Aberrant Anaplastic Lymphoma Kinase Expression Research Articles

Preclinical evidence for anaplastic lymphoma kinase inhibitors as novel therapeutic treatments for cholangiocarcinoma.

Bile duct cancer (cholangiocarcinoma, CCA) has a poor prognosis for patients, and despite recent advances in targeted therapies for other cancer types, it is still treated with standard chemotherapy. Anaplastic lymphoma kinase (ALK) has been shown to be a primary driver of disease progression in lung cancer, and ALK inhibitors are effective therapeutics in aberrant ALK-expressing tumors. Aberrant ALK expression has been documented in CCA, but the use of ALK inhibitors has not been investigated. Using CCA cell lines and close-to-patient primary cholangiocarcinoma cells, we investigated the potential for ALK inhibitors in CCA. ALK, cMET, and ROS1 expression was determined in CCA patient tissue by immunohistochemistry and digital droplet polymerase chain reaction, and that in cell lines was determined by immunoblot and immunofluorescence. The effect on cell viability and mechanism of action of ALK, cMet, and ROS1 inhibitors was determined in CCA cell lines. To determine whether ceritinib could affect primary CCA cells, tissue was taken from four patients with biliary tract cancer, without ALK rearrangement, mutation, or overexpression, and grown in three-dimensional tumor growth assays in the presence or absence of humanized mesenchymal cells. ALK and cMet but not ROS were both upregulated in CCA tissues and cell lines. Cell survival was inhibited by crizotinib, a c-met/ALK/ROS inhibitor. To determine the mechanism of this effect, we tested c-Met-specific and ALK/ROS-specific inhibitors, capmatinib and ceritinib, respectively. Whereas capmatinib did not affect cell survival, ceritinib dose-dependently inhibited survival in all cell lines, with IC50 ranging from 1 to 9 µM and co-treatments with gemcitabine and cisplatin further sensitized cells, with IC50 ranging from IC50 0.60 to 2.32 µM. Ceritinib did not inhibit cMet phosphorylation but did inhibit ALK phosphorylation. ALK was not mutated in any of these cell lines. Only ceritinib inhibited 3D growth of all four patient samples below mean peak serum concentration, in the presence and absence of mesenchymal cells, whereas crizotinib and capmatinib failed to do this. Ceritinib appeared to exert its effect more through autophagy than apoptosis. These results indicate that ceritinib or other ALK/ROS inhibitors could be therapeutically useful in cholangiocarcinoma even in the absence of aberrant ALK/ROS1 expression.

Malakoplakia with aberrant ALK expression by immunohistochemistry: a case report

BackgroundMalakoplakia is a rare inflammatory disease of the urogenital tract. There have been no reports of malakoplakia expressing anaplastic lymphoma kinase (ALK) to date. Here, we present one case of malakoplakia with aberrant ALK expression by immunohistochemistry and discuss the clinical significance.Case presentationA 65-year-old Chinese woman with a history of diabetes presented with solid masses in the liver and kidney and elevated lesions on the mucosal surface of the colon. Right nephrectomy and partial liver resection were performed. Microscopically, sheets of histiocytes with poor intercellular adhesion were seen, with Michaelis–Gutmann bodies present in both the intracellular and extracellular interstitium. CD10-, CD68-, and CD163-positive cells were present, with Michaelis–Gutmann bodies confirmed by staining with Alcian blue, periodic acid-Schiff (PAS), periodic acid-Schiff with diastase, Von Kossa, and Prussian blue. Aberrant ALK1 and ALK (D5F3) expression was observed in the cytoplasm and nucleus of cells. However, ALK gene mutation was not detected by fluorescence in situ hybridization or whole exome next-generation sequencing. NGS revealed nine individual somatic gene mutations: GOT1L1, GLIS2, SPOUT1, TMEM97, MUC3A, NSD2, SFXN5, ADAD1 and RAD50. The significance of the somatic gene mutations detected in this study is not clear, and the relationship between them and malakoplakia cannot be clarified by existing scientific studies. The pathological diagnosis was malakoplakia with aberrant ALK expression by immunohistochemistry. The antibiotics imipenem and vancomycin were started based on the results of drug sensitivity analysis and the patient was subsequently discharged. She experienced no discomfort during 30 months of follow-up.ConclusionThis is the first reported case of malakoplakia with aberrant ALK expression, it should be differentiated from ALK-positive histiocytosis to avoid misdiagnosis.

Anaplastic Lymphoma Kinase (ALK) Receptor Tyrosine Kinase: A Catalytic Receptor with Many Faces.

The anaplastic lymphoma kinase (ALK) receptor is a membrane-bound tyrosine kinase. The pathogenesis of several cancers is closely related to aberrant forms of ALK or aberrant ALK expression, including ALK fusion proteins, ALK-activated point mutations, and ALK amplification. Clinical applications of different ALK inhibitors represent significant progress in targeted therapy. Knowledge of different aspects of ALK biology can provide significant information to further the understanding of this receptor tyrosine kinase. In this mini-review, we briefly summarize different features of ALK. We also summarize some recent research advances on ALK fusion proteins in cancers.

The role of anaplastic lymphoma kinase in pediatric cancers.

The anaplastic lymphoma kinase (ALK) gene was initially identified as a fusion partner of the nucleophosmin gene in anaplastic large‐cell lymphoma with t(2;5)(p23;q35) translocation, and then described with different genetic abnormalities in a number of tumors. Although ALK is known to be involved in the pathogenesis of neuroblastoma through activating mutations or gene amplification, its role in the pathogenesis of other pediatric cancers is still elusive. In addition to neuroblastoma, the high‐grade amplification of ALK has been described in a subset of rhabdomyosarcoma cases. Normal ALK protein expression is restricted to the nervous systems of adult mammals, but the aberrant expression of ALK has been observed in a variety of pediatric cancers, including glioma and Ewing sarcoma. The discovery of oncogenic activation of ALK in neuroblastoma suggests that this cancer could be potentially treated with an ALK inhibitor, as could other cancers, such as non‐small‐cell lung cancer and anaplastic large‐cell lymphoma. However, cellular responses to mutant ALK are complex when compared to rearranged ALK, and treatment remains a challenge. This review focuses on the biology of ALK in pediatric cancers and possible therapeutic strategies for ALK‐associated tumors.

Aberrant expression of anaplastic lymphoma kinase in lung adenocarcinoma: Analysis of circulating free tumor RNA using one-step reverse transcription-polymerase chain reaction.

Lung adenocarcinoma patients harboring anaplastic lymphoma kinase (ALK) gene rearrangements respond well to approved ALK inhibitors. However, to date, limited evidence is available regarding whether using circulating free tumor mRNA to identify aberrant ALK expression is possible, and its feasibility remains to be clearly addressed. The present study evaluated ALK expression by a one-step reverse transcription‑polymerase chain reaction (PCR) assay on the circulating free tumor mRNA from12lung adenocarcinoma patients. Additionally, the present study tested for ALK rearrangements by fluorescence insitu hybridization (FISH) and immunohistochemistry. A molecular genetic characterization was performed on tumor tissues and plasma samples. Aberrant ALK expression was detected in2/12patients using mRNA purified from plasma specimens and the results agreed with the FISH and immunohistochemistry findings of solid biopsy samples. The detection of aberrant ALK expression on circulating free tumor RNA may be feasible using a one‑step real‑time PCR assay and may be particularly helpful when a solid biopsy sample is not available.

Aberrant Expression of Anaplastic Lymphoma Kinase in Ovarian Carcinoma Independent of Gene Rearrangement.

Ovarian carcinoma is the leading cause of death from gynecologic malignancies. The oncogenic role of anaplastic lymphoma kinase (ALK) is well characterized in many hematopoietic and solid tumors. ALK expression in ovarian carcinoma has been reported but the exact status of ALK protein and its association with clinicopathologic features requires further investigation. ALK expression was determined by immunohistochemistry in 110 primary ovarian carcinomas, including 85 cases of serous carcinoma and 25 cases of mucinous carcinoma. Fluorescence in situ hybridization (FISH) and real-time reverse transcription polymerase chain reaction (RT-PCR) were used for evaluating ALK translocation in ALK-positive ovarian carcinomas. Among 110 ovarian carcinomas, 23 (20.9%) cases were ALK positive by immunohistochemistry. All ALK-positive cases were ovarian high-grade serous carcinoma. ALK expression was detected in 23/85 (27.1%) ovarian serous carcinoma and 0/25 (0%) in ovarian mucinous carcinoma. None of the 23 ALK IHC-positive cases harbored ALK gene translocations by FISH or RT-PCR. ALK protein expression was associated with patient age, tumor stage, and histologic type. Specifically, the probability of ALK protein expression was significantly higher in high-grade serous carcinomas in older patients (above 50 y) with advanced disease (FIGO stage III and IV) compared with the low-grade serous and mucinous carcinomas in younger patients with relatively early disease. In conclusion, aberrant ALK expression is observed in ovarian serous carcinoma but not in mucinous carcinoma, is independent of gene translocation, and might be associated with progression and prognosis.

Aberrant anaplastic lymphoma kinase expression in high-grade pulmonary neuroendocrine carcinoma

AimsIn lung cancer with anaplastic lymphoma kinase (ALK) gene rearrangement, accurate diagnosis is essential to identify patients who can be treated with a specific kinase inhibitor. Sensitive ALK immunostaining can...

Identification of Anaplastic Lymphoma Kinase as a Potential Therapeutic Target in Ovarian Cancer

Ovarian cancer is the leading cause of death from gynecologic cancer. Improvement in the clinical outcome of patients is likely to be achieved by the identification of molecular events that underlie the oncogenesis of ovarian cancer. Here we show that the anaplastic lymphoma kinase (ALK) is aberrantly activated in ovarian cancer. Using an unbiased and global phosphoproteomic approach, we profiled 69 Chinese primary ovarian tumor tissues and found ALK to be aberrantly expressed and phosphorylated in 4 tumors. Genetic characterization of these ALK-positive tumors indicated that full-length ALK expression in two serous carcinoma patients is consistent with ALK gene copy number gain, whereas a stromal sarcoma patient carries a novel transmembrane ALK fusion gene: FN1-ALK. Biochemical and functional analysis showed that both full-length ALK and FN1-ALK are oncogenic, and tumors expressing ALK or FN1-ALK are sensitive to ALK kinase inhibitors. Furthermore, immunohistochemical analysis of ovarian tumor tissue microarray detected aberrant ALK expression in 2% to 4% serous carcinoma patients. Our findings provide new insights into the pathogenesis of ovarian cancer and identify ALK as a potential therapeutic target in a subset of serous ovarian carcinoma and stromal sarcoma patients.

MicroRNA 96 Is a Post-Transcriptional Suppressor of Anaplastic Lymphoma Kinase Expression

Anaplastic lymphoma kinase (ALK) constitutes a part of the oncogenic fusion proteins nucleophosmin-ALK and echinoderm microtubule-associated protein like 4-ALK, which are aberrantly expressed in a subset of T-cell anaplastic large-cell lymphoma and non-small-cell lung cancer, respectively. The expression of mutated, constitutively active ALK also occurs in a subset of neuroblastoma tumors. ALK is believed to play an important role in promoting tumor survival. Nevertheless, the mechanisms underlying the expression of ALK in cancer cells are not completely known. MicroRNA (miR) has been implicated in the regulation of the expression of both oncogenes and tumor suppressor genes. We tested the hypothesis that the expression of ALK could be regulated by miR. Three Internet-based algorithms identified miR-96 to potentially bind with the ALK 3'-untranslated region. Notably, miR-96 levels were markedly decreased in ALK-expressing cancer cell lines and primary human tumors compared with their normal cellular and tissue counterparts. Transfection of the cell lines with miR-96 decreased levels of the different forms of ALK protein, without significant effects on ALK mRNA. Furthermore, miR-96 decreased the phosphorylation of ALK target proteins, including Akt, STAT3, JNK, and type I insulin-like growth factor receptor, and it down-regulated JunB. These effects were associated with reduced proliferation, colony formation, and migration of ALK-expressing cancer cells. These data provide novel evidence that decreases in miR-96 could represent a mechanism underlying the aberrant expression of ALK in cancer cells.

Abstract 2220: Insight ALK ScreenTM, a highly sensitive and specific RT-qPCR first-line screening assay for comprehensive detection of all oncogenic anaplastic lymphoma kinase (ALK) fusions: clinical validation

Abstract More than 15 different ALK fusions (e.g., CLTC-ALK, multiple EML4-ALK variants, KIF5B-ALK, NPM-ALK, RanBP2-ALK, TFG-ALK, TPM4-ALK, others) cause malignancies including NSCLC, NHL, and inflammatory myofibroblastic tumors. Preliminary studies suggest several other cancer subsets may also express ALK fusions (e.g., breast, colorectal and esophageal cancers). Several ALK small-molecule inhibitors are in development, and patients treated with one clinical stage inhibitor have had marked antitumor responses. Immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) for ALK have been used to select patients likely to benefit from ALK inhibitor therapy; however, these methods are problematic. Both methods have suboptimal sensitivity for certain ALK fusions (e.g., EML4-ALK), and FISH is technically demanding and has a prolonged turn-around time (5-7 days). To address the unmet need for a highly sensitive, practical assay for clinical ALK detection, we developed Insight ALK Screen™, a real-time PCR test for ALK fusions and wild-type ALK upregulation. The test operates by qPCR of two regions corresponding to the ALK extracellular and intracellular kinase domains; for example, EML4-ALK or other ALK fusion events result in a ΔCt (change in threshold cycle) increase in transcripts encoding the ALK kinase domain over wild-type ALK expression levels.This assay design enables Insight ALK Screen to detect any ALK fusion regardless of the ALK fusion partner. We are validating Insight ALK Screen in comparison to ALK IHC and FISH using NSCLC FFPE specimens from the Clarient tumor bank. Eighty-two NSCLCs were screened using Insight ALK Screen, and 17 of the 82 (20.7%) showed either an ALK fusion or wild-type ALK upregulation. All 17 ALK-expressing NSCLCs were next tested using an EML4-ALK variant-specific RT-PCR assay; 6 (35.3%) of the 17 cases (7.3% of the original 82 cases) were shown to be EML4-ALK-positive. Ongoing studies are assessing the genetic mechanisms (e.g., other EML4-ALK variants not tested, other ALK fusions, ALK gene amplification, chromosome 2 polysomy, etc.) underlying the aberrant ALK expression in the other 11 of 17 NSCLCs; these data will be presented at the meeting together with complete IHC, FISH and clinical-molecular correlative information. Together with its ease-of-use, small tissue requirements, multiplex compatibility, quantitation of ALK expression levels, lack of bias for specific fusions, and quick turn-around (24-48 hrs), our preliminary results support Insight ALK Screen as the first-line ALK diagnostic method of choice. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2220. doi:10.1158/1538-7445.AM2011-2220

Abstract 1121: A novel KIF5B-ALK fusion gene variant with transforming potentials in non-small cell lung cancer

Abstract BACKGROUND: The anaplastic lymphoma kinase (ALK) gene is frequently involved in chromosomal translocations resulting in fusion genes with different partners found in various lympho-proliferative conditions. In non-small cell lung cancers (NSCLC), the fusion protein encoded by echinoderm microtubule-associated protein-like 4 (EML4)-ALK fusion gene was recently reported to confer oncogenic properties. This study aimed to identify other possible ALK fusion genes and analyze their oncogenic potentials. METHODS: Immunohistochemical analysis was used to screen for aberrant ALK expression in primary NSCLC. 5’ rapid amplification of cDNA ends was used to screen for potential novel 5’ fusion partners of ALK other than EML4-ALK. Reverse transcription polymerase chain reaction and fluorescence in situ hybridization were used to confirm the identity of 5’fusion partner. The genomic breakpoint was verified using genomic sequencing. Overexpression of the novel ALK fusion gene and variants 3a and 3b of EML4-ALK was performed to assess downstream signaling and functional effects. RESULTS: We identified a novel gene resulting from fusion of kinesin family member 5B (KIF5B) exon 15 to ALK exon 20 in a primary lung adenocarcinoma. Western blot of the clinical tumor tissues showed expression of a protein whose size correlated with that of the predicted KIF5B-ALK. Overexpression of KIF5B-ALK in mammalian cells led to STAT3 and AKT activation, enhanced cell proliferation, migration and invasion. These oncogenic properties were comparable to those of EML4-ALK variants. CONCLUSIONS: The discovery of the novel KIF5B-ALK variant with transforming functions further consolidates the role of aberrant ALK signaling in lung carcinogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1121. doi:10.1158/1538-7445.AM2011-1121

A novel KIF5B‐ALK variant in nonsmall cell lung cancer

The anaplastic lymphoma kinase (ALK) gene is involved frequently in chromosomal translocations, resulting in fusion genes with different partners found in various lymphoproliferative conditions. It was recently reported in nonsmall cell lung cancer (NSCLC) that the fusion protein encoded by echinoderm microtubule-associated protein-like 4-ALK (EML4-ALK) fusion gene conferred oncogenic properties. The objective of the current study was to identify other possible ALK fusion genes in NSCLC. Immunohistochemical analysis was used to screen for aberrant ALK expression in primary NSCLC. The authors used 5' rapid amplification of complementary DNA ends to screen for potential, novel 5' fusion partners of ALK other than EML4-ALK. Reverse transcriptase-polymerase chain reaction and fluorescence in situ hybridization analyses were used to confirm the identity of 5' fusion partners. The genomic breakpoint was verified using genomic sequencing. Overexpression of the novel ALK fusion gene and variants 3a and 3b of EML4-ALK was performed to assess downstream signaling and functional effects. The authors identified a novel gene resulting from the fusion of kinesin family member 5B (KIF5B) exon 15 to ALK exon 20 in a primary lung adenocarcinoma. Western blot analysis of clinical tumor tissues revealed the expression of a protein whose size correlated with that of the predicted KIF5B-ALK. Overexpression of KIF5B-ALK in mammalian cells led to the activation of signal transducer and activator of transcription 3 and protein kinase B and to enhanced cell proliferation, migration, and invasion. The discovery of the novel KIF5B-ALK variant further consolidated the role of aberrant ALK signaling in lung carcinogenesis.

Crizotinib inALK-Rearranged Inflammatory Myofibroblastic Tumor

Inflammatory myofibroblastic tumor (IMT) is a distinctive mesenchymal neoplasm characterized by a spindle-cell proliferation with an inflammatory infiltrate. Approximately half of IMTs carry rearrangements of the anaplastic lymphoma kinase (ALK) locus on chromosome 2p23, causing aberrant ALK expression. We report a sustained partial response to the ALK inhibitor crizotinib (PF-02341066, Pfizer) in a patient with ALK-translocated IMT, as compared with no observed activity in another patient without the ALK translocation. These results support the dependence of ALK-rearranged tumors on ALK-mediated signaling and suggest a therapeutic strategy for genomically identified patients with the aggressive form of this soft-tissue tumor. (Funded by Pfizer and others; ClinicalTrials.gov number, NCT00585195.).

IL-21 Contributes to JAK3/STAT3 Activation and Promotes Cell Growth in ALK-Positive Anaplastic Large Cell Lymphoma

Interleukin (IL)-21 has been reported to both stimulate cell growth and promote survival in benign lymphoid cells and several types of hematopoietic neoplasms. It induces JAK3/STAT3 signaling, a biologically important cellular pathway activated in most cases of anaplastic lymphoma kinase (ALK)-expressing anaplastic large cell lymphoma (ALK(+)ALCL). Therefore, we hypothesize that IL-21 may contribute to JAK3/STAT3 activation and cell growth in ALK(+)ALCL. By reverse transcription-PCR, we found consistent expression of IL-21 receptor (IL-21R) in all ALK(+)ALCL cell lines and frozen tumors examined. IL-21 was also consistently expressed in ALK(+)ALCL tumors, although its mRNA was detectable in only one of three cell lines tested. By immunohistochemistry, we examined 10 paraffin-embedded ALK(+)ALCL tumors; all cases were positive for both IL-21 and IL-21R in these neoplastic cells. IL-21 signaling is biologically significant in ALK(+)ALCL since the addition of recombinant IL-21 enhanced the activation of JAK3/STAT3 and significantly increased cell growth in ALK(+)ALCL cell lines. However, small interfering RNA down-regulation of IL-21R significantly decreased both STAT3 activation and cell growth. IL-21R expression is not linked to nucleophosmin-ALK since forced expression of nucleophosmin-ALK and small interfering RNA down-regulation of nucleophosmin-ALK did not significantly change the expression of either IL-21R or IL-21. Our findings thus support the enhancement of JAK3/STAT3 activation and cell growth in ALK(+)ALCL via IL-21 signaling. These results further support the concept that constitutive activation of STAT3 in these tumors is multifactorial.

ALK‐positive diffuse large B‐cell lymphoma

Anaplastic lymphoma kinase (ALK) positive diffuse large B-cell lymphoma (DLBCL) is a rare subtype of non-Hodgkins lymphoma. Five such cases have been described in children. We present a 9-year-old boy, in whom diagnosis of DLBCL has been established in addition to congenital multiple enchondromatosis. Immunohistopathological evaluation of tumor biopsy established the final diagnosis of ALK + DLBCL. The clathrin gene (CLTC)-ALK fusion underlying aberrant expression of ALK in the present case was demonstrated by interphase fluorescence in situ hybridization (FISH) using break-apart rearrangement probes for ALK and CLTC. The disease in this patient was highly resistant to applied chemotherapy regimens and to radiotherapy. Analysis of the disease course in our patient and review of other cases reported previously show that ALK + DLBCL can be an aggressive malignancy that can be cured with conventional chemotherapy protocols only at stage of localized disease.

ALK expression in extranodal anaplastic large cell lymphoma favours systemic disease with (primary) nodal involvement and a good prognosis and occurs before dissemination

Aims—In anaplastic large cell lymphoma (ALCL), the site of origin has been described as an important prognostic factor. Recently, a fusion protein containing anaplastic lymphoma kinase (ALK) was described in...

Inv(2)(p23q35) in anaplastic large-cell lymphoma induces constitutive anaplastic lymphoma kinase (ALK) tyrosine kinase activation by fusion to ATIC, an enzyme involved in purine nucleotide biosynthesis

AbstractThe non-Hodgkin lymphoma (NHL) subtype anaplastic large-cell lymphoma (ALCL) is frequently associated with a t(2;5)(p23;q35) that results in the fusion of the ubiquitously expressed nucleophosmin (NPM) gene at 5q35 to the anaplastic lymphoma kinase (ALK) gene at 2p23, which is not normally expressed in hematopoietic tissues. Approximately 20% of ALCLs that expressALK do not contain the t(2;5), suggesting that other genetic abnormalities can result in aberrant ALK expression. Here we report the molecular characterization of an alternative genetic means of ALK activation, the inv(2)(p23q35). This recurrent abnormality produces a fusion of the amino-terminus of 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase (ATIC), a bifunctional homodimeric enzyme that catalyzes the penultimate and final steps of de novo purine nucleotide biosynthesis, with the intracellular portion of the ALK receptor tyrosine kinase. RT-PCR analysis of 5 ALCL tumors that contained the inv(2) revealed identical ATIC-ALK fusion cDNA junctions in all of the cases. Transient expression studies show that theATIC-ALK fusion transcript directs the synthesis of an approximately 87-kd chimeric protein that is localized to the cytoplasm, in contrast to NPM-ALK, which typically exhibits a cytoplasmic and nuclear subcellular distribution. ATIC-ALK was constitutively tyrosine phosphorylated and could convert the IL-3–dependent murine hematopoietic cell line BaF3 to cytokine-independent growth. Our studies demonstrate an alternative mechanism for ALK involvement in the genesis of NHL and suggest that ATIC-ALK activation results from ATIC-mediated homodimerization. In addition, expected decreases in ATIC enzymatic function in ATIC-ALK–containing lymphomas may render these tumors more sensitive to antifolate drugs such as methotrexate.