Anaplastic Lymphoma Kinase Gene Status 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.

ALK fusion typing and response to crizotinib in ALK+ lung cancer-naive patients.

e21008 Background: Anaplastic lymphoma kinase (ALK) is one of the major oncogenic driver genes in lung cancer, and fusion as the main mutation type, of which EML4 is the most common fusion ligand. With the development of next-generation sequencing (NGS), some other rare fusion ligands and complex fusions have been discovered. The prognosis of patients with EML4-ALK fusion, non EML4-ALK fusion and multiple ALK fusion was different. Methods: We retrospectively analyzed NGS (including tissue, blood, or other body fluid samples) in 61 patients with ALK[+] lung cancer, including 59 patients with NSCLC and 2 patients with SCLC. Grouping was determined according to whether it was EML4-ALK fusion and multiple fusion. Results: In our data, the partner fused to the ALK gene are diverse, including EML4, KIF5B, TSN and others. Among them, EML4 was the most common ligand in ALK[+] NSCLC, accounting for 93.2% (55/59). Among the different fusion sites of EML4-ALK, V1 (E13:A20) and V3 (E6:A20) mutations were the most common, accounting for 45.5% (25/55) and 41.8% (23/55), respectively. Several novel ALK fusion partners were discovered in this study, including MCFD2, LINC01251, MVP17, CREG2, LINCO1498 and so on. In addition, the incidence of intergenic sequence-ALK fusions was 16.4% (10/61), such as ANXA4-ALK(A[intergenic]:A20), CREG2-ALK (C[intergenic]:A20), HCN1-ALK(H[intergenic]:A20), TSN-ALK(T[intergenic]:A20), RSAD2-ALK(R [intergenic]:A20) and so on. 19.0% (12/61) of the patient had multiple fusion, of which only one patient had multiple fusion of EML4-ALK (V2+V5'+V3a/b), and the remaining patients had co-fusion of EML4-ALK and non EML4-ALK (Table). V3 were more likely to coexist with non EML4-ALK fusions than V1 or other variants (P = 0.047). Multiple fusions were more likely to occur with non EML4-ALK fusions (P < 0.000). Compared with EML4-ALK fusion, mPFS treated with crizotinib in non EML4-ALK fusion were significantly shorter (18.2 vs. 8.5 months, P = 0.035). In the context of crizotinib treatment, multiple fusions were associated with worse mPFS compared with single fusions (13.8 vs. 8.4 months, P = 0.008). In addition, For SCLC patients with ALK fusion (n = 2), the PFS of crizotinib treatment were 8.0 and 30.6 months, respectively. Conclusions: Due to the heterogeneity of tumor development resulting in the diversity of ALK fusions, non EML4-ALK fusions and multiple fusions imply a poorer response to crizotinib. ALK gene status prior to ALK-TKI therapy helps predict drug efficacy and patient prognosis.[Table: see text]

Clinicopathological Features in Elderly ALK-rearranged Non-small Cell Lung Cancer Patients.

To clarify the clinicopathological features in elderly anaplastic lymphoma kinase (ALK) rearranged non-small cell lung cancer (NSCLC) patients. A retrospective study was performed in 129 ALK rearranged NSCLC patients diagnosed between April 2008 and March 2019 in fifteen Institutions of the Ibaraki prefecture, Japan. Median age of patients was 63 years. In 59 patients aged 65 and older, the proportions of patients with advanced stage and those treated with ALK-tyrosine kinase inhibitor (TKI) were lower than those younger than 65 years. There was no difference in overall survival (OS) between the two age groups. Among the elderly patients, no difference was observed in OS between the patients aged 65-69 and those aged 70 and older. In 89 patients treated with TKI, no significant differences were observed in the progression-free survival of TKIs and OS between patients aged 65 and older and those younger than 65, respectively. Evaluation of ALK gene status and TKI treatment are desirable even for elderly patients.

P1.09-22 Detection of ALK Gene Rearrangement in FFPE Tissues of Non-Small Cell Lung Cancer Using in Situ RNA Hybridization

Approximately 4-7% of non-small cell lung cancer (NSCLC) harbor anaplastic lymphoma kinase (ALK) gene rearrangements which lead to overexpression of ALK fusion protein and constitutive activation of the kinase. The tumor with ALK gene rearrangements is sensitive to its inhibitor, so identification of this molecular feature from the patients accurately is important. Fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) are conventional methods for testing ALK gene rearrangement and protein overexpression respectively; however it is challenge for distinguishing some rare variations by FISH, or determining the equivocal stain by IHC. A morphology related method under bright field about rearranged mRNA expression (RNAscope) of ALK gene fusion was developed, which is helpful to realize the biological behavior of ALK gene, together with FISH (break apart FISH probe kit, Abbott) and IHC (D5F3 clone, Ventana). We explored 7 cases of formalin-fixed paraffin-embedded (FFPE) samples from NSCLC patients by RNAscope® 2.5 Red assay Three of seven samples were found with ALK positive by RNAscope, which matched with FISH and IHC results. In these 3 ALK positive samples, exon 19-29 of ALK gene transcribed RNA signals were revealed by probe-Hs-ALK E1-E18 (score 1 to 2 in >50% cells), whereas exon 1-18 transcribed RNA signals were not shown by probe-Hs-ALK E19-E29, which confirmed by FISH results (with break apart signals). In 2 of 4 ALK RNAscope negative cases, ALK RNA signals were detected by both probe-Hs-ALK E1-E18 (score 2) and probe-Hs-ALK E19-E29 (score 2) in small regions (showed a small number of signals), which may indicate that ALK gene amplification and also confirmed by FISH results (with more than 2 fusion signals). And these 2 typical ALK RNAscope negative samples were considered close to be disomy by FISH. Our results demonstrate that RNAscope® 2.5 Red assay is a reliable and precise method to detect ALK mRNA expression caused by ALK gene fusion and also a one to discover the ALK gene amplification as well in FFPE samples of NSCLC, even though latter’s clinical significant is not clear. RNAscope® 2.5 Red assay is a both sensitive and specific method to provide information on the spatial distribution of ALK gene status and morphologic characteristic of tumors simultaneously under the bright field microscope.

Association between EGFR, ALK and KRAS Gene Status and Synchronous Distant Organ Metastasis in Non-small Cell Lung Cancer

肺癌是我国恶性肿瘤的首位死亡疾病，据统计大约57%的肺癌患者就诊时已经出现了远处转移，临床预后较差。抗肺癌转移是当前治疗晚期转移性肺癌的新方向和思路。既往研究表明肿瘤的生物学改变在一定程度上能够影响肿瘤的转移行为和侵袭扩散模式，而目前的基础及临床研究尚未阐明导致肺癌相关信号转导途径中发生特异性器官转移的分子机制，有关驱动基因突变与器官转移之间相关性的研究也较为罕见。本篇综述旨在对近几年有关非小细胞肺癌表皮生长因子受体（epidermal growth factor receptor, EGFR）、间变性淋巴瘤激酶（anaplastic lymphoma kinase, ALK）、Kristen鼠肉瘤病毒原癌基因同源体（V-Ki-ras2 Kirsten rat sarcoma viral oncogene homologue, KRAS）驱动基因表达的特点以及与转移器官分布之间相关性的文献进行小结。

Association between the ALK Gene Status and the Efficacy of First-line Pemetrexed Chemotherapy in Patients with Advanced Lung Adenocarcinoma

背景与目的间变性淋巴瘤激酶（anaplastic lymphoma kinase, ALK）是非小细胞肺癌（non-small cell lung cancer, NSCLC）的重要驱动基因之一，多项研究显示培美曲塞在ALK阳性肺癌中的疗效存在争议。本研究旨在继续探索以培美曲塞为基础的化疗在ALK阳性和阴性肺腺癌患者中的疗效。方法回顾性分析郑州大学第一附属医院2015年1月-2016年4月经组织病理学证实的98例表皮生长因子受体（epidermal growth factor receptor, EGFR）、鼠类肉瘤病毒癌基因（kirsten rat sarcoma viral oncogene, KRAS）、鼠类肉瘤滤过性毒菌致癌同源体B1（V-rafmurine sarcoma viral oncogene homolog B1, BRAF）均为阴性的晚期肺腺癌患者的临床资料。分析ALK基因状态、临床特征、化疗疗效及无疾病进展生存期（progression-free survival, PFS）之间的关系。结果98例患者均进行了ALK基因检测，ALK基因断裂融合34例（34.7%），未发生断裂融合64例（65.3%）。全部患者均接受一线培美曲塞联合铂类的化疗，客观缓解率（objective response rate, ORR）为21.4%，疾病控制率（disease control rate, DCR）为84.7%。ALK阳性肺腺癌患者的ORR和DCR均高于阴性患者（41.2% vs 10.9%, χ2=23.389, P < 0.001; 91.2% vs 81.3%, χ2=4.153, P=0.042），差异有统计学意义。ALK基因状态与年龄、性别、吸烟史、临床分期均无明显关系。ALK阳性肺腺癌的中位PFS为7.1个月（95%CI: 6.1-8.1），阴性4.7个月（95%CI: 3.818-5.582），二者的PFS差异有统计学意义（χ2=13.269, P < 0.001）。Cox回归多因素分析显示：培美曲塞联合铂类化疗的PFS与性别、年龄、吸烟、分期、与铂类药物的种类均无明显关系，ALK基因断裂融合是PFS相关的唯一变量（HR=0.392, 95%CI: 0.243-0.634, P < 0.001）。结论ALK阳性相比ALK阴性肺腺癌患者一线应用以培美曲塞为基础的化疗有更大的临床获益。

An Anaplastic Lymphoma Kinase Immunohistochemistry–Negative but Fluorescence In Situ Hybridization–Positive Lung Adenocarcinoma Is Resistant to Crizotinib

IntroductionOncogenic fusion of anaplastic lymphoma kinase (ALK) with echinoderm microtubule associated protein like 4 protein or other partner genes occurs in 3% to 6% of lung adenocarcinomas. Although fluorescence in situ hybridization (FISH) is the accepted standard for detecting anaplastic lymphoma receptor tyrosine kinase gene (ALK) gene rearrangement that gives rise to new fusion genes, not all ALK FISH–positive patients respond to ALK inhibitor therapies. We report here an ALK FISH–positive patient-derived xenograft (PDX) that was nonresponsive to crizotinib therapy. MethodsThe PDX patient human lung cancer (PHLC402) was established in NOD/SCID mice from a patient with resected pT4N1M0 lung adenocarcinoma. ALK gene status was investigated using the standard FISH break-apart assay, reverse-transcriptase quantitative polymerase chain reaction, RNA sequencing and immunohistochemical assay using the 5A4 antibody. PHLC402 was treated with crizotinib (50 mg/kg) by daily oral gavage. ResultsALK FISH assay was positive in both the primary patient tumor and PDX, which were negative for ALK protein expression by immunohistochemical analysis. ALK fusion product was not detected by RNA sequencing and reverse-transcriptase quantitative polymerase chain reaction comparing the 5′ and 3′ ALK transcript levels. Crizotinib treatment of PHLC402 grown in mice resulted in no tumor response. ConclusionALK protein expression may be necessary for ALK FISH–positive lung cancer to be responsive to ALK inhibitor therapy.

ALK gene expression status in pleural effusion predicts tumor responsiveness to crizotinib in Chinese patients with lung adenocarcinoma.

ObjectiveThe relationship between anaplastic lymphoma kinase (ALK) expression in malignant pleural effusion (MPE) samples detected only by Ventana immunohistochemistry (IHC) ALK (D5F3) and the efficacy of ALK-tyrosine kinase inhibitor therapy is uncertain. MethodsVentana anti-ALK (D5F3) rabbit monoclonal primary antibody testing was performed on 313 cell blocks of MPE samples from Chinese patients with advanced lung adenocarcinoma, and fluorescence in situ hybridization (FISH) was used to verify the ALK gene status in Ventana IHC ALK (D5F3)-positive samples. The follow-up clinical data on patients who received crizotinib treatment were recorded. ResultsOf the 313 MPE samples, 27 (8.6%) were confirmed as ALK expression-positive, and the Ventana IHC ALK (D5F3)-positive rate was 17.3% (27/156) in wild-type epidermal growth factor receptor (EGFR) MPE samples. Twenty-three of the 27 IHC ALK (D5F3)-positive samples were positive by FISH. Of the 11 Ventana IHC ALK (D5F3)-positive patients who received crizotinib therapy, 2 patients had complete response (CR), 5 had partial response (PR) and 3 had stable disease (SD). ConclusionsThe ALK gene expression status detected by the Ventana IHC ALK (D5F3) platform in MPE samples may predict tumor responsiveness to crizotinib in Chinese patients with advanced lung adenocarcinoma.

Evaluation of tyrosine kinase receptors in brain metastases of clear cell renal cell carcinoma reveals cMet as a negative prognostic factor.

Brain metastases (BMs) of clear cell renal cell carcinoma (ccRCC) are associated with a dismal prognosis, with limited treatment options. Tyrosine kinases are relevant 'druggable' biomarkers. The aim of this study was to evaluate the tyrosine kinase receptors anaplastic lymphoma kinase (ALK), epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor-α (PDGFRA) and cMet in a large series of ccRCC BMs. ALK, EGFR, PDGFRA and cMet protein expression was determined by immunohistochemistry in 53 ccRCCs BMs and 12 matched primary tumours. ALK and MET gene status and copy number alterations of chromosome 7 were studied with fluorescence in-situ hybridization (FISH). Data on the expression of hypoxia-inducible factor 1α (HIF1α) and Ki67 and microvessel density were available from previous studies. ALK was negative in all analysed specimens. EGFR was overexpressed in 41 of 51 (80.4%) BMs and in seven of eight primary tumours, PDGFRA was overexpressed in all BMs except one and in all primary tumours, and cMet was expressed in 26 of 50 (52%) BMs and in two of seven primary tumours, and did not correlate with MET amplification or polysomy 7. cMet was the only parameter associated with significantly shorter BM-specific survival (median 8 months versus 33 months, P = 0.005, Cox regression). EGFR, PDGFRA and cMet are commonly overexpressed in ccRCC BMs. cMet overexpression correlates with significantly shorter BM-specific survival.

ALK Testing in Lung Adenocarcinoma: Technical Aspects to Improve FISH Evaluation in Daily Practice

Anaplastic lymphoma kinase (ALK) gene rearrangement characterizes a subgroup of patients with lung adenocarcinoma who may benefit from ALK inhibitors. Fluorescence in situ hybridization (FISH) with a break-apart/split-signal strategy is the gold standard to investigate ALK. The cutoff to define ALK positivity has been settled at 15% or greater. A subset of patients has ALK borderline status, showing 15% ± 5% positive cells. Several aspects, both biological and technical, might influence signals evaluation, making FISH interpretation a challenging task. To improve ALK evaluation, we classified the different FISH patterns on the basis of the type of the split signals, namely short, long, far away, and deleted. We investigated ALK gene status by FISH in 244 lung adenocarcinomas and in a series of ALK negative cell lines samples, collected in three Institutions. ALK positive profile was found in 12% of patients; long, deleted, and far-away splits were the primary patterns observed. ALK borderline profile characterized 10% of samples; long and deleted splits were significantly more frequent in those borderline finally classified as ALK positive, whereas short split were mostly detected in those borderline patients finally classified as ALK negative (p = 3.4 × 10). In the ALK negative control series, short split was the predominant pattern. Concordance was observed among different operators and probes for both samples and controls. Difficulties in ALK FISH signal interpretation might be bypassed using this detailed scoring system, which is highly reproducible, helps clarify borderline samples (according to split type), and provides experimental evidence that 15% is a reasonable cutoff to overcome the assay-dependent background noise.

ALK translocation detection in non-small cell lung cancer cytological samples obtained by TBNA or EBUS-TBNA.

As the diagnosis of non-small cell lung cancer (NSCLC) is based on cytology in around 70% of cases, it is important to use the same material for molecular analyses. Fluorescence insitu hybridization (FISH) is the only approved test for the detection of the translocation and inversion of anaplastic lymphoma kinase (ALK), but the optimal procedures for the fixation or staining of the sample before FISH evaluation have not been established. We investigated whether ALK gene status determined by FISH in a prospectively enrolled case series of patients was affected by fixation and staining. One hundred and fifteen cytological samples were obtained by transbronchial needle aspiration (TBNA) or endobronchial ultrasound (EBUS)-TBNA from 109 patients with NSCLC. All samples were evaluated for epidermal growth factor receptor (EGFR) mutation by pyrosequencing and for ALK rearrangement by FISH. Specimens for ALK determination had been fixed with Cytofix(®) and/or Carnoy's solution or 10% formalin (cell blocks) and variously stained. Sixteen (14%) of the 115 samples were mutated for EGFR and 99 (86%) showed wild-type EGFR status. Of these 115 samples, 79 (69%) were negative for echinoderm microtubule-associated protein like 4 (EML4)-ALK translocation, nine (8%) were positive and 27 (23%) were unevaluable. In particular, 19 (26%) of the 72 Papanicolaou-stained smears fixed with Cytofix were unevaluable because of inadequate samples or cell overlapping; neither of the two May-Grünwald-Giemsa-stained samples were evaluable. Ten of 17 smears used for rapid on-site evaluation (ROSE) and immediately post-fixed in Carnoy's solution or 80% alcohol were evaluable. In this series, smears were unevaluable as a result of inadequate samples, cell overlapping or lack of fixation performed immediately after FNA.

Clinicopathologic and prognostic significance of c-MYC copy number gain in lung adenocarcinomas.

Background:c-MYC copy number gain (c-MYC gain) has been associated with aggressive behaviour in several cancers. However, the role of c-MYC gain has not yet been determined in lung adenocarcinomas classified by genetic alterations in epidermal growth factor receptor (EGFR), KRAS, and anaplastic lymphoma kinase (ALK) genes. We investigated the clinicopathologic and prognostic significance of c-MYC gain for disease-free survival (DFS) and overall survival (OS) according to EGFR, KRAS, and ALK gene status and stages in lung adenocarcinomas.Methods:In 255 adenocarcinomas resected in Seoul National University Bundang Hospital from 2003 to 2009, fluorescence in situ hybridisation (FISH) with c-MYC probe and centromeric enumeration probe 8 (CEP8) was analysed using tissue microarray containing single representative core per each case. EGFR (codon 18 to 21) and KRAS (codon 12, 13, and 61) mutations were analysed by polymerase chain reaction and direct sequencing method from formalin-fixed, paraffin-embedded tissue sections. ALK rearrangement was determined by FISH method. c-MYC gain was defined as >2 copies per nucleus, chromosome 8 gain as ⩾3 copies per nucleus, and gain of c-MYC:CEP8 ratio (hereafter, c-MYC amplification) as ⩾2.Results:We observed c-MYC gain in 20% (51 out of 255), chromosome 8 gain in 5.5% (14 out of 255), c-MYC amplification in 2.4% (6 out of 255), EGFR mutation in 49.4% (118 out of 239), KRAS mutation in 5.7% (7 out of 123), and ALK rearrangement in 4.9% (10 out of 205) of lung adenocarcinomas. c-MYC gain was observed in 19% (22 out of 118) of patients with lung adenocarcinomas with an EGFR mutation, but not in any patients with a KRAS mutation, or an ALK rearrangement. c-MYC gain (but not chromosome 8 gain or c-MYC amplification) was an independent poor-prognostic factor in the full cohort of lung adenocarcinoma (P=0.022, hazard ratio (HR)=1.71, 95% confidence interval (CI), 1.08–2.69 for DFS; P=0.032, HR=2.04, 95% CI, 1.06–3.91 for OS), as well as in stage I subgroup (P=0.023, HR=4.70, 95% CI, 1.24–17.78 for DFS; P=0.031, HR=4.65, 95% CI, 1.15–18.81 for OS), and in EGFR-mutant subgroup (P=0.022; HR=2.14; 95% CI, 1.11–4.10 for DFS).Conclusions:c-MYC gain (but not chromosome 8 gain or c-MYC amplification) was an independent poor-prognostic factor for DFS and OS in lung adenocarcinomas, both in full cohort and stage I cancer, and possibly for DFS in EGFR-mutant adenocarcinomas. Additional studies are required to determine if patients with lung adenocarcinoma with c-MYC gain are candidates for additional first-line treatment to mitigate their increased risk for disease progression and death.

ALK/EML4 Fusion Gene May Be Found in Pure Squamous Carcinoma of the Lung

The report of cases of lung squamous cell cancers harboring anaplastic lymphoma kinase (ALK) gene rearrangements raises the question whether this histologic subtype should be also evaluated for such molecular predictive test. A consecutive series of 40 lung pure squamous cell carcinomas were analyzed for ALK gene status by fluorescence in situ hybridization. Squamous differentiation was validated using an immunohistochemical panel including n-p63 (p40), cytokeratin (CK) 5/6, sex-determining region Y (SRY)-Box2 (SOX2), thyroid transcription factor 1, CK7, and Napsin-A. Squamous differentiation was confirmed in all tumors as they stained positive for n-p63 and CK5/6 and negative for thyroid transcription factor 1 and Napsin-A. One of 40 cases (2.5%) showed an ALK rearrangement on fluorescence in situ hybridization analysis. ALK translocation may be found in lung pure squamous cell carcinomas. Our data suggest the opportunity to test ALK rearrangements on biopsy samples harboring squamous cell cancer differentiation.

Differential expression of CRKL and AXL genes in lung adenocarcinoma subtypes according to the epidermal growth factor receptor and anaplastic lymphoma kinase gene status.

Non-small-cell lung cancer (NSCLC) is the most common cause of cancer-related mortality. Adenocarcinoma (AC) is the predominant histological type of NSCLC; however, AC consists of several subtypes. It has not yet been determined whether there is a correlation of CRKL and AXL expression with epidermal growth factor receptor (EGFR) and anaplastic lymphoma kinase (ALK) gene status in lung AC. We assayed exons 18 through 21 of the EGFR gene by direct sequencing; ALK rearrangement and the expression of CRKL and AXL were detected by immunostaining. A total of 212 cases of AC were included in this study, diagnosed using the novel classification system established by the International Association for the Study of Lung Cancer, the American Thoracic Society and the European Respiratory Society in 2011, including 69 acinar ACs, 17 lepidic predominant ACs (LPAs), 63 papillary, 14 mucinous, 17 micropapillary and 32 solid ACs. Of the 212 cases, 101 harbored EGFR mutations. The most common subtypes carrying delK745-S753 were papillary and acinar ACs. ALK rearrangement was found in 23 cases (11%) of lung ACs. Acinar and solid ACs were the most frequent subtypes with ALK aberrance, particularly in acinar ACs with cribriform structure (4/5 cases, 80%). The expression of CRKL was significantly different among the AC subtypes (P=0.01), with the highest and lowest expression levels of CRKL protein in papillary ACs and LPAs, respectively (P<0.05). AXL expression was also significantly different among the AC subtypes (P=0.002) and was correlated with lymph node infiltration in acinar ACs. ACs with EGFR mutations exhibited high levels of AXL protein expression compared to those without mutations (P<0.001). Acinar AC with cribriform structure is a distinct subtype that frequently harbors ALK rearrangement. The activation of AXL may be one of the factors contributing to the invasion of acinar and micropapillary ACs.

New Methods for ALK Status Diagnosis in Non–Small-Cell Lung Cancer: An Improved ALK Immunohistochemical Assay and a New, Brightfield, Dual ALK IHC–In Situ Hybridization Assay

The demonstration of anaplastic lymphoma kinase (ALK) positivity in non-small-cell lung cancer (NSCLC) has been hindered by the technical complexity and interpretative challenges of fluorescence in situ hybridization methods for detection of ALK gene rearrangement and by the inadequate sensitivity of existing immunohistochemistry (IHC) methods for ALK protein detection. In this study, we sought to increase the sensitivity of ALK IHC detection and to develop a brightfield assay for concurrent detection of ALK protein expression and ALK gene rearrangement. We developed a horseradish peroxidase-based IHC detection system using the novel, nonendogenous hapten 3-hydroxy-2-quinoxaline (HQ) and tyramide. We also developed a dual gene protein ALK assay combining a brightfield break-apart in situ hybridization ALK assay with another sensitive IHC method using the novel, nonendogenous hapten 5-nitro-3-pyrazole. We examined the sensitivity and accuracy of these methods using surgically resected NSCLC cases examined with ALK fluorescence in situ hybridization. The new HQ-tyramide IHC detection system offered readily interpretable staining with substantially greater sensitivity than conventional ALK IHC, and produced heterogeneous and homogeneous patterns of ALK protein staining among ALK-positive NSCLC surgical cases. The new 5-nitro-3-pyrazole-based IHC detection system was similar in ALK detection sensitivity to the HQ-tyramide IHC system and was compatible with the brightfield in situ hybridization assay. The new HQ-tyramide IHC reagent system allows more sensitive assessment of ALK protein status in NSCLC cases. The new ALK gene-protein assay allows the concurrent visualization of ALK gene and ALK protein status in single cells, allowing more accurate ALK status determination even in heterogeneous specimens.

Amplification but not translocation of anaplastic lymphoma kinase is a frequent event in oesophageal cancer

IntroductionTranslocations of anaplastic lymphoma kinase (ALK) to various fusions partners and formation of oncogenic fusions proteins have been demonstrated in a variety of human malignancies. These fusion-proteins are potential pharmaceutically targets. Aim of this study was to investigate ALK gene status in a large cohort of squamous cell carcinoma (SCC) and adenocarcinoma (AC) of the oesophagus. Materials and Methods117 SCCs and 136 ACs were included into this study. ALK and EML4 gene status were evaluated by fluorescence in situ hybridisation (FISH) using a triple colour break apart single fusion probe and a probe against 2p11. ALK and EML4 protein expression was determined by immunohistochemistry. Data on expression of ALK downstream effector tyrosine-705 phosphorylated STAT3 (pSTAT3) were available from a previous study. ResultsFISH was performed successfully in 251 cases. All cases were negative for ALK translocations, while 14/135 (12.1%) of SCCs and 14/116 (10.4%) of ACs showed ALK amplifications. Concomitant EML4 amplifications were present in 27/28 cases with ALK amplifications. Three cases (two SCC, one with additional ALK &EML4 amplification and one AC) showed EML4 translocations not involving ALK. None of the tumours with ALK amplification showed ALK protein expression, and no correlation with clinical parameters, survival or pSTAT3 expression was observed. ConclusionsWhile ALK translocations are not present in oesophageal cancer, ALK amplifications are common events with comparable rates in SCC and AC. Since ALK amplified breast cancer cells were shown to respond to ALK inhibitors, ALK amplified oesophageal cancers might be considered as possible candidates for therapies targeting ALK.

Multiparametric molecular characterization of pulmonary sarcomatoid carcinoma reveals a nonrandom amplification of anaplastic lymphoma kinase (ALK) gene

BackgroundGenetic alterations for targeting therapy are largely unexplored issues in pulmonary sarcomatoid carcinoma (PSC), a life-threatening tumor subset. MethodsEGFR, HER2, KRAS, p53, CTNNB1, BRAF and PIK3CA mutations were assessed by direct sequencing, ALK, EGFR and HER2 gene status by fluorescence in situ hybridization (FISH), and ALK protein expression by immunohistochemistry (IHC) in 20 pleomorphic carcinomas (PLC), two pulmonary blastomas (PB) and one carcinosarcoma (CS). Surgical specimens and, in case of positivity, the corresponding preoperative biopsies were analyzed. Furthermore, 51 consecutive metastatic lung adenocarcinomas (MELAD) were used as controls for FISH and IHC assays of ALK gene. ResultsWhile no rearrangements of ALK were detected in PSC, relevant amplification was identified 5/23 (22%) surgical specimens and paired biopsies (four PLC and one PB, two females and three males, four current and one never smoker, aged 30–83 years). Considering tumor heterogeneity, the percentage of ALK amplified tumor cells ranged from 11% to 43%, with a mean gene copy gain (GCG±SD) of 6.9±0.8 and no signal differences between the epithelial (6.5±0.9) and the sarcoma-like components (6.8±0.9) of tumors. In the remaining 18 non-amplified PSC, the relevant value was 2.9±0.5 in 1–80% tumor cells (p<0.001). ALK amplification was closely associated with chromosome 7 (EGFR) or 17 (HER2) polysomy (p<0.001). Out of 51 MELAD, 10 were ALK-rearranged (p=0.026) and only one amplified (p=0.009). No amplified tumors, either PSC or MELAD, expressed the relevant protein by IHC, while the 10 ALK-rearranged MELAD were strongly positive. TP53, KRAS and CTNNB1 mutations accounted for 30%, 22%, and 4% of cases, respectively, with no significant relationship with ALK amplification. No mutations for EGFR, HER2, BRAF or PIK3CA gene were observed. ConclusionALK gene amplification is a nonrandom and clonally related event in a subset of PSC, but its biologic rationale deserves further investigation. KRAS mutation could represent a novel tool for therapy of such so deadly tumors with MEK inhibitors.

ALK chromosomal alterations in neuroendocrine tumors.

10585 Background: Neuroendocrine tumors (NETs) comprise a heterogeneous group of neoplasms containing biologically active peptides and amines in their cytoplasm. Anaplastic lymphoma kinase (ALK) translocation has recently emerged as a potent predictor of benefit for treatment with ALK inhibitors. The endpoint of this study was to analyze ALK translocations and gene copy number status in a series of neuroendocrine tumors and correlate these findings with clinico-pathological features. Methods: Paraffin-embedded specimens from gastroenteropancreatic (GEP) and lung neuroendocrine tumors from patients diagnosed between years 2004-2010 were retrospectively retrieved from the tumor bank at our Institution. ALK gene status was characterized by fluorescence in situ hybridization (FISH) with a break-apart ALK probe (Abbot Molecular Inc). Translocation and gene copy gain were defined as previously reported (Salido et al). FISH ALK-positive and a set of negative cases were evaluated by immunohistochemistry (ALK-1, Dako). Statistical analysis was carried to evaluate the association between ALK gene alterations and clinico-pathological features. This study was approved by the review board at our Institution. Results: One hundred and eleven neuroendocrine tumors were included in the study. In 42 cases the sample was insufficient/inconclusive FISH results, leaving a final number of 69 evaluable cases (43 GEP, 20 lung, 6 other). There was one GEP neuroendocrine tumor (appendix) with an EML4-ALK translocation in 15% of tumoral cells and one lung neuroendocrine tumor with an atypical ALK translocation in 60% of tumoral cells (unknown partner). Ten cases (7 GEP; 3 lung) showed an increase in ALK gene copy number (mean 5 copies; range: 3-10 copies) with the percentage of cells with ALK copy gain ranging between 18-65% (mean 36%). Immunohistochemistry revealed <10% of tumor cells with mild ALK staining in the specimen with EML4-ALK translocation. The low number of positive cases precluded the finding of statistical associations. Conclusions: Although rare, ALK translocations are present in neuroendocrine tumors. This reveals a potential new indication for ALK inhibitors that merits further investigation.