Anaplastic Lymphoma Kinase Protein Expression Research Articles

ALK-gene rearrangement: a comparative analysis on circulating tumour cells and tumour tissue from patients with lung adenocarcinoma

A subgroup of anaplastic lymphoma kinase (ALK)-rearranged lung tumours can respond to ALK inhibitors. Until now, the ALK status in circulating tumour cells (CTCs) isolated from patients with lung cancer has not been characterised. We assessed the ALK status in CTCs detected in patients with lung cancer and correlated the results to the ALK status defined in the corresponding tumour tissue. A total of 87 patients with lung adenocarcinoma showing CTCs isolated using the isolation by size of epithelial tumour cell method were screened for their ALK status both in tumour samples and in CTCs. ALK break-apart fluorescence in situ hybridisation (FISH) and immunoreactivity analyses using an anti-ALK antibody (5A4 clone) were carried out on CTCs and compared with the results obtained in the corresponding tissue specimens. A total of five patients showed ALK-gene rearrangement and strong ALK protein expression in CTCs and in the corresponding tumour samples. Both ALK-FISH and ALK immunoreactivity analyses show negative results in CTCs and corresponding tumour samples for 82 patients. Conclusions We demonstrated that the ALK status can be determined in CTCs isolated from patients with lung cancer by immunocytochemistry and FISH analyses. These results favour non-invasive, ALK-gene status pre-screening on a routine basis on CTCs isolated from patients with lung cancer and open new avenues for real-time monitoring for adapted targeted therapy.

ALK-positive anaplastic large cell lymphoma limited to the skin: clinical, histopathological and molecular analysis of 6 pediatric cases. A report from the ALCL99 study

Anaplastic large cell lymphomas are peripheral T-cell lymphomas that are characterized by a proliferation of large anaplastic blasts expressing CD30. In children, systemic anaplastic large cell lymphomas often present at advanced clinical stage and harbor translocations involving the anaplastic lymphoma kinase (ALK) gene leading to the expression of chimeric anaplastic lymphoma kinase (ALK)-fusion proteins. Primary cutaneous anaplastic large cell lymphoma is regarded as an ALK-negative variant confined to the skin and is part of the spectrum of primary cutaneous CD30-positive T-cell lymphoproliferative disorders. Thirty-three of 487 pediatric patients registered within the Anaplastic Large Cell Lymphoma-99 trial (1999 to 2006) presented with a skin limited CD30-positive lympho-proliferative disorder. In 23 of the 33 patients, material for international histopathological review was available, and the cases were studied for histopathological, immunophenotypical and clinical features as well as for breaks within the ALK gene. Five of 23 cases and one additional case (identified after closure of the trial) expressed ALK-protein. Complete staging excluded any other organ involvement in all children. Expression of ALK proteins was demonstrated by immunohistochemistry in all cases and the presence of breaks of the ALK gene was genetically confirmed in 5 evaluable cases. The histopathological and clinical picture of these skin-restricted ALK-positive lymphomas was indistinguishable from that of cutaneous anaplastic large cell lymphoma. Five children presented with a single skin lesion that was completely resected in 4 and incompletely resected in one. Three of these patients received no further therapy, 2 additional local radiotherapy, and one chemotherapy. All children remain in complete remission with a median follow up of seven years (range 1-8 years). We present 6 pediatric cases of ALK-positive primary cutaneous anaplastic large cell lymphomas. After thorough exclusion of systemic involvement, therapy confined to local measures seems to be sufficient to induce cure.

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.

MET and ALK in glioblastoma multiforme (GBM): Comparison of IHC and FISH.

2021 Background: GBM, the most common and lethal malignant primary brain tumor in adults, has an overall one-year life expectancy of 33.7%. Personalized medicine on the basis of predictive biomarkers remains elusive in GBM; however a recent case report showed clinical improvement after treatment with a mesenchymal-epithelial transition (MET) inhibitor. The authors of that report ascribe clinical response to the MET inhibitor, but the drug is also an inhibitor of anaplastic lymphoma kinase (ALK). We explored the co-expression, gain, or amplification of both MET and ALK by IHC and FISH in GBM. Methods: A convenience sample of 56 available tumors from gross-resected GBM cases within the Duke Brain Tumor tissue bank was stained for MET using clone 8F11 (Leica) and for ALK using clone 5A4 (Novocastra). FISH employed the LSI ALK (2p23) breakpoint-spanning dual-color DNA probe and the LSI D7S486 (MET)/CEP 7 dual color DNA probe (Vysis). Results: Of 56 GBM cases, MET was expressed in 69.6% and ALK in 17.9% by IHC. By FISH, gain or amplification was found in 100% of cases for MET and in 48.2% for ALK. Co-expression of MET and ALK by IHC was 14.3% and 48.2% by FISH. Co-expression by either IHC or FISH was also 48.2%. OS estimation for this cohort is premature, with 77% of patients still alive. Conclusions: MET and ALK are (co-)expressed in a significant proportion of GBM. IHC detection of MET and ALK did not correlate well with FISH results, suggesting that gene gain or amplification does not necessarily lead to abnormal MET and ALK protein expression or that IHC methods or antibodies used can be optimized. Prior studies reported MET amplification in GBM as low as 4%. Our study indicates that MET gain/amplification and protein expression may be much higher, having implications for MET-targeted therapy. Additionally, co-expression of ALK and MET has implications for dual inhibitors of these signaling pathways as well as resistance mechanisms. Our continued work will explore significance of MET and ALK as predictors of therapeutic response, with careful control for clinically relevant patient and disease characteristics. [Table: see text]

Inflammatory myofibroblastic tumor of the urinary bladder diagnosed by anaplastic lymphoma kinase immunostaining

Inflammatory myofibroblastic tumor (IMFT) of the urinary bladder is an unusual spindle cell lesion that exhibits cytologic atypia, infiltrative growth, and mitotic activity mimicking malignant tumors, such as leiomyosarcoma, rhabdomyosarcoma, and sarcomatoid carcinoma. Recently, anaplastic lymphoma kinase (ALK) gene translocations or ALK protein expression in IMFT has been reported, especially in patients of children and young adults. This lesion has been described in numerous locations in addition to the urinary bladder. The detection of ALK protein and ALK gene rearrangements are useful in distinguishing IMFT from spindle cell malignancies in the urinary bladder.

Anaplastic Lymphoma Kinase Aberrations in Rhabdomyosarcoma: Clinical and Prognostic Implications

The aim of this study is to investigate anaplastic lymphoma kinase (ALK) protein expression and underlying genetic aberrations in rhabdomyosarcoma (RMS), with special attention to clinical and prognostic implications. A total of 189 paraffin-embedded RMS tumor specimens from 145 patients were collected on tissue microarray. ALK protein expression was evaluated by immunohistochemistry. ALK gene (2p23) copy number and translocations were determined by in situ hybridization. cDNA sequencing of the receptor tyrosine kinase domain of the ALK gene was assessed in 43 samples. Strong cytoplasmic ALK protein expression was more frequently observed in alveolar RMS (ARMS) than in embryonal RMS (ERMS) (81% v 32%, respectively; P < .001). ALK gene copy number gain was detected in the vast majority of ARMS (88%), compared with 52% of ERMS (P < .001). ALK copy number correlated with protein expression in primary tumors (n = 107). We identified one point mutation (2%) and seven tumors harboring whole exon deletions (16%). In ERMS, specific ALK gain in the primary tumor correlated with metastatic disease (100% in metastatic disease v 29% in nonmetastatic disease; P = .004) and poor disease-specific survival (5-year disease-specific survival: 62% v 82% for nonspecific or no gain; P = .046). Because ALK aberrations on genomic and protein levels are frequently found in RMSs, in particular ARMS, and are associated with disease progression and outcome in ERMS, ALK may play a role in tumor biology and may provide a potential therapeutic target for these tumors. Future research should aim at the oncogenic role of ALK and the potential effect of ALK inhibitors in RMS.

Complete Radiological Response of Metastatic Anaplastic Lymphoma Kinase-Positive Signet Ring Lung Adenocarcinoma to Systemic Chemotherapy

Twenty-seven-year-old woman attended Accident and Emergency department complaining of right-sided pleuritic chest pain for the past 3 to 4 weeks. She had no significant medical or surgical history. Her chest x-ray examination showed bulky right-sided hilar mass, consolidation in the right lower lung zone with ipsilateral pleural effusion (Figures 1A, B). Her routine blood tests were normal except of mildly elevated inflammatory markers. She underwent bronchoscopy, which did not reveal intrabronchial lesions. Cytology from bronchioalveolar lavage sample showed abnormal cells with signet ring features (Figure 2A). Transbronchial biopsy of right hilar mass showed adenocarcinoma with signet ring features (Figure 2B) that was positive on immunohistochemistry for thyroid transcription factor 1 (3 ) (Figure 2C) and was consistent with primary lung adenocarcinoma. Signet ring variant of lung primary adenocarcinoma is rare and can be associated with a novel genetic translocation joining the echinoderm microtubule-associated protein-like 4 gene (EML4) and the anaplastic lymphoma kinase (ALK) gene, forming EML4-ALK fusion gene. As previously described, presence of EML4-ALK fusion gene is associated with expression of ALK protein (2 ), which was confirmed by immunohistochemistry using anti-ALK antibody (DAKO Company, Carpinteria, CA) (Figure 2D).1,2 ALK positivity was confirmed on fluorescence in situ hybridization (FISH) as described previously.3 Positron emission tomography/computed tomography scan showed extensive neck, mediastinal, and paraaortic lymphadenopathy with multiple necrotic, and metabolically active lesions in right hilum and right lung base, which was extending through diaphragm into the posterior segments of the liver (Figures 3A–D). She was commenced on systemic chemotherapy with carboplatin (area under the curve 6) and pemetrexed (500 mg/m) every 21 days for a total of six cycles. She tolerated chemotherapy very well, and restaging positron emission tomography/computed tomography scan showed remarkable radiological response with significant decrease both in size and activity within all metastatic sites (Figures 3A–J). She continued on maintenance pemetrexed chemotherapy (500 mg/m every 21 days) for six more cycles with no evidence of disease progression 10 months after diagnosis. Signet ring variant of primary lung adenocarcinoma is very rare and is associated with EML4-ALK gene fusion that comprises approximately 5% of unselected patients with non-small cell lung cancer (NSCLC).1 In selected population of patients who are younger than 40 years, never/light smokers, and have nonsquamous histology, incidence of EML4ALK gene fusion may be as high as 13%.1 EML4-ALK positivity is associated with lack of response to epidermal growth factor receptor tyrosine kinase inhibitors.1 Recently published results of phase I/II clinical trial with orally bioavailable inhibitor (PF-0341066; crizotinib) of the ALK and c-Met/Hepatocyte Growth Factor receptor (HGFR) tyrosine kinases in patients with metastatic NSCLC who harbor EML4-ALK fusion gene showed remarkable response rates with minimal toxicity.3 Currently, crizotinib is investigated in two phase III randomized trials as a second-line treatment in patients with advanced/metastatic NSCLC with EML4-ALK gene fusion. We report herein complete radiological response to systemic chemotherapy with carboplatin and pemetrexed in this rare variant of NSCLC.

Abstract 4347: Anaplastic lymphoma kinase (ALK) expression is an independent prognostic factor in neuroblastoma patients and correlates well with ALK inhibitor response in vitro

Abstract Introduction: ALK mutations are one of the most common genetic aberrations occurring in 3-11% of the neuroblastoma (NBL) patients and are considered oncogenic. We determined the role of ALK protein levels on survival of NBL patients. Secondly, we tested in vitro responsiveness to ALK inhibitors in NBL cell lines with and without ALK mutation. Results: Formalin-Fixed, Paraffin-Embedded material of 71 NBL, 12 ganglioneuroblastoma (GNBL) and 20 ganglioneuroma (GN) patients was stained for total ALK protein by immunohistochemistry (IHC). The percentage of ALK positive cells in IHC was stratified in 4 Ccategories; Category 1 (n=40): &amp;lt; 20% ALK-positive cells, category 2 (n=25): 20-50% ALK positive cells, category 3 (n=25): 50-75% ALK positive cells, and category 4 (n=13): 75-100% ALK positive cells. These categories correlate significantly with overall survival in the spectrum of NBL, GNBL and GN (p=0.002, Logrank test). ALK mutations were identified in 2/63 NBL (3.2%) and 2/11 GNBL (18.2%) patients by sequencing. Importantly, in NBL and GNBL patients, ALK protein expression remains a significant factor in a multivariate model (Cox Proportional Hazard model) including the COG risk stratification (p&amp;lt;0.000), which encompasses stage and NMYC status (see table). Further, we studied the role of ALK levels in the response to ALK inhibitors in vitro in NBL cell lines. ALK inhibitor response (TAE684) correlates well with ALK protein levels (r=−0.813, p=0.001). ALK point mutated cell lines show higher ALK levels and a better ALK inhibitor response (LC50 values 14.9 fold lower, p=0.004) than WT and amplified cell lines. In conclusion: ALK protein expression is an independent predictor of overall survival in NBL patients and ALK inhibitor response in vitro. ICH staining with ALK antibody is reliable and correlates well with prognosis. 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 4347. doi:10.1158/1538-7445.AM2011-4347

Screening of Anaplastic Lymphoma Kinase Rearrangement by Immunohistochemistry in Non-small Cell Lung Cancer: Correlation with Fluorescence In Situ Hybridization

The use of a standard immunohistochemistry (IHC) assay to detect the anaplastic lymphoma kinase (ALK) protein in lung cancer is challenging. There are no universally accepted, evidence-based guidelines on identifying patients with ALK-rearranged lung cancer using IHC. We retrospectively reviewed 465 resected specimens of non-small cell lung cancer using a tissue microarray as a test set. ALK protein expression using IHC with 5A4 monoclonal antibody (Novocastra) and ALK gene rearrangement using fluorescence in situ hybridization (FISH) with dual-color break-apart probes (Abbott molecular) were examined. Immunoreactivity was scored as 0, 1, 2, or 3, and the results were compared with the FISH results. A diagnostic algorithm was derived from the correlation of the IHC and FISH results and applied to an additional 187 adenocarcinoma samples used as a validation set. In the test set, ALK protein expression was detected in 40 patients (40/465, 8.6%), consisting of IHC scores of 1 (n = 14), 2 (n = 10), and 3 (n = 16), whereas 19 patients (19/453, 4.2%) were FISH-positive. All the FISH-positive patients were assigned IHC scores of 2 or 3. All the patients with ALK IHC scores of 3 were FISH-positive, those with scores of 0 or 1 were FISH-negative, and those with scores of 2 were FISH variable. In the validation set, ALK protein expression was detected in 14 patients (scores of 1, n = 2; scores of 2, n = 6; and scores of 3, n = 6), of which nine patients (9/187, 4.8%) were FISH-positive. All the patients with IHC scores of 0 or 1 were FISH-negative, and those with scores of 3 were FISH- positive. Among the patients with IHC scores of 2, three (3/6, 50%) were FISH-positive. The sensitivity and specificity of IHC was 100% and 95.8%, respectively. These data supported an IHC scoring algorithm in which ALK IHC scores of 0, 1, or 3 were highly compatible with FISH results, and IHC scores of 2 were variable. Based on these findings, the IHC assay using the 5A4 antibody reliably detected non-small cell lung cancer with ALK rearrangement and may be useful as a screening method to identify these tumors.

Increased ALK Gene Copy Number and Amplification are Frequent in Non-small Cell Lung Cancer

Translocation of the anaplastic lymphoma kinase (ALK) gene is involved in the tumorigenesis of a subset of non-small cell lung carcinomas (NSCLCs) and identifies patients sensitive to ALK inhibitors. ALK copy number changes and amplification, which plays an oncogenic role in tumors such as neuroblastoma, are poorly characterized in NSCLC. We aimed to study the prevalence of ALK copy number changes and their correlation to ALK protein expression, epidermal growth factor receptor (EGFR) status, and clinicopathological data in patients with NSCLC. ALK status was evaluated by fluorescence in situ hybridization (FISH). Specimens with ALK translocation were studied for echinoderm microtubule-associated protein-like 4 (EML4), KIF5B, and TFG status. ALK expression was assessed by immunohistochemistry. EGFR gene and protein status were evaluated in adenocarcinomas. Survival analysis was performed. One hundred seven NSCLC cases were evaluated. There were two cases of EML4-ALK translocation and one with an atypical translocation of ALK. Both cases of EML4-ALK translocation had ALK protein expression, whereas in the rest, ALK was undetected. Eleven cases (10%) exhibited ALK amplification and 68 (63%) copy number gains. There was an association between ALK amplification and EGFR FISH positivity (p < 0.0001) but not with prognosis. In conclusion, EML4-ALK translocation is a rare event in NSCLC. The study reveals a significant frequency of ALK amplification and its association with EGFR FISH positivity in lung adenocarcinomas. Based on these findings, a potential role of ALK amplification in the response to ALK inhibitors alone or combined with EGFR inhibitors in NSCLC merits further studies.

Exon scanning by reverse transcriptase–polymerase chain reaction for detection of known and novel EML4–ALK fusion variants in non–small cell lung cancer

Chromosomal inversions within chromosome 2p, resulting in fusions between the echinoderm microtubule-associated protein-like 4 (EML4) and anaplastic lymphoma kinase (ALK) genes, are a recent focus of treatment options for non-small cell lung cancer. Thirteen EML4-ALK fusion variants have been identified, affecting eight EML4 exons. We have developed an exon scanning approach using multiplex reverse transcriptase-polymerase chain reaction (RT-PCR) to amplify known and potential variants involving the first 22 EML4 exons. A total of 55 formalin-fixed, paraffin-embedded lung cancer tumors were screened, of which 5 (9%) were positive for EML4-ALK fusions. Four positive cases harbored known fusion variants: variant 3a, 3b, or both in three cases and variant 1 in one case. The fifth positive specimen harbored two novel variants, designated 8a and 8b, involving exon 17 of EML4. Fluorescence in situ hybridization confirmed the presence of EML4-ALK fusions in three of the four RT-PCR-positive specimens with sufficient tissue for examination, and also confirmed absence of fusions in all 19 RT-PCR-negative specimens tested. Immunohistochemistry analysis confirmed ALK protein expression in the sample containing the novel 8a and 8b variants. This RT-PCR-based exon scanning approach avoids the limitations of screening only for previously identified EML4-ALK fusions and provides a simple molecular assay for fusion detection in a clinical diagnostics setting.

Cytomorphology of ALK+ anaplastic large cell lymphoma displaying spindle cells mimicking a sarcomatous tumor: Report of a case

Anaplastic large cell lymphoma (ALCL) is a lymphoma of T-cell or null-cell lineage characterized by molecular abnormalities involving anaplastic lymphoma kinase (ALK) gene and expression of ALK protein and CD30. Cytologic diagnosis of this entity is often a challenge and diagnostic difficulties are encountered when cytomorphology deviates from the usual descriptions. The reports of FNAC diagnosis of ALCL are rather limited. Histology and immunohistochemistry are usually required to confirm the diagnosis. We discuss a case of ALCL suspected on cytology in which a differential diagnosis of soft tissue sarcoma was also considered because of the presence of spindle-shaped and strap cells. Histology and immunohistochemistry confirmed the diagnosis of ALK+ ALCL with tumor cells displaying strong immunoreactivity for leucocyte common antigen, CD3, ALK-1 and CD30. We emphasize that the cytological features which may help in distinguishing sarcomatoid ALCL from soft tissue sarcoma are the presence of "hallmark cells," lymphoglandular bodies and admixed mature small lymphocytes.

Anaplastic lymphoma kinase immunoreactivity correlates with ALK gene rearrangement and transcriptional up-regulation in non–small cell lung carcinomas

Recently, the fusion gene EML4-ALK was identified in non-small cell lung carcinoma, which could be a potential therapeutic target. We investigated the prevalence of anaplastic lymphoma kinase protein expression in these tumors by immunohistochemistry and correlated the results with data from ALK molecular studies. Gene expression profiling was performed on 35 adenocarcinomas to identify cases with ALK gene up-regulation, which was correlated with protein overexpression by immunohistochemistry. Immunohistochemistry was also performed on an independent cohort consisting of 150 adenocarcinomas and 150 squamous cell carcinomas to evaluate the utility of anaplastic lymphoma kinase immunostaining as a screening tool. Florescence in situ hybridization for the ALK locus and reverse transcriptase-polymerase chain reaction for EML4-ALK were performed on tumors positive for anaplastic lymphoma kinase by immunohistochemistry. Transcriptional up-regulation of ALK was identified in 2 (6%) of 35 adenocarcinomas by gene expression profiling. These 2 cases were positive for anaplastic lymphoma kinase by immunohistochemistry, whereas the remaining 33 cases were completely negative. In the independent cohort, anaplastic lymphoma kinase immunostaining was positive in 1 of 150 squamous cell carcinomas and in 3 of 150 adenocarcinomas. The 6 cases positive for anaplastic lymphoma kinase by immunohistochemistry showed evidence of ALK locus rearrangement by florescence in situ hybridization but were negative for EGFR and KRAS mutation. The presence of EML4-ALK fusion transcript was confirmed in 2 cases by reverse transcriptase-polymerase chain reaction. In conclusion, anaplastic lymphoma kinase immunoreactivity in non-small cell lung carcinomas was associated with transcriptional up-regulation, ALK locus rearrangement, and the presence of EML4-ALK fusion transcript. Anaplastic lymphoma kinase immunohistochemistry may have utility as a screening tool or as a surrogate marker for the molecular techniques to detect the EML4-ALK fusion gene in these tumors.

ALK Mutation Analysis in Patients of the German Neuroblastoma Study

Activating mutations in the gene encoding anaplastic lymphoma kinase (ALK) have recently been observed in familial neuroblastoma suggesting that ALK mutations may contribute to the pathogenesis of this malignancy. In sporadic neuroblastoma, a subgroup of tumors harbors either mutations or amplifications of the ALK gene (George et al., Nature 2008). Nevertheless, the spectrum and incidence of mutations in sporadic NB has only partially been addressed, and no data has been available for patients within the German Neuroblastoma Study. We here re-sequenced the kinase-domain encoding exons of the ALK gene in 280 well-documented patients of the German Neuroblastoma Study Cohort. In addition, the ALK gene copy number was analysed by aCGH or real-time PCR. Data regarding the correlation of ALK gene alterations with clinical patient parameters, as well as correlations of the ALK gene copy number with ALK mRNA and protein expression levels will be presented.

Expression of ALK protein, mRNA and fusion transcripts in anaplastic large cell lymphoma

Systemic anaplastic large cell lymphoma (ALCL) can be divided into two subgroups, anaplastic lymphoma kinase (ALK)-positive and ALK-negative, based on the expression of ALK protein. Expression of this protein is due to genetic alterations of ALK at 2p23. Overall, observations on ALK protein, ALK mRNA, ALK-associated genetic alterations and their relationships, to one another are not often reported in the literature. In this study, we investigated the expression of ALK protein, mRNA and fusion transcripts involving ALK and their relationships in ALCL and analyzed formalin-fixed, paraffin-embedded tissues. Forty-five human cases were analyzed with immunohistochemistry for the ALK protein and RT-PCR for ALK mRNA and seven kinds of ALK involved fusion transcripts. Our results showed that the expression of ALK protein, ALK mRNA and ALK fusion transcripts were significantly related to one another (P < 0.01). Consistent with the expression of ALK protein, patients presenting with ALK mRNA or ALK involved fusion transcripts were significantly younger than those lacking ALK gene alteration (P < 0.01). This study demonstrates expression of both ALK protein and ALK mRNA are positively correlated with expression of ALK-associated fusion transcripts. Combined detection of ALK protein, ALK mRNA and ALK fusion transcripts can complement each other to aid in the diagnosis of ALCL.

Breast cancer update – analysis of knowledge of Brazilian breast surgeons

Anaplastic large cell lymphoma (ALCL), anaplastic lymphoma kinase (ALK)-positive (ALK+ ALCL) is an aggressive CD30-positive T-cell lymphoma that exhibits a chromosomal translocation involving the ALK gene and the expression of ALK protein. No particular risk factor has been clearly identified for ALCL. ALK+ ALCL shows a broad morphologic spectrum, but all cases contain a variable proportion of cells with eccentric, horseshoe- or kidney-shaped nuclei often with an eosinophilic region near the nucleus (hallmark cells). Five morphologic patterns can be recognized. ALK+ ALCL occurs in young subjects (median age ∼35 years), with male predominance, and frequently presents at an advanced stage, with systemic symptoms and extranodal involvement. Near 40% of patients are low risk according to the International Prognostic Index (IPI). Overall, the prognosis of ALK+ ALCL is remarkably better than other T-cell lymphomas. The IPI and the PIT scores in general predict survival in patients with ALK+ ALCL. Standard first-line treatment for ALK+ ALCL consists of doxorubicin-containing polychemotherapy, which is associated with an overall response rate of ∼90%, a 5-year relapse-free survival of ∼60%, and a 5-year overall survival of 70%. Excellent results have been reported with a variety of anthracycline-based chemotherapy regimens including CHOP, CHOEP or MACOP-B. Consolidative high-dose chemotherapy and autologous stem cell transplantation (HDC/ASCT) has also been evaluated in patients in first remission with favourable results, however, superiority to standard chemotherapy is unproven and this approach remains investigational. Following universally accepted guidelines for the treatment of failed aggressive lymphomas, HDC/ASCT can effectively salvage a proportion of patients with relapsed or refractory ALK+ ALCL. Recently, the development of novel therapies targeting CD30 and ALK appear promising.

Diffuse large B-cell lymphoma with expression of anaplastic lymphoma kinase protein: clinicopathologic and immunohistochemical study of 5 cases

To study the clinicopathologic features of diffuse large B-cell lymphoma (DLBCL) with expression of anaplastic lymphoma kinase (ALK) protein. Nine hundred and forty-five (945) cases of DLBCL (including 177 consultation cases) diagnosed according to the 2001 World Health Organization classification of tumors of hematopoietic and lymphoid tissues were enrolled into the study. Immunohistochemical study for anti-ALK-11 was performed using LSAB technique. The ALK-positive cases were further confirmed by immunohistochemical study using EnVision technique. Only ALK-positive cases by EnVision technique were further analyzed by immunostaining for antigens including CD20, CD3, CD30, EMA, granzyme-B, TIA-1 and PC. Immunoglobulin heavy chain gene rearrangement study was also performed and follow-up data collected. There were altogether 5 (4 males and 1 female) cases of DLBCL showing expression of ALK protein. The age of the patients ranged from 34 to 72 years. All were primary nodal DLBCL. One case belonged to clinical stage I, 2 in stage II and 2 in stage III. The duration of follow up ranged from 4 to 32 months. Three patients subsequently died and the longest survival was 32 months. Morphologic subtypes included centroblastic 2, anaplastic 1, immunoblastic with plasmacytoid differentiation 1 and plasmablastic 1. Immunohistochemically, 4 cases were CD20 positive (including 2 centroblastic, 1 anaplastic and 1 immunoblastic cases). The plasmablastic case expressed kappa light chain and was negative for CD20. Rearrangement of immunoglobulin heavy chain gene was demonstrated in all 5 cases studied. As for ALK protein staining, a mixed membranous and cytoplasmic (1 immunoblastic case), granular cytoplasmic (2 centroblastic and 1 anaplastic cases) and mixed nuclear and cytoplasmic (1 plasmablastic case) patterns were observed. Expression of ALK protein is a rare phenomenon in DLBCL and can be seen in centroblastic, anaplastic, immunoblastic and plasmablastic subtypes. It is often associated with aggressive clinical behavior and worse prognosis. A new pattern of ALK protein expression, mixed membranous and cytoplasmic, is reported.

Expression of anaplastic lymphoma kinase and survivin proteins in anaplastic large cell lymphoma and its significance

To study the expression of anaplastic lymphoma kinase (ALK) and survivin proteins in anaplastic large cell lymphoma (ALCL) and there clinical significance. The morphologic characteristics were studied by routine light microscopy. Immunohistochemical staining for ALK and survivin proteins was performed using LSAB method. ALK protein was positive in 51 cases (63%) and negative in 30 cases (37%) of the 81 cases of ALCL studied. The prognosis of patients with ALK protein expression was better than those without ALK expression (P < 0.05). As for survivin protein, there were various degrees of expression in all the 77 ALCL cases studied. High level of survivin protein expression was observed in 33 cases (42.9%), while low level of expression was seen in 44 cases (57.1%). The expression of survivin protein did not correlate with that of ALK protein (P > 0.05). The survival rate was significantly lower in patients with high survivin protein expression (P < 0.05). In cases with ALK protein expression, the prognosis was less favorable if there was also high co-expression of survivin protein (P < 0.05). In ALK protein negative cases, prognosis did not significantly correlate with the expression of survivin protein (P > 0.05). In addition, multivariate analysis confirmed the prognosis value of ALK protein expression, survivin protein expression and constitutional symptoms. Survivin protein expression can serve as an independent prognostic predictor of unfavorable clinical outcome in patients with ALCL, especially when ALK protein is positive.

Ablation of oncogenic ALK is a viable therapeutic approach for anaplastic large-cell lymphomas

AbstractAnaplastic large-cell lymphomas (ALCLs) carry chromosome translocations in which the anaplastic lymphoma kinase (ALK) gene is fused to several partners, most frequently, the NPM1 gene. We have demonstrated that the constitutive activation of ALK fusion proteins results in cellular transformation and lymphoid neoplasia. Herein, we specifically down-regulated ALK protein expression by using small hairpin RNA (shRNA) targeting a sequence coding for the catalytic domain of ALK. The ablation of ALK leads to the down-modulation of known ALK downstream effectors, cell growth arrest, and reversion of the transformed phenotype of ALK+ mouse embryonic fibroblasts in vitro and in vivo. In human ALCL cells lentiviral-mediated ALK knock-down leads to G1 cell-cycle arrest and apoptosis in vitro and tumor growth inhibition and regression in vivo. Using a specific approach we have demonstrated that the survival and growth of ALK+ ALCLs are strictly dependent on ALK activation and signaling. Therefore, ALK is a viable target for therapeutic intervention and its inactivation might represent a pivotal approach for the treatment of ALK lymphomas and other ALK-dependent human tumors.

ALK-1 Expression in Inflammatory Myofibroblastic Tumor of the Urinary Bladder

Inflammatory myofibroblastic tumor (IMT) of the bladder is an uncommon myofibroblastic spindle cell proliferation. Because of its cytologic features and infiltrative nature, it may be difficult to distinguish histologically from sarcomatous proliferations such as sarcomatoid urothelial carcinoma, leiomyosarcoma, and embryonal rhabdomyosarcoma. Recently, anaplastic lymphoma kinase (ALK) gene translocations or ALK protein expression in IMT has been reported, especially in patients of relatively young ages. However, there are only a few reports mentioning IMT of the bladder. We sought to determine the frequency of ALK expression among IMTs of the urinary bladder. We examined 16 cases of IMTof the bladder in 14 patients to elucidate the incidence of ALK-1 expression by immunohistochemistry and its diagnostic usefulness. The age of patients with IMT ranged from 18 to 76 years, with an average age of 42.8 years. The tumors from 10 of 14 patients (12 of 16 cases) were positive for ALK-1. ALK-1-positive cases ranged in age from 18 to 73 years (mean, 39.2 years; median, 38 years) and ALK-1-negative cases from 41 to 76 years (mean, 41.5 years; median, 44.5 years). Two locally recurrent cases were positive for ALK-1 in both the primary and recurrent lesion. ALK-1 immunostaining was detected only in the cytoplasm, with granular or subplasmalemmal linear features, suggesting ALK gene translocation. ALK-1 immunostaining was also performed in 8 sarcomatoid urothelial carcinomas, 5 genitourinary leiomyosarcomas, and 2 stromal tumors of uncertain malignant potential of the prostate, all of which were negative. These results support that ALK-1 immunostaining is useful to differentiate IMT from other malignant spindle cell neoplasms of the bladder. There were no histologic differences between ALK-1 positive and negative IMTs.