Anaplastic Lymphoma Kinase Immunohistochemistry Research Articles

Immunohistochemical expression of anaplastic lymphoma kinase in neuroblastoma and its relations with some clinical and histopathological features.

Anaplastic lymphoma kinase (ALK) mutations have been identified as a prominent cause of some familial and sporadic neuroblastoma (NB). ALK expression in NB and its relationship with clinical and histopathological features remains controversial. This study investigated ALK expression and its potential relations with these features in NB. Ninety cases of NB at the Department of Pathology, University of Medicine and Pharmacy at Ho Chi Minh City, Viet Nam from 01/01/2018 to 12/31/2021, were immunohistochemically stained with ALK (D5F3) antibody. The ALK expression and its relations with some clinical and histopathological features were investigated. The rate of ALK expression in NB was 91.1%. High ALK expression (over 50% of tumor cells were positive with moderate-strong intensity) accounted for 65.6%, and low ALK expression accounted for 34.4%. All the MYCN-amplified NB patients had ALK immunohistochemistry positivity, most cases had high ALK protein expression. The undifferentiated subtype of NB had a lower ALK-positive rate than the poorly differentiated and differentiated subtype. The percentages of ALK positivity were significantly higher in more differentiated histological types of NB (p = .024). There was no relation between ALK expression and: age group, sex, primary tumor location, tumor stage, MYCN status, clinical risk, Mitotic-Karyorrhectic Index, prognostic group, necrosis, and calcification. ALK was highly expressed in NB. ALK expression was not related to several clinical and histopathological features. More studies are needed to elucidate the association between ALK expression and ALK gene status and to investigate disease progression, especially the oncogenesis of ALK-positive NB.

Concordance of Immunohistochemistry and Fluorescence In Situ Hybridization in the Detection of Anaplastic Lymphoma Kinase (ALK) and Ros Proto-oncogene 1 (ROS1) Gene Rearrangements in Non-Small Cell Lung Carcinoma: A 4.5-Year Experience Highlighting Challenges and Pitfalls.

ALK and ROS1 rearrangements are essential biomarkers to be tested in advanced lung adenocarcinomas. While D5F3 Ventana assay is a companion diagnostic for anaplastic lymphoma kinase-targeted therapy, immunohistochemistry is only a screening tool for detecting ROS1 rearrangement. Confirmation by cytogenetic or molecular techniques is necessary. To evaluate the utility of ALK and ROS1 fluorescence in situ hybridization as a complement to immunohistochemistry in routine predictive biomarker testing algorithms. The study was ambispective, spanning 4.5 years during which lung adenocarcinoma samples were subjected to EGFR mutation testing by real-time polymerase chain reaction, and ALK/ROS1 rearrangement testing by immunohistochemistry (Ventana D5F3 assay for anaplastic lymphoma kinase protein; manual assay with D4D6 clone for Ros proto-oncogene 1 protein). Fluorescence in situ hybridization was performed in all anaplastic lymphoma kinase equivocal and Ros proto-oncogene 1 immunopositive cases. Of 1874 samples included, EGFR mutations were detected in 27% (481 of 1796). Anaplastic lymphoma kinase immunohistochemistry was positive in 10% (174 of 1719) and equivocal in 3% (58 of 1719) of samples tested. ALK fluorescence in situ hybridization showed 81% (77 of 95) concordance with immunohistochemistry. Ros proto-oncogene 1 immunopositivity was noted in 13% (190 of 1425) of cases, with hybridization-confirmed rearrangements in 19.3% (26 of 135) of samples, all of which showed diffuse, strong- to moderate-intensity, cytoplasmic staining in tumor cells. Ros proto-oncogene 1 protein overexpression without rearrangement was significantly common in EGFR-mutant and ALK-rearranged adenocarcinomas. Immunostaining is a robust method for ALK-rearrangement testing, with fluorescence in situ hybridization adding value in the rare equivocal stained case. ROS1-rearrangement testing is more cost-effective if immunohistochemistry is followed by fluorescence in situ hybridization after excluding EGFR-mutant and ALK-rearranged adenocarcinomas.

Renal Inflammatory Myofibroblastic Tumor in an Infant: Case Report with Review of Literature

AbstractInflammatory myofibroblastic tumor (IMT) is an intermediate-grade neoplasm of myofibroblastic lineage occurring due to a cytogenetic clonal abnormality of chromosome 2p23. Pediatric renal IMTs are rare and infant renal IMTs are almost anecdotal. We herein report a 1-year-old female child who presented with a firm mass in the right lumbar region. Biopsy, and later, surgical resection revealed a tumor composed of spindle cells with intermixed plasma cells. On immunohistochemistry (IHC), the lesional cells were positive for smooth muscle actin and anaplastic lymphoma kinase (ALK). A diagnosis of IMT was made based on morphology and IHC. Diagnosis of renal IMTs become challenging especially in a tiny biopsy wherein clear cell sarcoma of kidney, Wilms tumor with predominant mesenchymal component, congenital mesoblastic nephroma, and metanephric stromal tumor are the differential diagnoses in this age group. Renal IMTs generally have better prognosis as compared to extrarenal IMTs. Approximately, 50 to 60% of these tumors harbor ALK gene rearrangement as demonstrated by positivity for ALK IHC. ALK inhibitors like crizotinib or ceritinib can be given for advanced metastatic tumors.

Tyrosine kinase alterations in colorectal cancer with emphasis on the distinct clinicopathological characteristics.

Tyrosine kinase (TK) alterations, such as anaplastic lymphoma kinase (ALK) fusion, neurotrophic tyrosine receptor kinase (NTRK) fusion, c-ros oncogene 1 (ROS1) fusion and mesenchymal-epithelial transition factor (MET) exon 14 skipping, have been reported in colorectal cancers (CRC). We have previously reported CRCs with NTRK fusion among our cohort. However, their clinicopathological features have not been fully elucidated. Tissue microarray (TMA)-based immunohistochemistry (IHC) was performed on 951 CRC lesions from 944 patients. IHC was evaluated as positive or negative for ALK and ROS1 and 0 to 3+ for c-MET. For ALK and ROS1 IHC-positive cases, RNA-based imbalanced gene expression assays, Archer FusionPlex assays and reverse transcription-polymerase chain reaction (RT-PCR) followed by Sanger sequencing were performed. For c-MET IHC 3+ cases, RT-PCR followed by Sanger sequencing were performed. ALK IHC was positive in three cases (0.2%) and all showed imbalanced ALK gene expression. The following ALK fusions were confirmed: EML4 (exon 21)::ALK (exon 20), EML4 (exon 6)::ALK (exon 19) and HMBOX1 (exon 6)::ALK (exon 20). Two showed microsatellite instability-high/mismatch repair (MMR)-deficient, and all were located in the right colon. ROS1 IHC was positive in one case; however, imbalanced expression and ROS1 fusion was negative. Forty-two cases (4.4%) showed c-MET IHC3+. MET exon 14 skipping was confirmed in nine cases. All cases were microsatellite stable/MMR-proficient, and eight were located in the left colon and rectum. CRCs with these TK alterations had distinct clinicopathological features. Together with our previous study, 15 cases (1.6%) harboured targetable TK alterations (three NTRK fusion, three ALK fusion, nine MET exon 14 skipping).

Novel HIVEP1-ALK fusion in a patient with lung adenocarcinoma demonstrating sensitivity to alectinib: a case report

BackgroundAnaplastic lymphoma kinase (ALK) fusion is an important oncogenic driver in non-small cell lung cancer (NSCLC). Reports on the intergenic region (IGR) as an ALK fusion partner are rare. Here, we report the case of a patient with advanced NSCLC harboring a human immunodeficiency virus type I enhancer binding protein 1 (HIVEP1)-ALK fusion that responded effectively to alectinib.Case DescriptionA 60-year-old non-smoking male was referred with a 3-month history of productive cough secondary to lung adenocarcinoma metastatic to mediastinal lymph nodes, brain, liver, and bone (T2N3M1c, stage IVB). Next-generation sequencing identified an IGR (upstream HIVEP1-) ALK fusion, and immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) results were consistent with an ALK-positive tumor. The patient was subsequently started on alectinib, with no obvious adverse reaction. After 1 month of therapy, the patient achieved significantly remission of the clinical symptoms and had led to an ongoing partial response (PR) lasting >33 months.ConclusionsOur experience highlights the efficacy of alectinib in a patient with HIVEP1-ALK fusion positive NSCLC with multiple metastases including brain disease, and the need for multiple genetic testing methods to verify the oncogenicity of ALK fusions prior to treatment. It could provide useful guidance for the treatment of similar cases in the future.

Quantitative Multiplexed Proteomics Could Assist Therapeutic Decision Making in Non-Small Cell Lung Cancer Patients with Ambiguous ALK Test Results.

Simple SummaryTherapeutic guidance in non-small cell lung cancer (NSCLC) patients with discordant anaplastic lymphoma kinase (ALK) fluorescent in situ hybridization (+) where immunohistochemistry (IHC) (−) results are challenging. Selected reaction monitoring (SRM) quantitative multiplexed proteomics could detect ALK protein in NSCLC samples with delayed fixation where a conventional IHC method failed. ALK protein detection by the SRM method was associated with good responses on ALK inhibitors. It also could detect various predictive proteins for conventional chemotherapy at the same time, and combined results were related to clinical outcomes in this population. The SRM may provide additional information for therapeutic decision making in NSCLC patients with ambiguous ALK test results.Therapeutic guidance in non-small cell lung cancer (NSCLC) tumors that are positive for anaplastic lymphoma kinase (ALK) fluorescent in situ hybridization (FISH), but negative for ALK immunohistochemistry, is still challenging. Parallel routine screening of 4588 NSCLC cases identified 22 discordant cases. We rechecked these samples using ALK antibodies and selected reaction monitoring (SRM) quantitative multiplexed proteomics screening multiple protein targets, including ALK and MET for the ALK tyrosine kinase inhibitor (TKI), and FR-alpha, hENT1, RRM1, TUBB3, ERCC1, and XRCC1 for chemotherapy. The presence of ALK (31.8%), MET (36.4%), FR-alpha (72.7%), hENT1 (18.2%), RRM1 (31.8%), TUBB3 (72.9%), ERCC1 (4.5%), and a low level of XRCC1 (54.4%) correlated with clinical outcomes. SRM was more sensitive than the ALK D5F3 assay. Among the eight cases receiving ALK TKI, four cases with ALK or MET detected by SRM had complete or partial responses, whereas four cases without ALK or MET showed progression. Twenty-seven treatment outcomes from 20 cases were assessed and cases expressing more than half of the specific predictive proteins were sensitive to matching therapeutic agents and showed longer progression-free survival than the other cases (p < 0.001). SRM showed a potential role in therapeutic decision making in NSCLC patients with ambiguous ALK test results.

A case of concomitant EGFR/ALK alteration against a mutated EGFR background in early-stage lung adenocarcinoma.

Rare cases of lung adenocarcinoma (LUAD) with concomitant epidermal growth factor receptor (EGFR) mutation and anaplastic lymphoma kinase (ALK) translocation have been reported. However, their clonal and evolutional relationship remains unclear. We report a case of early-stage EGFR-mutated LUAD with a focal concomitant EGFR/ALK alteration. A 63-year-old male underwent lobectomy to remove a 1.9-cm-sized lung nodule, which was diagnosed with EGFR-mutated LUAD. ALK immunohistochemistry (IHC) showed focal positivity within the part of the tumor characterized by lepidic pattern, also confirmed by fluorescence in-situ hybridization (FISH). Targeted next-generation sequencing was performed separately on the ALK IHC/FISH-positive and -negative areas. EGFR L833V/L858R mutations were detected in both areas, whereas EML4 (echinoderm microtubule-associated protein-like 4)-ALK translocations was confirmed only in the ALK IHC/FISH-positive area, suggesting the divergence of an EGFR/ALK co-altered subclone from the original EGFR-mutant clone. Our study suggests that concurrent alterations of EGFR and ALK can arise via divergent tumor evolution, even in the relatively early phases of tumorigenesis.

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.

Staining Performance of ALK and ROS1 Immunohistochemistry and Influence on Interpretation in Non–Small-Cell Lung Cancer

Selection of non-small-cell lung cancer patients for treatment relies on the detection of expression of anaplastic lymphoma kinase (ALK) and ROS proto-oncogene 1 (ROS1) protein by immunohistochemistry (IHC). We evaluated staining performance for different IHC protocols and laboratory characteristics, and their influence on ALK and ROS1 interpretation during external quality assessment schemes between 2015 and 2018. Participants received five formalin-fixed, paraffin-embedded cases for staining by their routine protocol, whereafter at least two pathologists scored them simultaneously under a multihead microscope and awarded a graded expert staining score (ESS) from 1 to 5 points based on staining quality. European Conformity in Vitro Diagnostic kits (such as D5F3) revealed a better ALK ESS compared with laboratory-developed tests. ESS was indifferent to the applied antibody dilution or a recent protocol change. Lower ESSs were observed for higher antibody incubation times and temperatures. ESS for various ROS1 protocols were largely similar. Overall, for both markers, ESS improved over time and for repeated external quality assessment participation but was independent of laboratory setting or experience. Except for ROS1, ESS positively correlated with laboratory accreditation. IHC stains with lower ESS correlated with increased error rates in ALK and ROS1 interpretation and analysis failures. Laboratory characteristics differently affected staining quality and interpretation, and laboratories should assess both aspects, and less common protocols need improvement in staining performance.

Abstract 3191: Detection of molecular drivers in inflammatory myofibroblastic tumor: study on archival tissue from EORTC 90101 “CREATE” phase II clinical trial

Abstract 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 (&amp;gt;15% of cells) or absence (&amp;lt;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.

Unproductive Effects of ALK Gene Amplification and Copy Number Gain in Non-Small-Cell Lung Cancer. ALK Gene Amplification and Copy Gain in NSCLC.

Background: The Anaplastic Lymphoma Kinase (ALK) gene is known to be affected by several genetic alterations, such as rearrangement, amplification and point mutation. The main goal of this study was to comprehensively analyze ALK amplification (ALK-A) and ALK gene copy number gain (ALK-CNG) in a large cohort of non-small-cell lung cancer (NSCLC) patients in order to evaluate the effects on mRNA and protein expression. Methods: ALK locus number status was evaluated in 578 NSCLC cases by fluorescence in situ hybridization (FISH). In addition, ALK immunohistochemistry and ALK mRNA in situ hybridization were performed. Results: Out of 578 cases, 17 cases showed ALK-A. In addition, 14 cases presented ALK-CNG and 72 cases presented chromosome 2 polyploidy. None of those carrying ALK-A and -CNG showed either ALK immunohistochemical expression or ALK mRNA expression through in situ hybridization. We observed a high frequency of extra copies of the ALK gene. Conclusions: Our findings demonstrated that ALK-A is not involved in mRNA production and consequently is not involved in protein production; these findings support the hypothesis that ALK-A might not play a role in the pathogenesis of NSCLC, underlining the absence of a specific clinical application.

Screening for anaplastic lymphoma kinase (ALK) gene rearrangements in non-small-cell lung cancer in New Zealand.

Lung cancer is a major cause of death in New Zealand. In recent years, targeted therapies have improved outcomes. To determine the uptake of anaplastic lymphoma kinase (ALK) testing, and the prevalence, demographic profile and outcomes of ALK-positive non-small-cell lung cancer (NSCLC), in New Zealand, where no national ALK-testing guidelines or subsidised ALK tyrosine kinase inhibitor (TKI) therapies are available. A population-based observational study reviewed databases to identify patients presenting with non-squamous NSCLC over 6.5 years in northern New Zealand. We report the proportion tested for ALK gene rearrangements and the results. NSCLC samples tested by fluorescence in situ hybridisation were retested by next generation sequencing and ALK immunohistochemistry. A survival analysis compared ALK-positive patients treated or not treated with ALK TKI therapy. From a total of 3130 patients diagnosed with non-squamous NSCLC, 407 (13%) were tested for ALK gene rearrangements, and patient selection was variable and inequitable. Among those tested, 34 (8.4%) had ALK-positive NSCLC. ALK-positive disease was more prevalent in younger versus older patients, non-smokers versus smokers and in Māori, Pacific or Asian ethnic groups than in New Zealand Europeans. Fluorescence in situ hybridisation, ALK immunohistochemistry and next generation sequencing showed broad concordance for detecting ALK-positive disease under local testing conditions. Among patients with ALK-positive metastatic NSCLC, those treated with ALK TKI survived markedly longer than those not treated with ALK TKI (median overall survival 5.12 vs 0.55 years). Lung cancer outcomes in New Zealand may be improved by providing national guidelines and funding policy for ALK testing and access to subsidised ALK TKI therapy.

Attenuated isolated 3’ signal: A highly challenging therapy relevant ALK FISH pattern in NSCLC

ObjectivesTargeted therapies in the management of patients with lung cancer provide significantly better outcome compared to chemotherapy. Detection of the anaplastic lymphoma kinase (ALK) gene rearrangement has great predictive value for treatment with small molecule tyrosine kinase inhibitor (crizotinib and alectinib commonly). Fluorescent in situ hybridisation (FISH) assay is a basic diagnostic test designed for detecting ALK gene rearrangements. Although being considered as gold standard method by IASLC's guideline, it is often regarded as difficult and error prone. Our aim was to examine a unique atypical ALK FISH pattern, revealed during a systematic large-scale monitoring, which carries the great risk of misinterpretation, hence may result in loss of patients eligible for targeted therapy. Materials and MethodsTissue and cytology samples from nearly one thousand patients with advanced stage non-small cell lung cancer (NSCLC, n = 996) were routinely examined by ALK FISH and immunohistochemistry (Ventana ALK-D5F3-CDx assay). Anchored Multiplex PCR based Next Generation Sequencing (AMP-NGS) was used to detect fusion gene transcripts in ambiguous cases. ResultsFifty-nine (5,9%) of the cases were positive with ALK FISH test. Three cases showed atypical pattern with a significantly reduced sized red (3′) signal and complete loss of green signals. Digital signal measurement confirmed this finding, showing consistent attenuation of 3′ signals throughout the tumours. In all three cases AMP-NGS and ALK IHC verified the presence of a fusion gene and expressed oncoprotein, respectively. ConclusionApproximately 5% of the 59 ALK positive cases exhibited atypical attenuated isolated 3′ signal pattern. The immunohistochemistry and AMP-NGS examinations helped to clarify the presence of oncoprotein and the fusion gene, respectively. Our results emphasize the importance of extensive exploration of the genetic background of any unexpected FISH finding to avoid false diagnosis. This enables clinicians to indicate the adequate therapy with higher efficiency for patients suffering from NSCLC.

Uterine inflammatory myofibroblastic tumour: A case report

Inflammatory myofibroblastic tumour (IMT) is a mesenchymal neoplasm with intermediate biological potential, which may be under-recognised in the uterus.1–3 Morphological and immunophenotypic overlap exists between uterine IMT and some other uterine mesenchymal neoplasms, including smooth muscle tumours.2 Morphological features suggestive of IMT may be subtle or focal and include myxoid change, tapered nuclei, and a lymphoplasmacytic infiltrate.1 Chromosomal rearrangements involving the anaplastic lymphoma kinase (ALK) gene appear to be frequent in uterine IMT,1–3 and absent in other uterine mesenchymal neoplasms.2 Therefore, ALK immunohistochemistry (IHC) and ALK florescent in situ hybridisation (FISH) appear to be useful tools to confirm the diagnosis of uterine IMT.3 Although most uterine IMTs follow a benign clinical course, local recurrence and metastases have been observed in a small subset.1 Correct diagnosis is important to ensure accurate prognosis and optimal management, which may include a tyrosine kinase inhibitor. We present a case of uterine IMT with morphology and IHC suggestive of a leiomyosarcoma with myxoid change. ALK IHC was positive and an ALK rearrangement was demonstrated with FISH, confirming the diagnosis of uterine IMT. This case illustrates the importance of a low threshold for ALK testing in uterine mesenchymal tumours with morphological features suggestive of IMT.

Clinicopathological features and diagnostic methods of ALK fusion‑positive non‑small cell lung cancer in Korea.

Lung cancer is one of the most common malignancies and the leading cause of cancer-associated mortality in Korea. A significant amount of effort has been put into the development of new and more effective treatments and biological markers for the prediction of therapeutic responses, which has led to the identification of various genetic changes in cancer, that are the so-called ‘growth drivers’ of carcinogenesis. Certain genetic alterations have become new treatment targets, and it has been suggested that different mutations are associated with different clinicopathological characteristics and prognosis. The present study aimed to evaluate the status of the key ‘driver’ mutation anaplastic lymphoma kinase (ALK) fusion in Korean patients with non-small cell lung cancer (NSCLC) and its association with clinicopathological characteristics, including the presence of other genetic mutations. The present study also compared different methods for ALK fusion detection, including fluorescence in situ hybridization (FISH), immunohistochemistry (IHC) and next-generation sequencing (NGS) to evaluate which method is the most effective. A total of 482 patients with NSCLC who underwent ALK FISH analysis were evaluated for clinicopathological features, such as age, sex, smoking history, tumor stage, histological subtype, immunohistochemical profile, including ALK and EGFR mutation statuses, and survival. Some ALK FISH-positive and -negative cancers were newly submitted to NGS analysis for DNA and RNA alterations. The ALK fusion-positive tumors were associated with a younger age, female patients, frequent nodal metastases, advanced stage and shorter survival. Comparing the results of ALK FISH, IHC and NGS analyses, it was concluded that in practice, ALK testing should better be diversified concerning FISH and IHC, and NGS analysis would be a good alternative to FISH, with an additional advantage of being able to concurrently detect different mutations.

ALK immunohistochemistry positive, FISH negative NSCLC is infrequent, but associated with impaired survival following treatment with crizotinib

ObjectiveMetastasized non-small cell lung cancer (NSCLC) with an anaplastic lymphoma kinase (ALK) rearrangement is usually sensitive to a range of ALK-tyrosine kinase inhibitors. ALK-positive NSCLC have been identified in pivotal phase III trials with fluorescence in situ hybridization (ALK FISH+). These tumors are also expressing the fusion product (ALK immunohistochemistry (IHC)+). However, discrepant cases occur, including ALK IHC + FISH-. The aim of this study was to collect ALK IHC + cases and compare within this group response to crizotinib treatment of ALK FISH + cases with ALK FISH- cases. Materials and methodsIn this European prospective multicenter research study patients with Stage IV ALK IHC + NSCLC treated with crizotinib were enrolled. Tumor slides were validated centrally for ALK IHC and ALK FISH. ResultsRegistration of 3523 ALK IHC tests revealed a prevalence of 2.7% (n = 94) ALK IHC + cases. Local ALK FISH analysis resulted in 48 concordant (ALK IHC+/FISH+) and 16 discordant (ALK IHC+/FISH-) cases. Central validation revealed 37 concordant and 7 discordant cases, 5 of which had follow-up. Validation was hampered by limited amount of tissue in biopsy samples. The PFS at 1 year for ALK concordant and discordant was 58% and 20%, respectively (HR = 2.4; 95% CI: 0.78–7.3; p = 0.11). Overall survival was significantly better for concordant cases than discordant cases after central validation (HR=4.5; 95% CI= 1.2–15.9; p=0.010. ConclusionALK IHC + FISH- NSCLC is infrequent and associated with a worse outcome on personalized treatment. A suitable predictive testing strategy may be to screen first with IHC and then confirm with FISH instead of considering ALK IHC equivalent to ALK FISH according to the current guidelines.

P2.09-35 Proposal to Revise VENTANA ALK Scoring Interpretation Guide for Non-Small Cell Lung Carcinoma: Interpretation of ALK Heterogeneity

A small number of non-small cell lung cancer (NSCLC) cases showed heterogeneity of anaplastic lymphoma kinase (ALK) by VENTANA immunohistochemistry (IHC) in clinical practice. According to the ALK Scoring Interpretation Guide for VENTANA anti-ALK (D5F3), the presence of strong granular cytoplasmic staining in tumor cells (any percentage of positive tumor cells) is called positive for ALK. However, we know little about ALK heterogeneous cases. Multiple detection platforms are used to analyze molecular variability and pathological features in anticipation of clinical treatment decisions for such cases. A total of 2228 NSCLC cases with successful ALK IHC (VENTANA, D5F3, Roche) detection in Guangdong Provincial People`s Hospital were recruited between January 2012 and April 2018. Positive and negative system control and a negative agent control were established for each case. ALK IHC positivity was defined as the presence of strong granular cytoplasmic staining in 100% tumor cells; ALK IHC heterogeneity as the presence of strong granular cytoplasmic staining in 1-99% tumor cells; ALK IHC negativity as no tumor cells show strong granular cytoplasmic staining. Fluorescence in-situ hybridization (FISH) (Vysis ALK Break Apart FISH Probe Kit, Abbott) and next-generation sequencing (NGS) (OseqTMLung Cancer Gene Detection, BGI, China) was performed for cases showed ALK (D5F3) IHC heterogeneity. ALK (D5F3) double-blind review analysis showed 201 (9.0%) ALK-positive cases, 10 (0.4%) ALK-heterogeneous cases, and 2017 (90.5%) ALK-negative cases. The heterogeneity cases included 2 large cell neuroendocrine carcinomas, 1 lymphoid epithelioid carcinoma, and 7 squamous cell carcinomas. The percentages of tumor cells with strong granular cytoplasmic staining were 1% to 30%. The ALK FISH break apart signal of these ten cases were 0% to 12%, indicating ALK FISH negativity. Nine ALK-heterogeneous cases were successfully detected by NGS, and no ALKgene variations (including gene fusion, copy number variation, insertion/deletion or single nucleotide variation) were found. Immunohistochemical staining showed that some ALK-heterogeneous cases showed neuroendocrine differentiation.TableComparison of IHC, FISH and NGS results of the ALK-heterogeneous cases.Case No.GenderAgeBiopsy/surgeryDiagnosisTumor cell contentALK IHCALK FISHALK FISHALK NGSALK NGS8thTNMStrong positive staining tumor cellsBreak apart signalInterpretation resultALK fusionALK INDEL/SNV/CNV1M69Wedge resectionLarge cell neuroendocrine carcinoma70%2%0%NegativeNegativeNegativepT1bN0M02M55LobectomyLarge cell neuroendocrine carcinoma85%20%4%NegativeNegativeNegativepT2bN0M03F33LobectomyLymphoid epithelioid carcinoma60%5%0%NegativeNegativeNegativepT3N0M04M68LobectomySquamous cell carcinoma70%5%6%NegativeNegativeNegativepT3N0M05M69LobectomySquamous cell carcinoma70%5%12%NegativeNegativeNegativepT2aN0M06M52LobectomySquamous cell carcinoma80%1%0%NegativeNegativeNegativepT2aN1M07M73LobectomySquamous cell carcinoma90%5%2%NegativeNegativeNegativepT3N0M08M70LobectomySquamous cell carcinoma80%5%4%NegativeNegativeNegativepT2bN1M09M69BiopsySquamous cell carcinoma85%15%0%NegativeNegativeNegativesT3N1M010M60BiopsySquamous cell carcinoma85%30%0%NegativeNANAcT2bN3M0Abbreviations: INDEL, insertion and deletion; SNV, single nucleotide variation; CNV, copy number variation; NA, NGS was not performed due to insufficient tumor tissues. Open table in a new tab Abbreviations: INDEL, insertion and deletion; SNV, single nucleotide variation; CNV, copy number variation; NA, NGS was not performed due to insufficient tumor tissues. Multi-platform detection of ALK-heterogeneous cases did not show evidence of ALKgene variation, and the effect of ALK-TKI treatment was unknown. therefore, the VENTANA ALK scoring interpretation guide for non-small cell lung carcinoma should be revised. It is recommended that ALK-heterogeneous cases be defined as ALK (D5F3) uncertain equivocal cases. The molecular pathology report should clearly state that the clinical significance is unclear, and it is recommended to conduct further testing by FISH or NGS.

Fluorescence in Situ Hybridization (FISH) for Detecting Anaplastic Lymphoma Kinase (ALK) Rearrangement in Lung Cancer: Clinically Relevant Technical Aspects

In 2011, the Vysis Break Apart ALK fluorescence in situ hybridization (FISH) assay was approved by the United States Food and Drug Administration as a companion diagnostic for detecting ALK rearrangement in lung cancer patients who may benefit from treatment of tyrosine kinase inhibitor therapy. This assay is the current “gold standard”. According to updated ALK testing guidelines from the College of American Pathologists, the International Association for the Study of Lung Cancer and the Association for Molecular Pathology published in 2018, ALK immunohistochemistry is formally an alternative to ALK FISH, and simultaneous detection of multiple hot spots, including, at least, ALK, ROS1, RET, MET, ERBB2, BRAF and KRAS genes is also recommended while performing next generation sequencing (NGS)-based testing. Therefore, ALK status in a specimen can be tested by different methods and platforms, even in the same institution or laboratory. In this review, we discuss several clinically relevant technical aspects of ALK FISH, including pros and cons of the unique two-step (50- to 100-cell) analysis approach employed in the Vysis Break Apart ALK FISH assay, including: the preset cutoff value of ≥15% for a positive result; technical aspects and biology of discordant results obtained by different methods; and incidental findings, such as ALK copy number gain or amplification and co-existent driver mutations. These issues have practical implications for ALK testing in the clinical laboratory following the updated guidelines.

Anaplastic lymphoma kinase 5A4 immunohistochemistry as a diagnostic assay in lung cancer: A Canadian reference testing center's results in population-based reflex testing.

The presence of anaplastic lymphoma kinase (ALK) rearrangement predicts response to ALK tyrosine kinase inhibitor (TKI) therapy. Fluorescence in situ hybridization (FISH) was the initial reference standard to detect ALK rearrangement, but immunohistochemistry (IHC) using D5F3 has gained acceptance as an alternative diagnostic method. ALK IHC assays using other ALK antibodies have also been used as screening methods, but data supporting their utility as diagnostic tests have not been widely reported. Data from reflexive clinical ALK IHC test using the 5A4 clone concurrent with epidermal growth factor receptor (EGFR) mutation testing were analyzed. ALK IHC results were reported as negative (-), equivocal, or positive (+), with equivocal or positive staining validated by FISH break-apart probe testing. Treatment outcomes were reviewed for ALK IHC+ patients. Between 2012 and 2015, 146 (2.5%) cases were reported as ALK IHC+, 188 (3.2%) were reported as equivocal, and 5624 (94.4%) were reported as ALK IHC-. Of the ALK IHC+ cases, 131/143(91.6%) were ALK FISH+. Excluding 6 cases in which FISH was inconclusive or not performed, the positive predictive value was 95.6%, and the negative predictive value was 100%. Most specimens (n=5352 [89.6%]) were also successfully tested for EGFR. Clinical responses to ALK TKIs were noted in 49 ALK IHC+ patients, with a median progression-free survival of 9.9months. ALK 5A4 IHC can serve as a robust diagnostic test for ALK-rearranged lung cancer and is associated with treatment response and survival. Optimized tissue allocation resulted in high success rates of combined reflex EGFR and ALK testing.

A study of ALK-positive pulmonary squamous-cell carcinoma: From diagnostic methodologies to clinical efficacy

BackgroundHigh concordance has been observed between Ventana D5F3 ALK immunohistochemistry (IHC) and fluorescence in-situ hybridization (FISH) in lung adenocarcinoma (LADC). However, whether a similar conclusion can be applied to lung squamous-cell carcinoma (LSCC) has remained unclear. We therefore evaluated the ALK (anaplastic lymphoma kinase) status and the therapeutic effect of an ALK tyrosine kinase inhibitor (TKI) in IHC- or FISH-positive LSCC. Materials and methodsA total of 2403 LSCC patients from three institutions were screened for ALK aberration by IHC. All IHC-positive cases were subjected to FISH (with an approximately equal number of negative cases as a control group) and next-generation sequencing (NGS). Clinical efficacy was evaluated for the patients who received TKI therapy. ResultsIn 2403 cases of LSCC, 37 cases were identified as ALK-positive by IHC. After quality control, 28 cases were succeeded by FISH (six with insufficient tissue, three with lack of signals) and 13 by NGS (24 failed due to insufficient samples or poor DNA quality); the percentage of non-diagnostic tests was 24.3% (9/37) and 64.9% (24/37), respectively. Four cases (4/2394, 0.17%) analyzed by FISH were determined as ALK-positive. For the control group (40 ALK IHC), FISH demonstrated no samples with ALK gene fusion. The concordance between ALK IHC- and ALK FISH-positive results was 14.3% (4/28). In the 13 cases studied by NGS, two cases showed ALK-EML4 fusion (consistent with two FISH-positive results), and two cases were interpreted as harboring an ALK-association gene mutation. Among four patients (two FISH-positive and two IHC-positive only cases) receiving TKI therapy, two patients had stable disease and the other two had progressive disease. ConclusionsThe positive concordance rate of ALK IHC and FISH in LSCC is far less than that reported for LADC. Therefore, ALK IHC detection in LSCC cannot be used as a diagnostic method for ALK rearrangement.