Anaplastic Lymphoma Kinase Detection Research Articles

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.

Case report: ALK-positive histiocytosis presented as bilateral synchronous breast masses with long-term remission on crizotinib

ALK-positive histiocytosis (APH) is a rare type of histiocytic neoplasm with characteristic ALK (Anaplastic Lymphoma Kinase) gene translocation and fusion, with only 27 reported cases in the literature. In this study, we report the first case of synchronous bilateral breast involvement of ALK-positive histiocytosis on initial presentation in a 46-year-old Hispanic woman. APH was diagnosed by the confirmation of clonal histiocyte proliferation with ALK overexpression on IHC and the presence of KIF5B-ALK gene fusion from her breast and lung biopsies. The patient in our study is currently under complete and long-term remission with crizotinib treatment (an ALK inhibitor). This report expands on the clinical manifestation of APH, emphasizes the importance of ALK detection in histiocytic diseases, and provides the efficacy and long-term prognosis of the ALK inhibitor therapy for APH.

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

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

ALK rearrangement in TFE3-positive renal cell carcinoma: Alternative diagnostic option to exclude Xp11.2 translocation carcinoma

Anaplastic lymphoma kinase (ALK)-rearranged renal cell carcinoma (RCC) is a rare subtype of RCC with gene fusion involving ALK at 2p23. It was first included in the renal tumor classification system by WorldHealth organization (WHO) as a distinct emerging/provisional renal entity in 2016. To date, only a few cases of ALK-RCC have been reported. Here, we report an exceptional case of ALK-RCC in a 15-year-old girl and review the literature. The patient presented with gross hematuria and a tumor measured 7 cm × 6 cm was found in the left kidney by imaging examination. Then a laparoscopic radical nephrectomy combined with local lymph node dissection was performed. The pathologic stage of the tumor was pT1bN1Mx and postoperative pathology showed that the tumor corresponded to WHO/ISUP grade 3–4. Immunohistochemistry (IHC) demonstrated moderate nuclear expression of TFE3 protein. Interestingly, ALK gene rearrangement rather than TFE3 gene rearrangement was observed by fluorescence in situ hybridization (FISH). Now the girl is still alive without evidence of recurrence for 10 months follow-up. In conclusion, the positive expression of nuclear TFE3 in immunohistochemistry may be deceptive, the detection of ALK could be a diagnostic option if TFE3 was negative in FISH study. Large-scale and long-term studies are still needed to explore the biological behavior and molecular characteristic of ALK-RCC.

306P Detection of anaplastic lymphoma kinase (ALK) mutations using circulating tumour DNA (ctDNA) in advanced non-squamous non-small cell lung cancer (non-Sq-NSCLC) in Asia

306P Detection of anaplastic lymphoma kinase (ALK) mutations using circulating tumour DNA (ctDNA) in advanced non-squamous non-small cell lung cancer (non-Sq-NSCLC) in Asia

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

Clinical evaluation of the effectiveness of fusion-induced asymmetric transcription assay-based reverse transcription droplet digital PCR for ALK detection in formalin-fixed paraffin-embedded samples from lung cancer.

BackgroundAccurate detection of anaplastic lymphoma kinase (ALK) rearrangement is the prerequisite for anti‐ALK therapy for the patient with non‐small cell lung cancer (NSCLC). Fusion‐induced asymmetric transcription assay (FIATA)‐based reverse transcription droplet digital PCR (RT‐ddPCR) was developed and performed for ALK status survey in NSCLC samples.MethodsA total of 269 cases of formalin‐fixed paraffin‐embedded (FFPE) specimens from NSCLC, in which ALK status was confirmed by both fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC), were analyzed by FIATA‐based RT‐ddPCR.ResultsIn the ALK‐positive group, the 3′ ALK transcript copies range was 336.6–107 955.4, and the R3 [(the ratio of the 3′ ALK transcript copy numbers to the internal reference gene transcript copy numbers) × 100] was 17.23–672.77. In the ALK‐negative group, the 3′ ALK transcript copies range was 3.7–1370.6, and the R3 range was 0.10–15.57. The lowest R3 level in the ALK‐positive group was significantly higher than the highest R3 level in the ALK‐negative group. A positive correlation between the proportion of cancer cells in the tissue section and ALK RNA expression level (R3) was found (P < 0.05). There was no relationship between the percentage of FISH positive cells or FISH positive signal patterns and R3 level of the ALK gene. Compared with FISH and IHC, the clinical sensitivity and specificity of FIATA‐based RT‐ddPCR for ALK detection were 100%, respectively.ConclusionsAn absolute quantitative FIATA‐based RT‐ddPCR was developed and validated for ALK fusion detection in NSCLC. This method can rapidly, accurately, and objectively classify ALK types and help with individual therapy.

The Use of Three-Dimensional DNA Fluorescent In Situ Hybridization (3D DNA FISH) for the Detection of Anaplastic Lymphoma Kinase (ALK) in Non-Small Cell Lung Cancer (NSCLC) Circulating Tumor Cells.

Tumor tissue biopsy is often limited for non-small cell lung cancer (NSCLC) patients and alternative sources of tumoral information are desirable to determine molecular alterations such as anaplastic lymphoma kinase (ALK) rearrangements. Circulating tumor cells (CTCs) are an appealing component of liquid biopsies, which can be sampled serially over the course of treatment. In this study, we enrolled a cohort of ALK-positive (n = 8) and ALK-negative (n = 12) NSCLC patients, enriched for CTCs using spiral microfluidic technology and performed DNA fluorescent in situ hybridization (FISH) for ALK. CTCs were identified in 12/20 NSCLC patients ranging from 1 to 26 CTCs/7.5 mL blood. Our study revealed that 3D imaging of CTCs for ALK translocations captured a well-defined separation of 3′ and 5′ signals indicative of ALK translocations and overlapping 3′/5′ signal was easily resolved by imaging through the nuclear volume. This study provides proof-of-principle for the use of 3D DNA FISH in the determination of CTC ALK translocations in NSCLC.

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.

Feasibility of liquid biopsy using plasma and platelets for detection of anaplastic lymphoma kinase rearrangements in non-small cell lung cancer

PurposeFluorescence in situ hybridization (FISH) using tumor tissue is the gold standard for detection of anaplastic lymphoma kinase (ALK) rearrangement in non-small cell lung cancer (NSCLC). However, this method often is not repeatable due to difficulties in the acquisition of tumor tissues. Blood-based liquid biopsy using reverse transcription polymerase chain reaction (RT-PCR) is expected to be useful to overcome this limitation. Here, we investigated the feasibility of liquid biopsy using plasma and platelets for detection of ALK rearrangement and prediction of ALK inhibitor treatment outcomes.MethodsALK-FISH assays were performed in 1128 tumor specimens of NSCLC between January 2015 and June 2018. We retrospectively analyzed formalin-fixed paraffin-embedded (FFPE) tissues from previously confirmed FISH-positive (n = 199) and -negative (n = 920) cases. We recruited patients who had available tissue specimens and agreed to venous sampling. RNA was extracted from FFPE blocks, plasma, and platelets. Fusion RNA of echinoderm microtubule-associated protein-like 4 (EML4)-ALK was detected by quantitative PCR.ResultsThirty-three FISH-positive and 28 FISH-negative patients were enrolled. In validation, data compared with FISH, RT-PCR using FFPE tissues showed 54.5% sensitivity, 78.6% specificity, and 75.5% accuracy. Liquid biopsy had higher sensitivity (78.8%), specificity (89.3%) and accuracy (83.6%). Higher positivity for liquid biopsy was shown in subgroups with delayed (≥ 6 months from diagnosis) blood sampling (plasma, 85.7%; platelets, 87.0%). In 26 patients treated with crizotinib, the platelet-positive subgroup showed longer median duration of treatment (7.2 versus 1.5 months), longer median progression-free survival (5.7 months versus 1.7 months), a higher overall response rate (70.6% versus 11.1%), and a higher disease control rate (88.2% versus 44.4%) than the platelet-negative subgroup.ConclusionLiquid biopsy could have applications in the diagnosis of ALK-positive NSCLC, even when using RT-PCR, and platelets can be useful for predicting treatment outcomes of ALK inhibitors.

Expression of C-terminal ALK, RET, or ROS1 in lung cancer cells with or without fusion

BackgroundGenetic alterations, including mutation of epidermal growth factor receptor or v-Ki-ras2 kirsten rat sarcoma viral oncogene homolog and fusion of anaplastic lymphoma kinase (ALK), RET proto-oncogene (RET), or v-ros UR2 sarcoma virus oncogene homolog 1 (ROS1), occur in non-small cell lung cancers, and these oncogenic drivers are important biomarkers for targeted therapies. A useful technique to screen for these fusions is the detection of native carboxy-terminal (C-terminal) protein by immunohistochemistry; however, the effects of other genetic alterations on C-terminal expression is not fully understood. In this study, we evaluated whether C-terminal expression is specifically elevated by fusion with or without typical genetic alterations of lung cancer.MethodsIn 37 human lung cancer cell lines and four tissue specimens, protein and mRNA levels were measured by capillary western blotting and reverse transcription–PCR, respectively.ResultsCompared with the median of all 37 cell lines, mRNA levels at the C-terminus of all five of the fusion-positive cell lines tested (three ALK, one RET, and one ROS1) were elevated at least 2000-, 300-, or 2000-fold, respectively, and high C-terminal protein expression was detected. In an ALK fusion–positive tissue specimen, the mRNA and protein levels of C-terminal ALK were also markedly elevated. Meanwhile, in one of 36 RET fusion–negative cell lines, RET mRNA levels at the C-terminus were elevated at least 500-fold compared with the median of all 37 cell lines, and high C-terminal protein expression was detected despite the absence of RET fusion.ConclusionsThis study of 37 cell lines and four tissue specimens shows the detection of C-terminal ALK or ROS1 proteins could be a comprehensive method to determine ALK or ROS1 fusion, whereas not only the detection of C-terminal RET protein but also other methods would be needed to determine RET fusion.

A Comparative Study of Cell Block versus Biopsy for Detection of Epidermal Growth Factor Receptor Mutations and Anaplastic Lymphoma Kinase Rearrangement in Adenocarcinoma Lung.

Background:Lung cancer is a leading cause of deaths attributed to cancer worldwide. Epidermal growth factor receptor (EGFR) mutation and anaplastic lymphoma kinase (ALK) rearrangement are commonly found in patients of adenocarcinoma lung against, which targeted therapy is available. In this era of personalized medicine, it is a rationale to detect these molecular alterations in cases of lung carcinomas.Aims:The objectives were to compare the diagnostic efficacy of cytological samples for the detection of EGFR and ALK protein expression using immunocytochemistry in nonsmall cell lung carcinoma.Materials and Methods:We compared 22 cell blocks and biopsies for the detection of EGFR and ALK protein expression by immunohistochemistry (IHC). EGFR IHC was performed using EGFR Receptor (E746-A750 del Specific) (6B6) monoclonal antibody and ALK IHC was done using Ventana anti-ALK (D5F3) monoclonal primary antibody.Results:Two cases were found to be positive; 20 cases were negative for EGFR IHC both in biopsies and cell blocks. ALK IHC was positive in one case; negative in 21 cases. The results of IHC were also concordant for biopsies and cell blocks. The sensitivity and specificity were 100% for immunocytochemical detection of ALK and EGFR in cell blocks with respect to biopsies.Conclusion:We conclude that cell blocks can serve as a potential substitute for biopsies for detection of EGFR and ALK protein by immunocytochemistry, whenever patient presents with effusion and biopsy cannot be done or when tissue is not adequate.

A digital PCR based assay to detect all ALK fusion species

Accurate detection of Anaplastic lymphoma kinase (ALK) fusion in lung cancer cells is essential to screen for patients suitable for targeted drug treatments such as crizotinib. ALK fusion involves multiple genes and different exon junctions. When an ALK fusion results in overexpression of the ALK protein, a malignant transformation can occur. The challenge is to develop a single test that will detect ALK overexpression from all fusion combinations including novel/unidentified fusion partner(s). In this study, we evaluated two strategies for detecting ALK fusion events using digital PCR: 5′/3′ imbalance and ALK overexpression relative to a reference gene. Our data shows that the determination of ALK RNA expression levels is a better option when a reference gene is included in the assay. We further determined the threshold to call for positive or negative samples and evaluated the analytical specifications of the assay in 28 FFPE samples from Non-small cell lung cancer (NSCLC) patients with know ALK fusion status. We validated this threshold with 36 clinical samples with ALK status determined by IHC. Digital PCR ALK assay we developed had a concordance of 97.2% (35/36). Testing the assay on clinical samples demonstrated consistency with reference assays, suggesting a great potential for the dPCR assay to service the clinical detection needs.

Ligation-dependent RT-PCR: a new specific and low-cost technique to detect ALK, ROS, and RET rearrangements in lung adenocarcinoma

Detection of anaplastic lymphoma kinase (ALK), ROS proto-oncogene 1 (ROS1), and rearranged during transfection (RET) gene rearrangements in lung adenocarcinoma is usually performed by immunohistochemistry (IHC) screening followed by fluorescence in situ hybridization (FISH), which is an expensive and difficult technique. Ligation-dependent reverse transcription polymerase chain reaction (RT-PCR) multiplex technique can detect gene rearrangements using probes specifically hybridized to either side of the break point. PCR products are then sequenced by pyrosequencing or high throughput sequencing in order to identify the two genes involved. The reagent cost is <15 dollars per patient and results are available in 2 days. We have developed a 47-probe LD-RT-PCR kit especially for lung adenocarcinomas. Thirty-nine lung adenocarcinomas were studied: 24 ALK+, 14 ROS1+, and 1 RET+. ALK+ and ROS1+ were IHC+ (D5F3 Ventana for ALK and D4D6 Cell Signaling Technology for ROS1) and all cases were FISH+ (Vysis ALK Breakapart Probe Abbott for ALK, Zytolight SPEC ROS1 Dualcolor Breakapart Probe for ROS1 and Zytolight SPEC RET Dual Color Breakapart for RET); 14 wild type samples were included as negative controls. Using LD-RT-PCR, 15 rearrangements (63%) were detected in the ALK cases (gene partner: EML4 in all cases), 9 rearrangements (64%) in the ROS1 cases (gene partners: CD74 in 8 cases and SLC34A2 in 1 case) and 1 (100%) in the single RET case (gene partner: KIF5B). No rearrangement was found in the 14 negative control cases. Negative cases using LD-RT-PCR could be explained by the fact that some partner genes were not included in our assay and therefore could not be detected. Because it is an affordable, fast, and very simple technique, we propose using LD-RT-PCR when ALK immunostaining is positive. For LD-RT-PCR-negative cases, samples should then be analyzed by FISH.

ALK immunohistochemistry is highly sensitive and specific for the detection of ALK translocated lung adenocarcinomas: lessons from an audit of lung cancer molecular testing.

Background The approval of novel targeted treatments for epidermal growth factor receptor (EGFR)-positive and anaplastic lymphoma kinase (ALK)-positive non-small cell lung cancer has led to the increased requirement for mutation testing. Results We report our experience of ALK testing with immunohistochemistry (IHC) and fluorescence in-situ hybridisation (FISH) and present the prevalence of EGFR, Kirsten rat sarcoma 2 viral oncogene homolog (KRAS) and ALK mutations. From January 2011 to May 2014, we found mutation rates of EGFR, KRAS and ALK to be 10.4% (67/643), 35.8% (86/240) and 2.3% (7/304), respectively. ALK-rearrangements were found to be associated with never smokers (p < 0.001) and younger patients (≤ 50 years old) (p < 0.001). ALK IHC protein expression in tumour cells is 100% sensitive (7 IHC+/7 FISH+) and 96.6% specific (113 IHC-/117 FISH-) for ALK-rearrangements by FISH. ALK-rearranged tumours were wildtype for EGFR and KRAS. Conclusion Our findings support the use of ALK protein expression and KRAS mutation testing as part of the molecular diagnostic algorithm for lung adenocarcinomas.

Detection of Anaplastic Lymphoma Kinase (ALK) gene in Non-Small Cell lung Cancer (NSCLC) By CISH Technique

Background/Aim: Anaplastic Lymphoma Kinase (ALK) rearrangement has evaluated activity of NSCLC compared with other molecular subtypes (EGFR, KRAS). Many studies demonstrated that patients with ALK rearrangement positive NSCLC have improved with a good response rates and progression (free survival) when treated with either monotherapy or by a combination therapy compared with EGFR-mutated, KRAS/EGFR/ALK wild type or KRAS-mutated. The aim of this study was to detect and study the signal pattern of normal ALK and compare it to that of mutated ALK with gene rearrangement in cases of non-small cell lung cancer and Inflammatory conditions by implementing the CISH technique. In addition to correlate ALK signal pattern with the histopathological type and grade as well as the age and sex of the patients.Materials &amp; Methods: Forty patients with NSCLC and Inflammatory diseases were enrolled in a comparative cross sectional study. The tissue blocks were sectioned on non-charged slides for the preparation of routine H&amp;E staining. Positively charged slides were used for tissue sections prepared for chromogenic in situ hybridization procedure to detect ALK gene.Results: ALK gene signal break apart was detected in (18/20, 90%) of malignant cases; (0/20, 0%) of non-neoplastic lung lesions. There was a significant statistical difference in their distribution, p &lt; 0.05. While There was no significant association between any disease status and sex P value = 1.000NS. The CISH test was 100% sensitive using negative score as a cutoff point and 90.9% specific. The score was divided into three levels that categorized the cases, so there were six cases in score one (1-32%), nine cases in score two (33-67%) and three cases in score three (68-100%).Conclusion: Detection of ALK rearrangement in the early diagnosis of NSCLC is highly sensitive and can save a lot of efforts in planning chemotherapy regimens. Results were very promising in identifying this mutation by a sensitive and highly specific test. The ALK gene rearrangement could be an early mutation and it is needed as an initiating step for the carcinogenesis process. The presence of a double gene mutation, however, could be the cause of a higher-grade cancer.

Development of a high-throughput and sensitive assay of fusion genes in lung cancer by array-based MALDI-TOFMS.

ALK (anaplastic lymphoma kinase gene), ROS1 (ros proto-oncogene 1) and RET (ret proto-oncogene) fusions are oncogenic drivers in non-small cell lung cancer (NSCLC). Methods like fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) are highly sensitive but subjectively analyzed, labor intensive, expensive and unsuitable for multiple fusion gene screening. This study aimed to establish a high-throughput, sensitive and cost-effective screening method (array-based matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, array-based MALDI-TOFMS) for ALK, ROS1 and RET fusion detection. This method was established with three fusion gene positive cell lines (H2228, ALK positive; HCC78, ROS1 positive; LC-2/AD, RET positive) and negative samples. Then, 34 clinical samples were selected and detected by Sanger sequencing, next generation sequencing (NGS) and array-based MALDI-TOFMS. The results were compared and analyzed and Sanger sequencing was considered the standard. 7 cases showed ALK fusions, 1 case showed ROS1 fusions, no case showed RET fusions and 4 cases were both ALK and ROS1 fusions. Results showed that array-based MALDI-TOFMS was 100% concordant with Sanger sequencing and NGS 82.3%. In this study, we reported the utility of array-based MALDI-TOFMS in the assessment of ALK, ROS1 and RET fusions in routine lung biopsies of FFPE and fresh tissue specimens. Besides, this method may also be applied to the diagnosis, monitoring and prognosis of illness.

Multiplex ALK, RET, and ROS1 fusion mutation detection in FFPE from lung cancer patients by MALDI-TOF mass spectrometry.

e13103 Background: Approximately 3-7% of lung tumors harbor anaplastic lymphoma kinase (ALK) fusions in the subgroup of non-small cell lung cancer (NSCLC). In addition to echinoderm microtubule-associated protein-like 4 (EML4)-ALK fusion, TRK-fused gene (TFG)-ALK, kinesin family member 5B (KIF5B)-ALK and kinesin light chain 1 (KLC1)-ALK had been reported in lung cancer. On the other hand, RET proto-oncogene (RET) and ROS proto-oncogene 1 (ROS1) fusion proteins also have prevalence in lung cancer. Food and Drug Administration (FDA)-approved several target drugs are available to treat patients with fusion mutations. Therefore, the diagnosis of ALK, RET or ROS1 fusion genes shows quite important. However, nowadays methods of detecting fusions such as fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) are limited to technique, low sensitivity, sample quality as well as subtype classification. Methods: We established nucleotide MALDI-TOF mass spectrometry based multiplex detection platform to distinguish major types including 9 types of EML4-ALK, 5 types of ALK, 5 types of RET and 8 types ROS1 fusions. Results: The detection limitation was about less 1% mutant cells among wild-type cells. In the pilot testing, we used 2 patients’ cell cDNA and 4 patients’ lung FFPE samples cDNA, which had been diagnosed as ALK fusion before, to be detected by this panel, and then identified their variant types successfully. Furthermore, one patient harbored CCDC6-RET fusion mutation was identified by our platform and confirmed by Sanger Sequencing. Conclusions: Taken together, this new panel has high sensitivity and allows little and poor quality samples for detecting. The correlation between clinical characteristics and fusion subtypes can be further investigated by utilizing this platform in the future. Also, the detection panel can be revised based on clinical needs by removing/adding probes.

Detection of ALK and KRAS Mutations in Circulating Tumor DNA of Patients With Advanced ALK-Positive NSCLC With Disease Progression During Crizotinib Treatment

BackgroundIn patients with anaplastic lymphoma kinase (ALK)-positive non–small-cell lung cancer (NSCLC), disease progression occurs after a median of 9 to 10 months of crizotinib treatment. Several mechanisms of resistance have been identified and include ALK mutations and amplification or the activation of bypassing signaling pathways. Rebiopsy in NSCLC patients represents a critical issue and the analysis of circulating cell-free DNA (cfDNA) has a promising role for the identification of resistance mechanisms. Patients and MethodsTwenty patients with advanced ALK-positive NSCLC were enrolled after disease progression during crizotinib treatment; cfDNA was analyzed using digital droplet polymerase chain reaction (BioRad, Hercules, CA) for ALK (p.L1196M, p.G1269A, and p.F1174L) and Kirsten rat sarcoma (KRAS) (codons 12 and 13) mutations. ResultsALK secondary mutations (p.L1196M, p.G1269A, and p.F1174L) were identified in 5 patients; 1 patient had 2 ALK mutations (p.L1196M and p.G1269A). Overall, 10 patients presented KRAS mutations (7 p.G12D, 2 p.G12V, and 1 p.G12C mutations, respectively). In 3 patients KRAS mutations were associated with ALK mutations. cfDNA was monitored during the treatment with second-generation ALK inhibitors and the amount of ALK as well as KRAS mutations decreased along with tumor regression. ConclusionALK and KRAS mutations are associated with acquired resistance to crizotinib in ALK-positive NSCLC. In particular, ALK acquired mutations can be detected in plasma and could represent a promising tumor marker for response monitoring.

A story of ALK variants and the efficacy of ALK inhibitors: moving toward precision oncology

In the era of precision medicine, rearrangement of the anaplastic lymphoma kinase ( ALK ) gene has been proven to be a targetable oncogenic driver in 3–7% of patients with advanced non-small cell lung cancer (NSCLC) (1). Multiple clinical trials have demonstrated the superiority of ALK inhibitors compared with chemotherapy for treating patients with ALK -rearranged NSCLC; however, the responses to ALK inhibitors have varied in each study (2-7). Fluorescence in situ hybridization (FISH) or VENTANA anti-ALK (D5F3) immunohistochemistry, which are widely used as standard tests for ALK detection for enrollment in clinical trials, are unable to distinguish between the different variants or fusion partners of the ALK gene. The impact of ALK variants on the heterogeneity of the response to ALK inhibitors has not been fully elucidated.