gene rearrangement by FISH (0/8) with ALK copy number ranging from 1.6 to 2.1. Sixty-seven percent of IMT were ALK positive by IHC (10/15 D5F3, 8/15 5A4, 7/15 ALK1), and 9 of the 10 cases were positive for ALK gene rearrangement by FISH. All follicular dendritic cell sarcomas were negative for ALK by IHC (D5F3 and 5A4). Our results indicate that ALK expression in AFH is common, particularly with the highly sensitive D5F3 and 5A4 antibodies and enhanced detection systems, and may be a potential source of diagnostic confusion with IMT. The underlying mechanism of ALK expression in AFH is unclear, although it does not appear to be from ALK rearrangement or amplification.

ALK and EGFR expression by immunohistochemistry are associated with Merkel cell polyomavirus status in Merkel cell carcinoma.

AIMS: Merkel cell carcinoma, a rare cutaneous neuroendocrine tumour of the skin, can be categorised into two groups according to Merkel cell polyomavirus (MCV) presence. MCV-negative tumours are more aggressive and frequently associated with gene mutations. Some of the genes are potential therapeutic targets. We have previously reported EGFR mutations in six of 27 MCC tumours and overexpression of ALK and EZH2 at mRNA level in MCC tumours. In this study, we sought to determine expression of ALK, EGFR and EZH2 in MCC samples and assess their correlation to MCV status and clinical parameters. METHODS AND RESULTS: Tissue microarrays were utilised and stained with primary antibodies. Staining data were statistically compared to patient sex, tumour location and development of metastasis and MCC-specific death; 112 tumours and their corresponding patient data were included. We found strong expression of ALK in 51% and strong expression of EZH2 in 76% of the tumours. There was evident correlation of ALK expression with MCV-positivity. Expression of EGFR was infrequent, presenting only in seven MCV-negative tumours. None of the proteins associated with development of metastasis or MCC specific death. CONCLUSIONS: ALK and EZH2 expression are frequent in MCC and ALK expression correlates to MCV positivity. EGFR positive tumours might respond to EGFR inhibiting treatment.

ALK-positive anaplastic large-cell lymphoma with primary bone involvement: A rare case and review of the literature.

Primary bone lymphoma (PBL) is an uncommon type of extranodal lymphoma involvement. An anaplastic large-cell lymphoma (ALCL) is an extremely rare type of PBL, and it remains unclear whether ALCLs that primarily involve the bone exhibit favourable or unfavourable biological behaviour, and whether they are similar to ALCLs in general, or not. We reported a case of ALK-positive ALCL with primary bone involvement, and reviewed the clinicopathological features of 22 previously reported cases. An ALCL with primary bone involvement mostly affects younger patients with a preponderant towards the involvement of axial-bone. The prognosis of an ALCL that primarily involves bone is unfavourable, compared with PBL generally. The ALK-positive ALCLs in PBLs had less decedents than the ALK-negative ALCLs with a statistical non-significance (p=0.198).

Targeting ALK in pediatric RMS does not induce antitumor activity in vivo.

PURPOSE: The anaplastic lymphoma kinase (ALK) has been demonstrated to be a valid clinical target in diseases such as anaplastic large cell lymphoma and non-small cell lung cancer. Recent studies have indicated that ALK is overexpressed in pediatric rhabdomyosarcoma (RMS) and hence we hypothesized that this kinase may be a suitable candidate for therapeutic intervention in this tumor. METHODS: We evaluated the expression of ALK in a panel of pediatric RMS cell lines and patient-derived xenografts (PDX), and sensitivity to ALK inhibitors was assessed both in vitro and in vivo. RESULTS: Essentially, all RMS lines were sensitive to crizotinib, NVP-TAE684 or LDK-378 in vitro, and molecular analyses demonstrated inhibition of RMS cell proliferation following siRNA-mediated reduction of ALK expression. However, in vivo PDX studies using ALK kinase inhibitors demonstrated no antitumor activity when used as single agents or when combined with standard of care therapy (vincristine, actinomycin D and cyclophosphamide). More alarmingly, however, crizotinib actually accelerated the growth of these tumors in vivo. CONCLUSIONS: While ALK appears to be a relevant target in RMS in vitro, targeting this kinase in vivo yields no therapeutic efficacy, warranting extreme caution when considering the use of these agents in pediatric RMS patients.

Exposure-response analysis of alectinib in crizotinib-resistant ALK-positive non-small cell lung cancer.

PURPOSE: Alectinib is a selective and potent anaplastic lymphoma kinase (ALK) inhibitor that is active in the central nervous system (CNS). Alectinib demonstrated robust efficacy in a pooled analysis of two single-arm, open-label phase II studies (NP28673, NCT01801111; NP28761, NCT01871805) in crizotinib-resistant ALK-positive non-small-cell lung cancer (NSCLC): median overall survival (OS) 29.1 months (95% confidence interval [CI]: 21.3-39.0) for alectinib 600 mg twice daily (BID). We investigated exposure-response relationships from final pooled phase II OS and safety data to assess alectinib dose selection. METHODS: A semi-parametric Cox proportional hazards model analyzed relationships between individual median observed steady-state trough concentrations (Ctrough,ss) for combined exposure of alectinib and its major metabolite (M4), baseline covariates (demographics and disease characteristics) and OS. Univariate logistic regression analysis analyzed relationships between Ctrough,ss and incidence of adverse events (AEs: serious and Grade ≥ 3). RESULTS: Overall, 92% of patients (n = 207/225) had Ctrough,ss data and were included in the analysis. No statistically significant relationship was found between Ctrough,ss and OS following alectinib treatment. The only baseline covariates that statistically influenced OS were baseline tumor size and prior crizotinib treatment duration. Larger baseline tumor size and shorter prior crizotinib treatment were both associated with shorter OS. Logistic regression confirmed no significant relationship between Ctrough,ss and AEs. CONCLUSION: Alectinib 600 mg BID provides systemic exposures at plateau of response for OS while maintaining a well-tolerated safety profile. This analysis confirms alectinib 600 mg BID as the recommended global dose for patients with crizotinib-resistant ALK-positive NSCLC.

Impact of EML4-ALK Variant on Resistance Mechanisms and Clinical Outcomes in ALK-Positive Lung Cancer.

Purpose Advanced anaplastic lymphoma kinase ( ALK) fusion-positive non-small-cell lung cancers (NSCLCs) are effectively treated with ALK tyrosine kinase inhibitors (TKIs). However, clinical outcomes in these patients vary, and the benefit of TKIs is limited as a result of acquired resistance. Emerging data suggest that the ALK fusion variant may affect clinical outcome, but the molecular basis for this association is unknown. Patients and Methods We identified 129 patients with ALK-positive NSCLC with known ALK variants. ALK resistance mutations and clinical outcomes on ALK TKIs were retrospectively evaluated according to ALK variant. A Foundation Medicine data set of 577 patients with ALK-positive NSCLC was also examined. Results The most frequent ALK variants were EML4-ALK variant 1 in 55 patients (43%) and variant 3 in 51 patients (40%). We analyzed 77 tumor biopsy specimens from patients with variants 1 and 3 who had progressed on an ALK TKI. ALK resistance mutations were significantly more common in variant 3 than in variant 1 (57% v 30%; P = .023). In particular, ALK G1202R was more common in variant 3 than in variant 1 (32% v 0%; P < .001). Analysis of the Foundation Medicine database revealed similar associations of variant 3 with ALK resistance mutation and with G1202R ( P = .010 and .015, respectively). Among patients treated with the third-generation ALK TKI lorlatinib, variant 3 was associated with a significantly longer progression-free survival than variant 1 (hazard ratio, 0.31; 95% CI, 0.12 to 0.79; P = .011). Conclusion Specific ALK variants may be associated with the development of ALK resistance mutations, particularly G1202R, and provide a molecular link between variant and clinical outcome. ALK variant thus represents a potentially important factor in the selection of next-generation ALK inhibitors.

Genomic heterogeneity of ALK fusion breakpoints in non-small-cell lung cancer.

In lung adenocarcinoma, canonical EML4-ALK inversion results in a fusion protein with a constitutively active ALK kinase domain. Evidence of ALK rearrangement occurs in a minority (2-7%) of lung adenocarcinoma, and only ~60% of these patients will respond to targeted ALK inhibition by drugs such as crizotinib and ceritinib. Clinically, targeted anti-ALK therapy is often initiated based on evidence of an ALK genomic rearrangement detected by fluorescence in situ hybridization (FISH) of interphase cells in formalin-fixed, paraffin-embedded tissue sections. At the genomic level, however, ALK rearrangements are heterogeneous, with multiple potential breakpoints in EML4, and alternate fusion partners. Using next-generation sequencing of DNA and RNA together with ALK immunohistochemistry, we comprehensively characterized genomic breakpoints in 33 FISH-positive lung adenocarcinomas. Of these 33 cases, 29 (88%) had detectable DNA level ALK rearrangements involving EML4, KIF5B, or non-canonical partners including ASXL2, ATP6V1B1, PRKAR1A, and SPDYA. A subset of 12 cases had material available for RNA-Seq. Of these, eight of eight (100%) cases with DNA rearrangements showed ALK fusion transcripts from RNA-Seq; three of four cases (75%) without detectable DNA rearrangements were similarly negative by RNA-Seq, and one case was positive by RNA-Seq but negative by DNA next-generation sequencing. By immunohistochemistry, 17 of 19 (89%) tested cases were clearly positive for ALK protein expression; the remaining cases had no detectable DNA level rearrangement or had a non-canonical rearrangement not predicted to form a fusion protein. Survival analysis of patients treated with targeted ALK inhibitors demonstrates a significant difference in mean survival between patients with next-generation sequencing confirmed EML4-ALK rearrangements, and those without (20.6 months vs 5.4 months, P<0.01). Together, these data demonstrate abundant genomic heterogeneity among ALK-rearranged lung adenocarcinoma, which may account for differences in treatment response with targeted ALK inhibitors.

Epithelioid fibrous histiocytoma: molecular characterization of ALK fusion partners in 23 cases.

Epithelioid fibrous histiocytoma is a rare and distinctive cutaneous neoplasm. Most cases harbor ALK rearrangement and show ALK overexpression, which distinguish this neoplasm from conventional cutaneous fibrous histiocytoma and variants. SQSTM1 and VCL have previously been shown to partner with ALK in one case each of epithelioid fibrous histiocytoma. The purpose of this study was to examine a large cohort of epithelioid fibrous histiocytomas by next-generation sequencing to characterize the nature and prevalence of ALK fusion partners. A retrospective archival review was performed to identify cases of epithelioid fibrous histiocytoma (2012-2016). Immunohistochemistry was performed to confirm ALK expression. Targeted next-generation sequencing was applied on RNA extracted from formalin-fixed paraffin-embedded tissue to identify the fusion partners. Twenty-three cases fulfilled inclusion criteria. The mean patient age was 39 years (range, 8-74), there was no sex predilection, and >75% of cases involved the lower extremities. The most common gene fusions were SQSTM1-ALK (N=12; 52%) and VCL-ALK (N=7; 30%); the other four cases harbored novel fusion partners (DCTN1, ETV6, PPFIBP1, and SPECC1L). The pattern of ALK immunoreactivity was usually granular cytoplasmic (N=12; 52%) or granular cytoplasmic and nuclear (N=10; 43%); the case containing an ETV6 fusion partner showed nuclear staining alone. There was no apparent relationship between tumor morphology and the ALK fusion partner. In summary, SQSTM1 and VCL are the most common ALK fusion partners in epithelioid fibrous histiocytoma; DCTN1, ETV6, PPFIBP1, and SPECC1L represent rare fusion partners. The proteins encoded by these genes play diverse roles in scaffolding, cell adhesion, signaling, and transcription (among others) without clear commonalities. These findings expand the oncogenic promiscuity of many of these ALK fusion genes, which drive neoplasia in tumors of diverse lineages with widely varied clinical behavior. This is the first documented account of ETV6-ALK and SPECC1L-ALK translocations in neoplasms.

Prevalence and clinical association of gene mutations through multiplex mutation testing in patients with NSCLC: results from the ETOP Lungscape Project.

Background: Reported prevalence of driver gene mutations in non-small-cell lung cancer (NSCLC) is highly variable and clinical correlations are emerging. Using NSCLC biomaterial and clinical data from the European Thoracic Oncology Platform Lungscape iBiobank, we explore the epidemiology of mutations and association to clinicopathologic features and patient outcome (relapse-free survival, time-to-relapse, overall survival). Methods: Clinically annotated, resected stage I-III NSCLC FFPE tissue was assessed for gene mutation using a microfluidics-based multiplex PCR platform. Mutant-allele detection sensitivity is >1% for most of the ∼150 (13 genes) mutations covered in the multiplex test. Results: Multiplex testing has been carried out in 2063 (76.2%) of the 2709 Lungscape cases (median follow-up 4.8 years). FFPE samples mostly date from 2005 to 2008, yet recently extracted DNA quality and quantity was generally good. Average DNA yield/case was 2.63 µg; 38 cases (1.4%) failed QC and were excluded from study; 95.1% of included cases allowed the complete panel of mutations to be tested. Most common were KRAS, MET, EGFR and PIK3CA mutations with overall prevalence of 23.0%, 6.8%, 5.4% and 4.9%, respectively. KRAS and EGFR mutations were significantly more frequent in adenocarcinomas: PIK3CA in squamous cell carcinomas. MET mutation prevalence did not differ between histology groups. EGFR mutations were found predominantly in never smokers; KRAS in current/former smokers. For all the above mutations, there was no difference in outcome between mutated and non-mutated cases. Conclusion: Archival FFPE NSCLC material is adequate for multiplex mutation analysis. In this large, predominantly European, clinically annotated stage I-III NSCLC cohort, none of the mutations characterized showed prognostic significance.