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Original article
Patterns of ALK expression in different human cancer types Pierre Tennstedt,1 Gundula Strobel,2 Charlotte Bölch,2 Tobias Grob,2 Sarah Minner,2 Sawinee Masser,2 Ronald Simon2 1
Martini-Clinic, University Medical Center HamburgEppendorf, Hamburg, Germany 2 Institute of Pathology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany Correspondence to Dr Ronald Simon, Institute of Pathology, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany; [email protected] PT and GS contributed equally. Received 22 November 2013 Revised 10 January 2014 Accepted 12 January 2014 Published Online First 29 January 2014
ABSTRACT Aims Oncogenic gene fusions involving the anaplastic lymphoma kinase (ALK) tyrosine kinase have been identified in several haematopoietic and sporadically also in solid tumour types. Preliminary results from clinical trials suggest that patients with ALK fusion positive cancers might optimally benefit from the tyrosine kinase inhibitor crizotinib, but a comprehensive analysis of solid tumour types for ALK fusion and fusion associated expression is lacking. Methods In order to identify human solid cancers carrying ALK alterations, we performed real-time PCR screening of 1000 tumour samples representing 29 different tumour entities. ALK-positive samples were then transferred into a tissue microarray format and subjected to ALK break-apart fluorescence in situ hybridisation (FISH) analysis and ALK immunohistochemistry (IHC) analysis. Results ALK expression was detected by real-time PCR in 260 of 896 (29%) interpretable tumour samples. FISH analysis was successful in 189 of 260 arrayed cancers but did not detect ALK rearrangement. There was also no ALK expression detectable by IHC. Conclusions Different levels of ALK expression can be found in various cancer types using sensitive methods like real-time PCR. However, such low-level expression is independent from oncogenic ALK fusions and cannot be detected with less-sensitive methods like IHC. ALK fusion is a rare event in human solid cancers.
TMP3,19–21 TMP4,21–24 ATIC,25–28 CLTC,29–32 MYH9,33 SEC31L1,34 RANBP2,35 CARS,14 36 KIF5B37 and EML4,38 39 have been identified in individual cases of lymphomas, inflammatory myofibroblastic tumours (IMT), oesophageal cancers, breast cancers, colorectal cancers and non-small cell lung cancers (NSCLC). Preclinical studies have shown that continued ALK signalling is essential for survival of cancer cells carrying ALK fusions, sensitising these tumours for anti-ALK therapies.40 In 2012, the tyrosine kinase inhibitor crizotinib was approved for the treatment of ALK fusion-positive NSCLC, which makes up about 4% of these cancers. Available efficacy and safety data are encouraging since most patients with ALK-positive lung cancers respond to crizotinib. At present, ALK fusion diagnosis is performed using a fluorescence in situ hybridisation (FISH) test detecting breakage of the ALK gene independently from the fusion partner. Other methods may include PCR or immunohistochemistry (IHC) tests to detect highlevel ALK expression that is typically associated with ALK fusion.41 42 A comprehensive analysis of solid human cancers for ALK fusion and overexpression is lacking. In order to identify additional tumour types that might develop ALK fusions and allow for anti-ALK therapies, we analysed 1000 tumour samples representing 24 different frequent solid tumour types by real-time PCR, IHC, FISH and conventional RT-PCR for ALK rearrangement and expression.
INTRODUCTION
To cite: Tennstedt P, Strobel G, Bölch C, et al. J Clin Pathol 2014;67: 477–481.
Anaplastic lymphoma kinase (ALK) is an orphan receptor tyrosine kinase (RTK) of the insulin receptor (IR) superfamily,1 2 which is believed to play a role in the development of various fetal tissues and the adult nervous system.1 3 4 In cancer cells, inactive ALK can be reactivated by various gene fusions, which typically link the promoters of transcriptionally active genes to the cytoplasmic portion of the normal ALK protein.5 The first ALK fusion, NPM-ALK, was identified in non-Hodgkin’s large cell lymphoma of haematopoietic tumours where it results from a t(2;5) (p23;q35) translocation.4 6 The oncogenic potential of NPM-ALK was demonstrated in mice models in which the ALK gene fusion induced tumour formation.7–9 Signalling pathways influenced by the NPM-ALK gene fusion mediate the induction of growth factor-independent proliferation, cellular transformation, protection from apoptosis and resistance to therapeutic drugs and gamma irradiation.10–13 Subsequently, a large number of additional gene rearrangements, resulting in aberrant ALK expression by bringing ALK under the control of the promoters of ALO17,14 TFG,15–17 MSN,18
Tennstedt P, et al. J Clin Pathol 2014;67:477–481. doi:10.1136/jclinpath-2013-201991
MATERIALS AND METHODS Tissue specimens Formalin-fixed, paraffin-embedded tissues were selected from the archive of the Institute of Pathology, University Medical Center HamburgEppendorf. A total of 1002 tissue samples representing 29 different tumour types and 40 normal tissues were included in the study. A detailed list of all tissues is given in table 1. One pathologist reviewed all haematoxylin-eosin-stained sections of all tissues and selected one block per tumour for RNA isolation. For tumour samples, areas with high tumour cell content (≥60% tumour cells) were marked on the slide. A hollow needle was used to take two tissue cylinders (diameter of 0.5 mm) from each tissue block.
RNA extraction and cDNA synthesis Punched tissue material was deparaffinised with xylene and 80% ethanol. After digestion with Proteinase K at 56°C overnight, total RNA was isolated using the RNeasy FFPE Kit (Qiagen) in a fullautomated nucleic acid isolation device (QIAcube, 477
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Original article tissue core was punched from a representative tumour area and transferred in a TMA format as described before.43
Table 1 Tissue samples for ALK expression analysis Normal tissue Skin Lymph node Lung Oral cavity Breast Endometrium Ovary Vulvar Myometrium Liver Cancers Malignant melanoma Larynx carcinoma Lung cancer Oral cavity cancer Breast cancer Endometrium carcinoma Ovarian cancer Uterus cervix carcinoma Vulvar cancer Colon cancer Oesophageal cancer
2 2 2 2 1 2 2 2 2 3 10 40 133 56 53 31 33 28 39 50 50
Pancreas Stomach Kidney Prostate Testis Bladder Thyroid gland Brain Skeletal muscle Adipose tissue Liver cancer Pancreatic cancer Stomach cancer Renal cell cancer Prostate cancer Testis cancer Urinary bladder cancer Thyroid gland cancer Leiomyosarcoma Liposarcoma
1 2 2 2 3 2 2 2 2 2 50 40 50 59 48 59 55 40 42 36
ALK, anaplastic lymphoma kinase.
Qiagen). cDNA was synthesised in a 96-well plate format using the high-capacity cDNA reverse transcription kit (Applied Biosystems) following the manufacturer’s instructions. In total, 1 mg of total RNA was used for reverse transcription of all samples.
Real-time PCR analysis Real-time PCR was performed using the Lighcycler LC480 (Roche) detection system and the QuantiTect SYBR Green PCR Kit (Qiagen). For specific amplification of ALK and the housekeeping gene TATA box binding protein (TBP), the QuantiTect Primer Assay (Qiagen) was used. RT-PCR was performed in a 25 mL reaction using AmpliTaq Gold polymerase (Applied Biosystems, Darmstadt, Germany). For detection of EML4-ALK variants, the following were the primers: E13; A20, E14; ins11del49A20 and E14; del12A20: 50 -GGAGATGTTCTTACTGGAGACTCA-30 , E20; A20 and E21; A20: 50 -GCTACATCACACACCTT GACTGGT-30 , E6a/b; A20: 50 -CAGATGATAGCCGTAA TAAATTGT-30 , E2; A20 and E2; ins117A20: 5-GCC AACCAAGCCCTCGAGCAGT-30 , E18; A20: 50 - CTGGA TGCAGAAACCAGAGATCTA-30 , and 50 -TGCATGGCTTGC AGCTCCTGGTGC-30 as reverse primer for all fusion variants. PCR cycling conditions began with an initial denaturation step at 95°C for 10 min, followed by 35 cycles of 95°C for 20 s, 58°C annealing for 20 s and 72°C extension for 40 s, and a final extension step at 72°C for 7 min. The quality of the PCR products was confirmed by capillary electrophoresis on a QIAxcel system (Qiagen). Relative quantity of ALK expression was calculated by the 2−ΔΔCt method using median value in two normal tissue samples of each tumour type.
TMA construction A tissue microarray (TMA) was constructed from cases that showed ALK overexpression in real-time PCR analysis. One 478
Fluorescence in situ hybridisation A 4 mm TMA section was used for dual-colour FISH. For proteolytic slide pretreatment, a commercial kit was used ( paraffin pretreatment reagent kit; Vysis, Downers Glove, Illinois, USA). TMA sections were deparaffinised, air-dried and dehydrated in 70%, 85% and 100% ethanol, followed by denaturation for 5 min at 74°C in 70% formamid 2× SSC solution. A commercial break-apart probe (LSI ALK, Vysis) was used to detected ALK gene arrangements. Hybridisation was done overnight at 37°C in a humidified chamber, slides were washed and counterstained with 0.2 mmol/L 40 -6-diamidino-2-phenylindole in an antifade solution. ALK gene arrangement was defined as presence of split signals in more than 60% of cell nuclei. This definition was based on the results of a previous study detecting ERG breakage using a break-apart probe in prostate cancer tissues.44
Immunohistochemistry A freshly cut 4 mm TMA section was used for immunostaining. Antigen retrieval was performed by boiling using an autoclave in citrate buffer ( pH 7.8). A commercial available antibody (anti-ALK, CD246, clone ALK1, DAKO, Glostrup, Denmark) was used for detection of ALK protein expression. The Envision system (DAKO) was used to visualise the immunostaining. One pathologist evaluated immunohistochemical staining of ALK. Any detectable staining was regarded as positive for ALK expression.
RESULTS ALK mRNA expression in cancer tissues RT-PCR analysis was successful in 896/1002 tumour samples. The remaining 104 tumour samples were excluded from the analysis because no expression of the house-keeper gene (TBP) was detected, indicating a poor quality of the RNA in these samples. ALK expression was found in 260/896 (29.0%) interpretable tumour samples, including all analysed tumour entities. Tumour types with the most frequent ALK expression included thyroid cancer 32/39 ALK-positive (82%), seminoma (22/29, 76%), small cell lung cancer (9/17, 53%) and ovarian cancer (16/31, 52%). Tumour types with rare ALK positivity included squamous cell cancer of the oesophagus (1/20, 5%), prostate cancer (2/35, 6%) and cervical cancer (2/26, 8%). All results are summarised in table 2. Besides the overall expression frequency, the expression level varied between the different cancer types. The cancer types with strongest ALK expression (ie, ovarian cancer) had 18-fold higher expression levels as compared to prostate cancers, which represented the tumour type with the lowest expression. A comparison of the relative expression levels across all ALK-positive cancer types is given in figure 1.
EML4-ALK expression in lung cancer 38/129 analysed lung cancer showed ALK positivity by RT-PCR. In none of these ALK-positive tumour samples EML4-ALK expression was detectable.
ALK mRNA expression in normal tissues RT-PCR analysis was successful in 10/40 normal tissue samples. ALK expression was detected in normal breast, ovarian, testis, myometrium, prostate, lung and endometrial tissues. The relative level of expression is compared in figure 2. Tennstedt P, et al. J Clin Pathol 2014;67:477–481. doi:10.1136/jclinpath-2013-201991
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Original article Table 2 ALK positivity in 260/1002 analysed tumour samples
No.
Tumour type
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
Thyroid Seminoma Lung, SCLC Ovarian Renal, clear cell Vulva Mamma, ductal Melanoma Liposarcoma Mamma, lobulary Lung, squamous Stomach Lung, LCLC Non-seminoma Pancreatic Lung, adeno Colon Bladder, non-invasive Oral cavity Bladder, invasive Larynx Renal, papillary Endometrium Oesophagus, adeno Leiomyosarcoma Liver Cervix Prostate Oesophagus, squamous combined
19 20 21 22 23 24 25 26 27 28 29
n overall
n analysable
n ALK positive
% ALK positive
40 30 17 33 30 39 26 10 36 27 25 50 23 29 40 68 50 27
39 29 17 31 29 34 24 10 29 27 24 49 23 27 37 65 44 25
32 22 9 16 14 15 10 4 11 10 8 14 6 7 9 15 10 5
82.1 75.9 52.9 51.6 48.3 44.1 41.7 40.0 37.9 37.0 33.3 28.6 26.1 25.9 24.3 23.1 22.7 20.0
56 28 40 29 31 27 42 50 28 48 23
50 26 34 18 23 25 37 39 26 35 20
10 5 6 3 3 3 4 4 2 2 1
20.0 19.2 17.6 16.7 13.0 12.0 10.8 10.3 7.7 5.7 5.0
1002
896
260
29.0
ALK, anaplastic lymphoma kinase; LCLC, large-cell lung carcinoma; SCLC, small cell lung carcinoma.
ALK FISH break-apart analysis ALK FISH analysis was interpretable in 189 (73%) of the 260 arrayed cancer samples. Non-interpretable tumours included cases with lack of FISH signals or lack of tumour cells in the tissue spot. No ALK rearrangement was detected in any of the analysable tissues.
IHC analysis No positive staining was found in any of the arrayed cancers.
DISCUSSION Quantitative RT-PCR is the currently most sensitive method to measure mRNA expression in tissue samples. The results of our study demonstrate that at least low-level ALK expression can be found in about one-third of human tissue samples in the absence of genomic ALK fusions. Tumour types with most intense and frequent ALK expression in our study included thyroid cancers, seminomas, lung and ovarian cancer. A systematic analysis of human cancer types for ALK expression including multiple different tumour entities has not been performed before. Published data confirm PCR-detectable ALK expression in lung cancer, breast cancer, colorectal cancer, gastrointestinal stroma tumours,17 38 39 45–51 as well as cell lines from thyroid cancer, small cell lung cancer, breast carcinoma, melanoma, neuroblastoma, glioblastoma, astrocytoma, retinoblastoma, Ewing sarcoma, rhabdomyosarcoma, anaplastic large cell lymphoma and pre-B-cell leukaemia.52 The levels of ALK expression in most tumour types were not substantially different from the levels found in the corresponding normal tissues. For example, only slight alterations (ie, less than twofold) were seen in the ALK expression levels between normal and neoplastic tissues from breast, kidney, oral cavity, stomach or colon. This suggests that endogenous ALK expression is not a major driver of tumour development in these tumour types. Markedly higher expression levels (ie, more than eightfold increase in expression) in the tumours as compared to the corresponding normal tissues were found in four tumour types, including ovarian cancers, melanomas, lung cancers and
Figure 1 Anaplastic lymphoma kinase (ALK) overexpression in different tumour types standardised to ALK expression level in prostate cancer.
Tennstedt P, et al. J Clin Pathol 2014;67:477–481. doi:10.1136/jclinpath-2013-201991
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Original article
Take home messages ▸ Low-level anaplastic lymphoma kinase (ALK) expression can be found in various cancer types. ▸ Low-level ALK expression is independent of ALK activation genomic rearrangements. ▸ Specific qPCR assays for detection of therapeutically relevant ALK expression is recommended for selection of patients for anti-ALK therapy.
Figure 2 Anaplastic lymphoma kinase (ALK) expression levels in different normal tissue. ALK expression level (log2) was standardised to ALK expression in endometrium.
Contributors RS and TG were responsible for study concept and design. RS, PT and TG were responsible for analysis and interpretation of data. RS and PT were responsible for drafting of the manuscript. SM and TG were responsible for critical revision of the manuscript for important intellectual content. GS, CB, SM, SM and TG were responsible for administrative, technical or material support. Competing interests None.
thyroid cancers. These data demonstrate that endogenous lowlevel expression of wild-type ALK is common in the absence of ALK rearrangements. High-level ALK expression has so far only been reported from tumour types harbouring translocations, resulting in gene fusions bringing ALK under the control of strong promoters from the fusion partner genes. These include fusions between ALK and NPM,4 6 53 ALO17,14 TFG,15–17 MSN,18 TPM3,19–21 TPM4,21–24 ATIC,25–28 CLTC29–32 and MYH9,33 in anaplastic large cell lymphoma, SEC31L1,34 RANBP235 and CARS,14 36 in IMT, TPM4 in oesophagus squamous cell carcinomas,23 24 NPM53 and CLTC,22 in diffuse large B-cell lymphomas, as well as KIF5B,37 in lung and EML4 in lung,38 breast and colorectal cancers.39 We did not find ALK:EML4 rearrangements using a primer set specific for this particular alteration in our set of 38 lung cancers with detectable ALK expression by means of standard quantitative PCR. This was not unexpected given that the ALK: EML4 fusion is rare in lung cancer. Although it is possible that ALK expression in these tumours was caused by other gene fusions involving ALK, the overall low-expression level as well as the fact that none of these cancers stained positive for ALK protein by means of IHC suggests that our standard qPCR assay may have detected low-level wild-type ALK expression rather than abnormal ALK transcripts. Although these findings indicate a markedly superior sensitivity of our standard qPCR assay as compared to IHC for detection of ALK expression, it challenges the suitability of standard RT-PCR as a ‘sensitive’ method for expression analysis in a potential diagnostic setting aiming at the identification of patients eligible for anti-ALK treatment. In contrast, PCR assays employing primer sets specifically designed for detection of ALK fusions have been proven to be highly suitable for this purpose.54 55 At present, only FISH-based detection of ALK rearrangement is recommended for selection of patients for treatment with the ALK inhibitor crizotinib. This recommendation is based on clinical trials demonstrating that only patients with lung cancers harbouring genomic ALK translocations respond to crizitinib treatment.56–59 In summary, our study shows that low-level ALK expression can be found in the absence of ALK activation genomic rearrangements in various cancer types using a standard RT-PCR assay. Because this expression most likely constitutes physiological levels of ALK, the results of our study do not support the use of RT-PCR for selection of patients for anti-ALK therapies. 480
Patient consent Obtained. Ethics approval WS-049/09 Provenance and peer review Not commissioned; externally peer reviewed.
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Patterns of ALK expression in different human cancer types Pierre Tennstedt, Gundula Strobel, Charlotte Bölch, Tobias Grob, Sarah Minner, Sawinee Masser and Ronald Simon J Clin Pathol 2014 67: 477-481 originally published online January 29, 2014
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Original article
Patterns of ALK expression in different human cancer types Pierre Tennstedt,1 Gundula Strobel,2 Charlotte Bölch,2 Tobias Grob,2 Sarah Minner,2 Sawinee Masser,2 Ronald Simon2 1
Martini-Clinic, University Medical Center HamburgEppendorf, Hamburg, Germany 2 Institute of Pathology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany Correspondence to Dr Ronald Simon, Institute of Pathology, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany; [email protected] PT and GS contributed equally. Received 22 November 2013 Revised 10 January 2014 Accepted 12 January 2014 Published Online First 29 January 2014
ABSTRACT Aims Oncogenic gene fusions involving the anaplastic lymphoma kinase (ALK) tyrosine kinase have been identified in several haematopoietic and sporadically also in solid tumour types. Preliminary results from clinical trials suggest that patients with ALK fusion positive cancers might optimally benefit from the tyrosine kinase inhibitor crizotinib, but a comprehensive analysis of solid tumour types for ALK fusion and fusion associated expression is lacking. Methods In order to identify human solid cancers carrying ALK alterations, we performed real-time PCR screening of 1000 tumour samples representing 29 different tumour entities. ALK-positive samples were then transferred into a tissue microarray format and subjected to ALK break-apart fluorescence in situ hybridisation (FISH) analysis and ALK immunohistochemistry (IHC) analysis. Results ALK expression was detected by real-time PCR in 260 of 896 (29%) interpretable tumour samples. FISH analysis was successful in 189 of 260 arrayed cancers but did not detect ALK rearrangement. There was also no ALK expression detectable by IHC. Conclusions Different levels of ALK expression can be found in various cancer types using sensitive methods like real-time PCR. However, such low-level expression is independent from oncogenic ALK fusions and cannot be detected with less-sensitive methods like IHC. ALK fusion is a rare event in human solid cancers.
TMP3,19–21 TMP4,21–24 ATIC,25–28 CLTC,29–32 MYH9,33 SEC31L1,34 RANBP2,35 CARS,14 36 KIF5B37 and EML4,38 39 have been identified in individual cases of lymphomas, inflammatory myofibroblastic tumours (IMT), oesophageal cancers, breast cancers, colorectal cancers and non-small cell lung cancers (NSCLC). Preclinical studies have shown that continued ALK signalling is essential for survival of cancer cells carrying ALK fusions, sensitising these tumours for anti-ALK therapies.40 In 2012, the tyrosine kinase inhibitor crizotinib was approved for the treatment of ALK fusion-positive NSCLC, which makes up about 4% of these cancers. Available efficacy and safety data are encouraging since most patients with ALK-positive lung cancers respond to crizotinib. At present, ALK fusion diagnosis is performed using a fluorescence in situ hybridisation (FISH) test detecting breakage of the ALK gene independently from the fusion partner. Other methods may include PCR or immunohistochemistry (IHC) tests to detect highlevel ALK expression that is typically associated with ALK fusion.41 42 A comprehensive analysis of solid human cancers for ALK fusion and overexpression is lacking. In order to identify additional tumour types that might develop ALK fusions and allow for anti-ALK therapies, we analysed 1000 tumour samples representing 24 different frequent solid tumour types by real-time PCR, IHC, FISH and conventional RT-PCR for ALK rearrangement and expression.
INTRODUCTION
To cite: Tennstedt P, Strobel G, Bölch C, et al. J Clin Pathol 2014;67: 477–481.
Anaplastic lymphoma kinase (ALK) is an orphan receptor tyrosine kinase (RTK) of the insulin receptor (IR) superfamily,1 2 which is believed to play a role in the development of various fetal tissues and the adult nervous system.1 3 4 In cancer cells, inactive ALK can be reactivated by various gene fusions, which typically link the promoters of transcriptionally active genes to the cytoplasmic portion of the normal ALK protein.5 The first ALK fusion, NPM-ALK, was identified in non-Hodgkin’s large cell lymphoma of haematopoietic tumours where it results from a t(2;5) (p23;q35) translocation.4 6 The oncogenic potential of NPM-ALK was demonstrated in mice models in which the ALK gene fusion induced tumour formation.7–9 Signalling pathways influenced by the NPM-ALK gene fusion mediate the induction of growth factor-independent proliferation, cellular transformation, protection from apoptosis and resistance to therapeutic drugs and gamma irradiation.10–13 Subsequently, a large number of additional gene rearrangements, resulting in aberrant ALK expression by bringing ALK under the control of the promoters of ALO17,14 TFG,15–17 MSN,18
Tennstedt P, et al. J Clin Pathol 2014;67:477–481. doi:10.1136/jclinpath-2013-201991
MATERIALS AND METHODS Tissue specimens Formalin-fixed, paraffin-embedded tissues were selected from the archive of the Institute of Pathology, University Medical Center HamburgEppendorf. A total of 1002 tissue samples representing 29 different tumour types and 40 normal tissues were included in the study. A detailed list of all tissues is given in table 1. One pathologist reviewed all haematoxylin-eosin-stained sections of all tissues and selected one block per tumour for RNA isolation. For tumour samples, areas with high tumour cell content (≥60% tumour cells) were marked on the slide. A hollow needle was used to take two tissue cylinders (diameter of 0.5 mm) from each tissue block.
RNA extraction and cDNA synthesis Punched tissue material was deparaffinised with xylene and 80% ethanol. After digestion with Proteinase K at 56°C overnight, total RNA was isolated using the RNeasy FFPE Kit (Qiagen) in a fullautomated nucleic acid isolation device (QIAcube, 477
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Original article tissue core was punched from a representative tumour area and transferred in a TMA format as described before.43
Table 1 Tissue samples for ALK expression analysis Normal tissue Skin Lymph node Lung Oral cavity Breast Endometrium Ovary Vulvar Myometrium Liver Cancers Malignant melanoma Larynx carcinoma Lung cancer Oral cavity cancer Breast cancer Endometrium carcinoma Ovarian cancer Uterus cervix carcinoma Vulvar cancer Colon cancer Oesophageal cancer
2 2 2 2 1 2 2 2 2 3 10 40 133 56 53 31 33 28 39 50 50
Pancreas Stomach Kidney Prostate Testis Bladder Thyroid gland Brain Skeletal muscle Adipose tissue Liver cancer Pancreatic cancer Stomach cancer Renal cell cancer Prostate cancer Testis cancer Urinary bladder cancer Thyroid gland cancer Leiomyosarcoma Liposarcoma
1 2 2 2 3 2 2 2 2 2 50 40 50 59 48 59 55 40 42 36
ALK, anaplastic lymphoma kinase.
Qiagen). cDNA was synthesised in a 96-well plate format using the high-capacity cDNA reverse transcription kit (Applied Biosystems) following the manufacturer’s instructions. In total, 1 mg of total RNA was used for reverse transcription of all samples.
Real-time PCR analysis Real-time PCR was performed using the Lighcycler LC480 (Roche) detection system and the QuantiTect SYBR Green PCR Kit (Qiagen). For specific amplification of ALK and the housekeeping gene TATA box binding protein (TBP), the QuantiTect Primer Assay (Qiagen) was used. RT-PCR was performed in a 25 mL reaction using AmpliTaq Gold polymerase (Applied Biosystems, Darmstadt, Germany). For detection of EML4-ALK variants, the following were the primers: E13; A20, E14; ins11del49A20 and E14; del12A20: 50 -GGAGATGTTCTTACTGGAGACTCA-30 , E20; A20 and E21; A20: 50 -GCTACATCACACACCTT GACTGGT-30 , E6a/b; A20: 50 -CAGATGATAGCCGTAA TAAATTGT-30 , E2; A20 and E2; ins117A20: 5-GCC AACCAAGCCCTCGAGCAGT-30 , E18; A20: 50 - CTGGA TGCAGAAACCAGAGATCTA-30 , and 50 -TGCATGGCTTGC AGCTCCTGGTGC-30 as reverse primer for all fusion variants. PCR cycling conditions began with an initial denaturation step at 95°C for 10 min, followed by 35 cycles of 95°C for 20 s, 58°C annealing for 20 s and 72°C extension for 40 s, and a final extension step at 72°C for 7 min. The quality of the PCR products was confirmed by capillary electrophoresis on a QIAxcel system (Qiagen). Relative quantity of ALK expression was calculated by the 2−ΔΔCt method using median value in two normal tissue samples of each tumour type.
TMA construction A tissue microarray (TMA) was constructed from cases that showed ALK overexpression in real-time PCR analysis. One 478
Fluorescence in situ hybridisation A 4 mm TMA section was used for dual-colour FISH. For proteolytic slide pretreatment, a commercial kit was used ( paraffin pretreatment reagent kit; Vysis, Downers Glove, Illinois, USA). TMA sections were deparaffinised, air-dried and dehydrated in 70%, 85% and 100% ethanol, followed by denaturation for 5 min at 74°C in 70% formamid 2× SSC solution. A commercial break-apart probe (LSI ALK, Vysis) was used to detected ALK gene arrangements. Hybridisation was done overnight at 37°C in a humidified chamber, slides were washed and counterstained with 0.2 mmol/L 40 -6-diamidino-2-phenylindole in an antifade solution. ALK gene arrangement was defined as presence of split signals in more than 60% of cell nuclei. This definition was based on the results of a previous study detecting ERG breakage using a break-apart probe in prostate cancer tissues.44
Immunohistochemistry A freshly cut 4 mm TMA section was used for immunostaining. Antigen retrieval was performed by boiling using an autoclave in citrate buffer ( pH 7.8). A commercial available antibody (anti-ALK, CD246, clone ALK1, DAKO, Glostrup, Denmark) was used for detection of ALK protein expression. The Envision system (DAKO) was used to visualise the immunostaining. One pathologist evaluated immunohistochemical staining of ALK. Any detectable staining was regarded as positive for ALK expression.
RESULTS ALK mRNA expression in cancer tissues RT-PCR analysis was successful in 896/1002 tumour samples. The remaining 104 tumour samples were excluded from the analysis because no expression of the house-keeper gene (TBP) was detected, indicating a poor quality of the RNA in these samples. ALK expression was found in 260/896 (29.0%) interpretable tumour samples, including all analysed tumour entities. Tumour types with the most frequent ALK expression included thyroid cancer 32/39 ALK-positive (82%), seminoma (22/29, 76%), small cell lung cancer (9/17, 53%) and ovarian cancer (16/31, 52%). Tumour types with rare ALK positivity included squamous cell cancer of the oesophagus (1/20, 5%), prostate cancer (2/35, 6%) and cervical cancer (2/26, 8%). All results are summarised in table 2. Besides the overall expression frequency, the expression level varied between the different cancer types. The cancer types with strongest ALK expression (ie, ovarian cancer) had 18-fold higher expression levels as compared to prostate cancers, which represented the tumour type with the lowest expression. A comparison of the relative expression levels across all ALK-positive cancer types is given in figure 1.
EML4-ALK expression in lung cancer 38/129 analysed lung cancer showed ALK positivity by RT-PCR. In none of these ALK-positive tumour samples EML4-ALK expression was detectable.
ALK mRNA expression in normal tissues RT-PCR analysis was successful in 10/40 normal tissue samples. ALK expression was detected in normal breast, ovarian, testis, myometrium, prostate, lung and endometrial tissues. The relative level of expression is compared in figure 2. Tennstedt P, et al. J Clin Pathol 2014;67:477–481. doi:10.1136/jclinpath-2013-201991
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Original article Table 2 ALK positivity in 260/1002 analysed tumour samples
No.
Tumour type
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
Thyroid Seminoma Lung, SCLC Ovarian Renal, clear cell Vulva Mamma, ductal Melanoma Liposarcoma Mamma, lobulary Lung, squamous Stomach Lung, LCLC Non-seminoma Pancreatic Lung, adeno Colon Bladder, non-invasive Oral cavity Bladder, invasive Larynx Renal, papillary Endometrium Oesophagus, adeno Leiomyosarcoma Liver Cervix Prostate Oesophagus, squamous combined
19 20 21 22 23 24 25 26 27 28 29
n overall
n analysable
n ALK positive
% ALK positive
40 30 17 33 30 39 26 10 36 27 25 50 23 29 40 68 50 27
39 29 17 31 29 34 24 10 29 27 24 49 23 27 37 65 44 25
32 22 9 16 14 15 10 4 11 10 8 14 6 7 9 15 10 5
82.1 75.9 52.9 51.6 48.3 44.1 41.7 40.0 37.9 37.0 33.3 28.6 26.1 25.9 24.3 23.1 22.7 20.0
56 28 40 29 31 27 42 50 28 48 23
50 26 34 18 23 25 37 39 26 35 20
10 5 6 3 3 3 4 4 2 2 1
20.0 19.2 17.6 16.7 13.0 12.0 10.8 10.3 7.7 5.7 5.0
1002
896
260
29.0
ALK, anaplastic lymphoma kinase; LCLC, large-cell lung carcinoma; SCLC, small cell lung carcinoma.
ALK FISH break-apart analysis ALK FISH analysis was interpretable in 189 (73%) of the 260 arrayed cancer samples. Non-interpretable tumours included cases with lack of FISH signals or lack of tumour cells in the tissue spot. No ALK rearrangement was detected in any of the analysable tissues.
IHC analysis No positive staining was found in any of the arrayed cancers.
DISCUSSION Quantitative RT-PCR is the currently most sensitive method to measure mRNA expression in tissue samples. The results of our study demonstrate that at least low-level ALK expression can be found in about one-third of human tissue samples in the absence of genomic ALK fusions. Tumour types with most intense and frequent ALK expression in our study included thyroid cancers, seminomas, lung and ovarian cancer. A systematic analysis of human cancer types for ALK expression including multiple different tumour entities has not been performed before. Published data confirm PCR-detectable ALK expression in lung cancer, breast cancer, colorectal cancer, gastrointestinal stroma tumours,17 38 39 45–51 as well as cell lines from thyroid cancer, small cell lung cancer, breast carcinoma, melanoma, neuroblastoma, glioblastoma, astrocytoma, retinoblastoma, Ewing sarcoma, rhabdomyosarcoma, anaplastic large cell lymphoma and pre-B-cell leukaemia.52 The levels of ALK expression in most tumour types were not substantially different from the levels found in the corresponding normal tissues. For example, only slight alterations (ie, less than twofold) were seen in the ALK expression levels between normal and neoplastic tissues from breast, kidney, oral cavity, stomach or colon. This suggests that endogenous ALK expression is not a major driver of tumour development in these tumour types. Markedly higher expression levels (ie, more than eightfold increase in expression) in the tumours as compared to the corresponding normal tissues were found in four tumour types, including ovarian cancers, melanomas, lung cancers and
Figure 1 Anaplastic lymphoma kinase (ALK) overexpression in different tumour types standardised to ALK expression level in prostate cancer.
Tennstedt P, et al. J Clin Pathol 2014;67:477–481. doi:10.1136/jclinpath-2013-201991
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Original article
Take home messages ▸ Low-level anaplastic lymphoma kinase (ALK) expression can be found in various cancer types. ▸ Low-level ALK expression is independent of ALK activation genomic rearrangements. ▸ Specific qPCR assays for detection of therapeutically relevant ALK expression is recommended for selection of patients for anti-ALK therapy.
Figure 2 Anaplastic lymphoma kinase (ALK) expression levels in different normal tissue. ALK expression level (log2) was standardised to ALK expression in endometrium.
Contributors RS and TG were responsible for study concept and design. RS, PT and TG were responsible for analysis and interpretation of data. RS and PT were responsible for drafting of the manuscript. SM and TG were responsible for critical revision of the manuscript for important intellectual content. GS, CB, SM, SM and TG were responsible for administrative, technical or material support. Competing interests None.
thyroid cancers. These data demonstrate that endogenous lowlevel expression of wild-type ALK is common in the absence of ALK rearrangements. High-level ALK expression has so far only been reported from tumour types harbouring translocations, resulting in gene fusions bringing ALK under the control of strong promoters from the fusion partner genes. These include fusions between ALK and NPM,4 6 53 ALO17,14 TFG,15–17 MSN,18 TPM3,19–21 TPM4,21–24 ATIC,25–28 CLTC29–32 and MYH9,33 in anaplastic large cell lymphoma, SEC31L1,34 RANBP235 and CARS,14 36 in IMT, TPM4 in oesophagus squamous cell carcinomas,23 24 NPM53 and CLTC,22 in diffuse large B-cell lymphomas, as well as KIF5B,37 in lung and EML4 in lung,38 breast and colorectal cancers.39 We did not find ALK:EML4 rearrangements using a primer set specific for this particular alteration in our set of 38 lung cancers with detectable ALK expression by means of standard quantitative PCR. This was not unexpected given that the ALK: EML4 fusion is rare in lung cancer. Although it is possible that ALK expression in these tumours was caused by other gene fusions involving ALK, the overall low-expression level as well as the fact that none of these cancers stained positive for ALK protein by means of IHC suggests that our standard qPCR assay may have detected low-level wild-type ALK expression rather than abnormal ALK transcripts. Although these findings indicate a markedly superior sensitivity of our standard qPCR assay as compared to IHC for detection of ALK expression, it challenges the suitability of standard RT-PCR as a ‘sensitive’ method for expression analysis in a potential diagnostic setting aiming at the identification of patients eligible for anti-ALK treatment. In contrast, PCR assays employing primer sets specifically designed for detection of ALK fusions have been proven to be highly suitable for this purpose.54 55 At present, only FISH-based detection of ALK rearrangement is recommended for selection of patients for treatment with the ALK inhibitor crizotinib. This recommendation is based on clinical trials demonstrating that only patients with lung cancers harbouring genomic ALK translocations respond to crizitinib treatment.56–59 In summary, our study shows that low-level ALK expression can be found in the absence of ALK activation genomic rearrangements in various cancer types using a standard RT-PCR assay. Because this expression most likely constitutes physiological levels of ALK, the results of our study do not support the use of RT-PCR for selection of patients for anti-ALK therapies. 480
Patient consent Obtained. Ethics approval WS-049/09 Provenance and peer review Not commissioned; externally peer reviewed.
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Patterns of ALK expression in different human cancer types Pierre Tennstedt, Gundula Strobel, Charlotte Bölch, Tobias Grob, Sarah Minner, Sawinee Masser and Ronald Simon J Clin Pathol 2014 67: 477-481 originally published online January 29, 2014
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