ALK fusion and its association with other driver gene mutations in Finnish non‐small cell lung cancer patients

Screening of anaplastic lymphoma tyrosine kinase (ALK) gene fusions in non‐small cell lung cancer (NSCLC) patients enables the identification of the patients likely to benefit from ALK‐targeted therapy. Our aim was to assess the prevalence of ALK fusion in Finnish NSCLC patients, which has not been reported earlier, and to study the presence of ALK fusion in relation to clinicopathological characteristics and other driver gene mutations. A total of 469 formalin‐fixed paraffin‐embedded tumor tissue specimens from Finnish NSCLC patients were screened for ALK fusion by immunohistochemistry (IHC). For confirmation of IHC results, fluorescence in situ hybridization (FISH) was conducted for 171 specimens. Next‐generation sequencing was performed for all ALK‐positive specimens to characterize the association of ALK fusion with mutations in targeted regions of 22 driver genes. Of the 469 tumors screened, 11 (2.3%) harbored an ALK fusion, including nine adenocarcinomas and two large cell carcinomas. The IHC results for all 11 ALK‐positive and 160 random ALK‐negative specimens were confirmed by FISH. ALK fusion was significantly associated with never/ex‐light smoking history (P < 0.001) and younger age (P = 0.004). Seven ALK‐positive tumors showed additional mutations; three in MET, one in MET and CTNNB1, two in TP53, and one in PIK3CA. Our results show that ALK fusion is an infrequent alteration in Finnish NSCLC patients. Although the majority of ALK‐positive cases were adenocarcinomas, the fusion was also seen in large cell carcinomas. Further studies are needed to elucidate the clinical significance of the coexistence of ALK fusion with MET, TP53, CTNNB1, and PIK3CA mutations. © 2014 Wiley Periodicals, Inc.     

Oncogenic role of miR‐155 in anaplastic large cell lymphoma lacking the t(2;5) translocation

Anaplastic large cell lymphoma (ALCL) is a rare, aggressive, non‐Hodgkin's lymphoma that is characterized by CD30 expression and disease onset in young patients. About half of ALCL patients bear the t(2;5)(p23;q35) translocation, which results in the formation of the nucleophosmin‐anaplastic lymphoma tyrosine kinase (NPM–ALK) fusion protein (ALCL ALK⁺). However, little is known about the molecular features and tumour drivers in ALK‐negative ALCL (ALCL ALK^?), which is characterized by a worse prognosis. We found that ALCL ALK^?, in contrast to ALCL ALK⁺, lymphomas display high miR‐155 expression. Consistent with this, we observed an inverse correlation between miR‐155 promoter methylation and miR‐155 expression in ALCL. However, no direct effect of the ALK kinase on miR‐155 levels was observed. Ago2 immunoprecipitation revealed miR‐155 as the most abundant miRNA, and enrichment of target mRNAs C/EBPβ and SOCS1. To investigate its function, we over‐expressed miR‐155 in ALCL ALK⁺ cell lines and demonstrated reduced levels of C/EBPβ and SOCS1. In murine engraftment models of ALCL ALK^?, we showed that anti‐miR‐155 mimics are able to reduce tumour growth. This goes hand‐in‐hand with increased levels of cleaved caspase‐3 and high SOCS1 in these tumours, which leads to suppression of STAT3 signalling. Moreover, miR‐155 induces IL‐22 expression and suppresses the C/EBPβ target IL‐8. These data suggest that miR‐155 can act as a tumour driver in ALCL ALK^? and blocking miR‐155 could be therapeutically relevant. Original miRNA array data are to be found in the supplementary material (Table S1). © 2015 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.     

Molecular breakdown: a comprehensive view of anaplastic lymphoma kinase (ALK)‐rearranged non‐small cell lung cancer

Most anaplastic lymphoma kinase (ALK)‐rearranged non‐small cell lung cancers (NSCLCs) show good clinical response to ALK inhibitors. However, some ALK‐rearranged NSCLC patients show various primary responses with unknown reasons. Previous studies focused on the clinical aspects of ALK fusions in small cohorts, or were conducted in vitro and/or in vivo to investigate the function of ALK. One of the suggested theories describes how echinoderm microtubule‐associated protein‐like 4 (EML4)–ALK variants play a role towards different sensitivities in ALK inhibitors. Until now, there has been no integrated comprehensive study that dissects ALK at the molecular level in a large scale. Here, we report the largest extensive molecular analysis of 158 ALK‐rearranged NSCLCs and have investigated these findings in a cell line construct experiment. We discovered that NSCLCs with EML4–ALK short forms (variant 3/others) had more advanced stage and frequent metastases than cases with the long forms (variant 1/others) (p = 0.057, p < 0.05). In vitro experiments revealed that EML4–ALK short forms show lower sensitivity to ALK inhibitors than do long forms. Clinical analysis also showed a trend for the short forms showing worse PFS. Interestingly, we found that breakpoints of ALK are evenly distributed mainly in intron 19 and almost all of them undergo a non‐homologous end‐joining repair to generate ALK fusions. We also discovered four novel somatic ALK mutations in NSCLC (T1151R, R1192P, A1280V, and L1535Q) that confer primary resistance; all of them showed strong resistance to ALK inhibitors, as G1202R does. Through targeted deep sequencing, we discovered three novel ALK fusion partners (GCC2, LMO7, and PHACTR1), and different ALK fusion partners showed different intracellular localization. With our findings that the EML4–ALK variants, new ALK somatic mutations, and novel ALK‐fusion partners may affect sensitivity to ALK inhibitors, we stress the importance of targeted therapy to take the ALK molecular profiling into consideration. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Discordance between anaplastic lymphoma kinase status in primary non‐small‐cell lung cancers and their corresponding metastases

Aims: The anaplastic lymphoma kinase gene (ALK) has attracted considerable attention as a potential molecular target in non‐small‐cell lung cancer (NSCLC). However, it is unclear whether ALK alterations are acquired during the metastatic progression of NSCLC. Methods and results: ALK status and ALK expression were evaluated in a series of 67 primary NSCLCs and their corresponding metastatic lesions using fluorescence in‐situ hybridization and immunohistochemistry. ALK rearrangement was detected in 7.5% (5/67) of the primary tumours and in 9.0% (6/67) of the metastases (P < 0.001). ALK copy number gain (CNG) was detected in 1.5% (1/67) of the primary tumours and in 35.8% (24/67) of the metastases. Whereas ALK rearrangement was detected only in adenocarcinomas, CNG was identified in various histological subtypes of NSCLC. ALK expression was detected in 11.9% (8/67) of the primary tumours and in 25.4% (17/67) of the metastatic lesions. Conclusions: ALK alteration and ALK expression can be acquired during metastatic progression in NSCLC, and ALK CNG is associated with ALK expression.     

Anaplastic lymphoma kinase translocation is correlated with anaplastic lymphoma kinase expression and mutually exclusive with epidermal growth factor receptor mutation in Taiwanese non‐small cell lung cancer

The echinoderm microtubule‐associated protein‐like 4‐anaplastic lymphoma kinase (EML4‐ALK) fusion gene is an important biomarker for target therapy. The aim of this study is to better understand the clinical and molecular features of the EML4‐ALK fusion gene in lung cancer patients in Taiwan and therefore to generate an efficient algorithm for the detection of ALK translocation. In the first cohort, ALK translocation was identified in 1 adenocarcinoma from 100 lung cancer patients by using break apart fluorescent in situ hybridization (FISH). Next, we detected 6 ALK translocations in another 40 EGFR wild type adenocarcinomas but not in 40 cases with EGFR mutation. Histological analysis revealed that solid growth with signet‐ring cells or cribriform glands with extracellular mucin were noted in all the 7 ALK translocated cases. One ALK positive cancer with mucinous cribriform pattern had no ALK expression. ALK expression was correlated with ALK translocation (p < 0.001), but not with ALK gene copy number gain (CNG) (P = 0.838). ALK translocation was also mutually exclusive with EGFR mutation in Taiwanese non‐small cell lung cancer (P = 0.033). These results indicate that screening tests for EGFR mutation status and/or ALK expression could help efficiently select ALK translocated patients for target therapy.     

Expression of carbohydrate antigens, p80NPM/ALK, cytotoxic cell-associated antigens, and Epstein-Barr virus gene products in anaplastic large cell lymphomas

The expression of carbohydrate antigens, including sialyl Lewis X (SLEX) and BNH9 antigen, the nucleophosmin (NPM)-anaplastic lymphoma kinase (ALK) fusion protein (p80^NPM/ALK), cytotoxic cell-associated antigens, and Epstein-Barr virus (EBV) gene products in CD30+ anaplastic large cell lymphoma (ALCL) was investigated by immunohistochemistry and in situ hybridization (ISH) methods. The expression of SLEX and BNH9 antigen in ALCL was examined using CSLEX1 and BNH9, which specifically react with SLEX and oligosaccharides (H and Y haptens), respectively. SLEX was expressed in seven of 12 ALCL and BNH9 was positive for five of 12 ALCL. With respect to the relationship between SLEX and BNH9 expression in ALCL, some ALCL expressed both antigens, which suggests that they might have an increased or preserved activity of glycosyltransferase that is responsible for the synthesis of the type I or type II core sequences, although other ALCL expressed either SLEX or BNH9. To detect p80^NPM/ALK in ALCL, the sections were immunostained with an anti-p80 antibody. Three of 12 ALCL expressed the NPM/ALK-encoded p80 protein. All three ALCL positive for p80^NPM/ALK expressed SLEX and two of them were stained with BNH9, which raised the possibility that p80 over-expression may be involved in the aberrant expression of type I or type II chains with varying degrees of fucosylation or sialylation. While the expression of cytotoxic cell-associated antigens such as CD8, CD56 and T cell intercellular antigen 1 (TIA-1) in ALCL was Immunohistochemlcally examined, none of the 12 ALCL expressed CD56 and only one case expressed CD8. TIA-1 was expressed in seven of 12 ALCL. Four of five BNH9- positive cases expressed TIA-1, suggesting that BNH9-positive cases tended to have TIA-1. In situ hybridization studies using an EBV-encoded RNA-1 (EBER-1) probe were performed on 12 ALCL to detect EBV in the lymphoma cells. EBER-1 signals were detected in the small lymphocytes but not in the lymphoma cells of two ALCL. However, latent membrane protein 1 immunoreactivlty was found In one case. These results appear to indicate that there is no strong association between EBV and ALCL.     

Screening for ALK abnormalities in central nervous system metastases of non‐small‐cell lung cancer

Anaplastic lymphoma kinase (ALK) gene rearrangement was reported in 3%–7% of primary non‐small‐cell lung cancer (NSCLC) and its presence is commonly associated with adenocarcinoma (AD) type and non‐smoking history. ALK tyrosine kinase inhibitors (TKIs) such as crizotinib, alectinib and ceritinib showed efficiency in patients with primary NSCLC harboring ALK gene rearrangement. Moreover, response to ALK TKIs was observed in central nervous system (CNS) metastatic lesions of NSCLC. However, there are no reports concerning the frequency of ALK rearrangement in CNS metastases. We assessed the frequency of ALK abnormalities in 145 formalin fixed paraffin embedded (FFPE) tissue samples from CNS metastases of NSCLC using immunohistochemical (IHC) automated staining (BenchMark GX, Ventana, USA) and fluorescence in situ hybridization (FISH) technique (Abbot Molecular, USA). The studied group was heterogeneous in terms of histopathology and smoking status. ALK abnormalities were detected in 4.8% (7/145) of CNS metastases. ALK abnormalities were observed in six AD (7.5%; 6/80) and in single patients with adenosuqamous lung carcinoma. Analysis of clinical and demographic factors indicated that expression of abnormal ALK was significantly more frequently observed (P = 0.0002; χ² = 16.783) in former‐smokers. Comparison of IHC and FISH results showed some discrepancies, which were caused by unspecific staining of macrophages and glial/nerve cells, which constitute the background of CNS tissues. Their results indicate high frequency of ALK gene rearrangement in CNS metastatic sites of NSCLC that are in line with prior studies concerning evaluation of the presence of ALK abnormalities in such patients. However, they showed that assessment of ALK by IHC and FISH methods in CNS tissues require additional standardizations.     

Genetic instability and recurrent MYC amplification in ALK‐translocated NSCLC: a central role of TP53 mutations

The anaplastic lymphoma kinase (ALK) rearrangement defines a distinct molecular subtype of non‐small cell lung cancer (NSCLC). Despite the excellent initial efficacy of ALK inhibitors in patients with ALK+ lung cancer, resistance occurs almost inevitably. To date, there is no reliable biomarker allowing the identification of patients at higher risk of relapse. Here, we analysed a subset of 53 ALK+ tumours with and without TP53 mutation and ALK+ NSCLC cell lines by NanoString nCounter technology. We found that the co‐occurrence of early TP53 mutations in ALK+ NSCLC can lead to chromosomal instability: 24% of TP53‐mutated patients showed amplifications of known cancer genes such as MYC (14%), CCND1 (10%), TERT (5%), BIRC2 (5%), ORAOV1 (5%), and YAP1 (5%). MYC‐overexpressing ALK+ TP53‐mutated cells had a proliferative advantage compared to wild‐type cells. ChIP‐Seq data revealed MYC‐binding sites within the promoter region of EML4, and MYC overexpression in ALK+ TP53‐mutated cells resulted in an upregulation of EML4–ALK, indicating a potential MYC‐dependent resistance mechanism in patients with increased MYC copy number. Our study reveals that ALK+ NSCLC represents a more heterogeneous subgroup of tumours than initially thought, and that TP53 mutations in that particular cancer type define a subset of tumours that harbour chromosomal instability, leading to the co‐occurrence of pathogenic aberrations. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.     

Vocal cord inflammatory myofibroblastic tumor with mucoid deposits harboring TIMP3–ALK fusion: A potential diagnostic pitfall

A 35‐year‐old Japanese man who had experienced hoarseness for 10 years presented with a vocal cord lesion. A gross examination revealed a left vocal cord polyp occupying two‐thirds of the vocal space. The endoscopically resected lesion contained scattered atypical fibroblastic, stellate, or ganglion‐like cells with mucoid stroma. Vacuolated cells were also seen. Lymphoplasmacytic infiltrate was largely undetectable. A vocal cord polyp was first suspected, but well‐differentiated liposarcoma and inflammatory myofibroblastic tumor (IMT) were included in the differential diagnoses. The tumor cells were positive for anaplastic lymphoma kinase (ALK), calponin, and vimentin, and negative for other smooth muscle markers by immunohistochemistry. Structures resembling myofibroblasts were not observed by electron microscopy, which confirmed abundant rough endoplasmic reticulum in the tumor cells and accumulated lipid droplets in some tumor cells. ALK gene rearrangement was detected by fluorescence in situ hybridization, and TIMP3–ALK fusion was confirmed by 5′ rapid ampli?cation of cDNA ends. We diagnosed the lesion as an IMT, and an ALK‐rearranged stellate cell tumor may be postulated. This is the first report of a fusion partner gene of ALK in a case of laryngeal IMT.     

Novel PPP1CB‐ALK fusion in spindle cell tumor defined by S100 and CD34 coexpression and distinctive stromal and perivascular hyalinization

A novel group of S100‐ and CD34‐positive spindle cell tumors with distinctive stromal and perivascular hyalinization harboring recurrent gene fusions involving kinases including RAF1, BRAF, NTRK1/2/3, and RET have been recently reported. To our knowledge, no such cases harboring ALK rearrangements have been identified. We report a previously healthy 41‐year‐old male with a 12‐cm intramuscular shoulder mass. The tumor was composed of bland‐appearing spindled to epithelioid cells, arranged in a patternless pattern in a background of loose myxoid stroma containing striking amianthoid‐like stromal collagen and perivascular rings. In accordance with the previously reported tumors, the tumor cells showed diffuse immunopositivity with S100 and CD34, while lacking SOX10 expression. Targeted RNA‐based next‐generation sequencing identified a novel serine/threonine‐protein phosphatase PP1‐beta‐catalytic subunit (PPP1CB)‐ALK fusion gene. Although ALK break‐apart was not detected by FISH, likely due to a paracentric inversion of chromosome 2, the presence of the fusion was confirmed by Sanger sequencing showing a 10‐bp linker between exon 6 of PPP1CB and intron 19 of ALK while maintaining reading frame. Subsequent ALK‐1 immunostain exhibited diffuse cytoplasmic staining in the tumor cells. Our case expands the molecular genetic spectrum of the distinctive group of spindle cell tumors with CD34/S100+ immunophenotype, supporting the important role of various kinases as drivers of oncogenesis. Awareness of this entity including its unique morphologic and immunophenotypic features as well as its interchangeable kinase gene fusions is crucial for correct classification and potential targeted therapy, particularly in aggressive subsets.     

Detection of EML4-ALK fusion genes in non-small cell lung cancer patients with clinical features associated with EGFR mutations

EML4‐ALK fusion genes have been recognized as novel “driver mutations” in a small subset of non‐small cell lung cancers (NSCLC). The frequency of EML4‐ALK fusions in NSCLC patients who have clinical characteristics related to EGFR mutation remains unknown. We screened 102 Chinese patients with NSCLC based on one or more of the following characteristics: female, no or light smoking history, and adenocarcinoma histology. EML4‐ALK fusion genes were identified by RT‐PCR, whereas EGFR (Exons 18–21) and KRAS (Exons 1 and 2) mutations were detected by DNA sequencing. Eight specimens (8%) were positive for EML4‐ALK fusions, with seven being Variant 1 and one Variant 2. There were 44 (43%) and 17 (16%) patients harboring EGFR and KRAS mutations, respectively. Thirty‐one (31%) cases were wild type for EML4‐ALK, EGFR, and KRAS mutations. Of the eight patients with EML4‐ALK, none had an EGFR mutation, whereas a KRAS mutation was detected in one patient. Histologically, five of the EML4‐ALK positive tumors were adenocarcinoma and two were mixed adenosquamous carcinoma; only one was a squamous carcinoma. Our data support the conclusion that the EML4‐ALK fusion gene defines a new molecular subset of NSCLC with distinct pathologic features. © 2012 Wiley Peiodicals, Inc.     

Analysis of ALK,MYCN, and the ALK ligand ALKAL2 (FAM150B/AUGα) in neuroblastoma patient samples with chromosome arm 2p rearrangements

Gain of chromosome arm 2p is a previously described entity in neuroblastoma (NB). This genomic address is home to two important oncogenes in NB—MYCN and anaplastic lymphoma kinase (ALK). MYCN amplification is a critical prognostic factor coupled with poor prognosis in NB. Mutation of the ALK receptor tyrosine kinase has been described in both somatic and familial NB. Here, ALK activation occurs in the context of the full‐length receptor, exemplified by activating point mutations in NB. ALK overexpression and activation, in the absence of genetic mutation has also been described in NB. In addition, the recently identified ALK ligand ALKAL2 (previously described as FAM150B and AUGα) is also found on the distal portion of 2p, at 2p25. Here we analyze 356 NB tumor samples and discuss observations indicating that gain of 2p has implications for the development of NB. Finally, we put forward the hypothesis that the effect of 2p gain may result from a combination of MYCN, ALK, and the ALK ligand ALKAL2.     

Analysis of nucleophosmin–anaplastic lymphoma kinase (NPM‐ALK)‐reactive CD8+ T cell responses in children with NPM‐ALK+ anaplastic large cell lymphoma

Cellular immune responses against the oncoantigen anaplastic lymphoma kinase (ALK) in patients with ALK‐positive anaplastic large cell lymphoma (ALCL) have been detected using peptide‐based approaches in individuals preselected for human leucocyte antigen (HLA)‐A*02:01. In this study, we aimed to evaluate nucleophosmin (NPM)‐ALK‐specific CD8⁺ T cell responses in ALCL patients ensuring endogenous peptide processing of ALK antigens and avoiding HLA preselection. We also examined the HLA class I restriction of ALK‐specific CD8⁺ T cells. Autologous dendritic cells (DCs) transfected with in‐vitro‐transcribed RNA (IVT‐RNA) encoding NPM–ALK were used as antigen‐presenting cells for T cell stimulation. Responder T lymphocytes were tested in interferon‐gamma enzyme‐linked immunospot (ELISPOT) assays with NPM–ALK‐transfected autologous DCs as well as CV‐1 in Origin with SV40 genes (COS‐7) cells co‐transfected with genes encoding the patients’ HLA class I alleles and with NPM–ALK encoding cDNA to verify responses and define the HLA restrictions of specific T cell responses. NPM–ALK‐specific CD8⁺ T cell responses were detected in three of five ALK‐positive ALCL patients tested between 1 and 13 years after diagnosis. The three patients had also maintained anti‐ALK antibody responses. No reactivity was detected in samples from five healthy donors. The NPM–ALK‐specific CD8⁺ T cell responses were restricted by HLA‐C‐alleles (C*06:02 and C*12:02) in all three cases. This approach allowed for the detection of NPM–ALK‐reactive T cells, irrespective of the individual HLA status, up to 9 years after ALCL diagnosis.     

荧光原位杂交检测石蜡包埋间变性大细胞淋巴瘤病例中ALK基因转位及其意义

目的　比较荧光原位杂交 (fluorescence in situ hybridization,FISH)和免疫组织化学在检测间变性大细胞淋巴瘤 (anaplastic large cell lymphoma,AL CL )中间变性淋巴瘤激酶 (anaplastic lymphomakinase,AL K)基因转位及其融合蛋白中的作用 ,并探讨 FISH在石蜡包埋组织中的应用。方法　采用双色FISH和免疫组织化学检测 2 2例石蜡包埋 AL CL病例中 AL K基因转位及其融合蛋白。结果　通过调整组织切片的酶消化时间等优化措施 ,成功地在石蜡切片上进行了双色 FISH实验 ;FISH和免疫组织化学均在 6 0 % (12 /2 0 )系统性 AL CL中检测到 AL K基因转位或融合蛋白 ,在 2例皮肤原发 AL CL中未检测到基因转位或融合蛋白 ,两种方法的符合率为 10 0 %。结论　 (1)在检测 AL CL中有无 AL K基因转位时 ,AL K蛋白免疫组化由于其简单、快捷、价廉成为一般情况下的首选方法 ;在具备 FISH条件时 ,也可以将 FISH作为首选 ;(2 )通过优化实验条件 ,可以在石蜡包埋组织上成功地进行 FISH实验。     

A small cell variant of ALK-positive, CD8-positive anaplastic large cell lymphoma with primary subcutaneous presentation mimicking subcutaneous panniculitis-like T-cell lymphoma

ALK-positive anaplastic large cell lymphoma (ALK+ ALCL) is an uncommon non-Hodgkin's lymphoma of T-cell origin, the majority of which express CD4 and show frequent pan-T-cell antigen loss. While most cases of ALK+ ALCL have the common pattern characterized by anaplastic morphology with hallmark cells, a less common but well-recognized variant with a small cell pattern may pose a diagnostic challenge. We report a case of ALK+ ALCL with small cell morphology and CD8 subset restriction in a 53-year-old male patient who presented primarily with multiple recurrent subcutaneous nodules with histopathologic features simulating a subcutaneous panniculitis-like T-cell lymphoma (SPTCL). The case was initially diagnosed as SPTCL but was reconsidered as ALK+ ALCL when the incidental finding of CD30 positivity on a subsequent biopsy prompted an ALK immunostain, which turned out to be positive in the neoplastic T-cells. The diagnosis of ALK+ ALCL, small cell variant, was then confirmed by detection of an ALK gene rearrangement by FISH analysis. This report highlights a case of ALK+ ALCL with a deceiving clinical and histopathologic presentation, and emphasizes the value of immunohistochemical panel studies and genetic tests in such cases to avoid diagnostic errors.