Stains & CD markers

Resident / Fellow Advisory Board: Mario L. Marques-Piubelli, M.D.
Editorial Board Member: Christian M. Schürch, M.D., Ph.D.
A. Cristina Vargas, M.B.B.S., Ph.D.

Last author update: 7 March 2022
Last staff update: 7 March 2022

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PubMed Search: ALK pathology [title]

A. Cristina Vargas, M.B.B.S., Ph.D.
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Cite this page: Vargas AC. ALK. website. Accessed July 13th, 2024.
Definition / general
Essential features
  • ALK overexpression occurs as a result of diverse alterations, including translocation, mutation and amplification / polysomy, among others
  • Tumors harboring ALK translocations are potentially sensitive to ALK inhibitors
  • Pathologists should be aware of the frequency of ALK overexpression and translocations according to tumor type to maximize ALK screening in the appropriate clinical setting
  • Understanding the limitations of testing methodologies is important to avoid pitfalls in interpretation
  • ALK expression in itself is a defining feature of specific entities, which when present, enables tumor classification and subtyping in routine pathology practice.
  • Activation of ALK receptor requires ligand binding, which triggers homodimerization and transphosphorylation of its tyrosine kinase inhibitor (TKI) domain
  • ALK is an orphan receptor with no known ligand; heparin is one of the known activating ligands, which promotes ALK signaling through heparin sulfation, leading to ALK dimerization (Science Signaling 2015;8:ra6)
  • Downstream signaling pathways triggered by ALK include STAT3, ERK1 / ERK2, PLC and PI3K / AKT, which upon activation lead to cell proliferation and survival
  • In the inactive state, the ALK receptor promotes apoptosis via caspase 3 activation, leading to kinase inactivation
  • In ALK chromosomal rearrangements, the 3' portion of ALK, which contains the TKI domain, fuses with the 5' of a partner gene that provides the N-terminus with the dimerization domain
  • Fusion chimeric oncoproteins result in constitutive autophosphorylation of the ALK kinase, leading to uncontrolled cell proliferation and survival
  • References: Oncol Lett 2019;17:2020, Cancers (Basel) 2019;11:275
Diagrams / tables

Images hosted on other servers:

ALK signalling pathway

Clinical features
  • Large group of unrelated malignant and benign tumors express ALK; its expression is not a marker of malignant phenotype
  • Prognostic significance of ALK expression depends on tumor type, the underlying molecular mechanism of ALK expression and sensitivity to ALK inhibitors
  • ALK rearrangement predicts response to ALK inhibitors
  • Testing for ALK rearrangements is recommended in all patients with lung adenocarcinomas to guide therapy
  • ALK inhibitors are offered to any ALK rearranged tumor in the advanced or metastatic setting
  • Therapeutic inhibition of ALK fusion oncoproteins is achieved through small molecule TKI, with crizotinib being the first ALK TKI approved by the FDA
  • ALK driven resistance occurs due to secondary mutations in the kinase domain or gene amplification
  • Second (i.e. ceritinib and alectinib) and third (i.e. lorlatinib) generation TKI are used to overcome resistance, including emerging new targets of the ALK signaling pathway (i.e. STAT3, PI3K or ERK / MEK)
  • References: Cancers (Basel) 2019;11:275, Oncol Lett 2019;17:2020
Uses by pathologists
  • ALK immunohistochemistry (IHC) is used as a predictive biomarker of an underlying ALK translocation (or other gene alteration), to identify patients who can potentially benefit from ALK inhibitors
  • Presence of ALK positivity by IHC in the context of specific histological features allows tumor classification in routine pathology practice
  • References: J Clin Pathol 2021 Apr 19 [Epub ahead of print], Arch Pathol Lab Med 2018;142:321
Microscopic (histologic) images

Contributed by A. Cristina Vargas, M.B.B.S., Ph.D., Patricia Guzman, M.D., Fiona Bonar, M.B.B.Ch., Alison Cheah, M.B.B.S. and Martin Jones, M.B.B.S.

ALK rearranged lung adenocarcinoma

ALK IHC in lung adenocarcinoma

Anaplastic large cell lymphoma


Anaplastic large cell lymphoma


Uterine IMT

ALK IHC on uterine IMT

ALK-EML4 spindle cell tumor

ALK-EML4 spindle cell tumor ALK

Undifferentiated pleomorphic sarcoma with ALK

Undifferentiated pleomorphic sarcoma-like ALK

Epithelioid fibrous histiocytoma

ALK IHC in epithelioid fibrous histiocytoma



Positive staining - normal
  • ALK expression in normal tissue is limited to restricted zones of the brain, peripheral nervous system and testis
Positive staining - disease

Table: ALK gene fusion partners according to tumor entity

Tumor type Most common ALK gene fusion partners References
Lung adenocarcinoma EML4 (80%), NPM1, CLTC, TPM3, RANBP2, STRN, ATIC, KIF5B, TPM4, TFG, HIP1, SQSTM1, A2M, KLC1, TPR J Clin Pathol 2021 [Epub ahead of print], Am J Surg Pathol 2011;35:1226, Oncol Lett 2019;17:2020
Anaplastic large cell lymphoma (ALCL) NPM1 (80%), TPM3, ATIC, TFG, CLTC, MSN, TPM4, MYH9, ALO17, TRAF1, EEF1G, PAPBC1, SQSTM1 Science 1994;263:1281, Semin Diagn Pathol 2020;37:57, Oncol Lett 2019;17:2020
ALK+ primary cutaneous ALCL NPM1, TRAF1, ATIC, TPM3 Am J Surg Pathol 2020;44:776
Inflammatory myofibroblastic tumor (IMT) TPM3, TPM4, RANBP2, TFG, CARS, ATIC LMNA, PRKAR1A, CLTC, FN1, SEC31A, EML4, DCTN1, PPFIBP1, FN1 Am J Surg Pathol 2015;39:957, Oncol Lett 2019;17:2020
Epithelioid IMT RANBP2 Am J Surg Pathol 2011;35:135
Spitz tumors DCTN1, TPM3, NPM1, TPR Am J Surg Pathol 2015;39:581
Thyroid carcinoma STRN, EML4, TFG, GTF2IRD1, CCDC149, ITSN2, CTSB, PPP1R21, DCTN1 Am J Surg Pathol 2015;39:652, Endocr Relat Cancer 2019;26:803, Oncol Lett 2019;17:2020
Renal cell carcinomas TPM3, EML4 , STRN, VCL Genes Chromosomes Cancer 2016;55:442, Oncol Lett 2019;17:2020
Peritoneal mesothelioma STRN, ATG16L1, TPM1 JAMA Oncol 2018;4:235, Am J Surg Pathol 2021;45:653
ALK+ histiocytosis TPM3, KIF5B Mod Pathol 2019;32:598, Am J Surg Pathol 2021;45:347
Salivary gland carcinomas MSN (myoepithelial), CTNNA1 (secretory), STRN, MYO18A (intraductal), HNRNPH3, EML4 (salivary duct) Virchows Arch 2021;478:933, Am J Surg Pathol 2020;44:962
Colorectal adenocarcinoma EML4, TPM4, CAD, SPTBN1, SLMAP, DIAPH2, LOC101929227, DIAPH2, STRN Am J Surg Pathol 2020;44:1224, Oncol Lett 2019;17:2020
Pancreatic adenocarcinoma EML4, STRN J Natl Compr Canc Netw 2017;15:555
Leiomyosarcoma STK32B, IGFBP5, ACTG2, TNS1, KANK2 Mol Cancer Res 2019;17:676
Mesenchymal spindle cell tumors (S100+ / SOX10- / CD34+) EML4, PPP1CB, CLIP1 Genes Chromosomes Cancer 2021;60:282, Genes Chromosomes Cancer 2018;57:611
Non neural granular cell tumor (NNGCT) DCTN1, SQSTM1 Am J Surg Pathol 2018;42:1133
Epithelioid fibrous histiocytoma VCL, SQSTM1 Mod Pathol 2015;28:904
ALK+ large B cell lymphoma CLTC, NPM1, SEC31A, SQSTM1, RANBP2, IGL Am J Surg Pathol 2017;41:25

  • ALK overexpression as a result of mutations or other gene alterations
    • Medulloblastomas can harbor ALK somatic or germline mutations (e.g. p.M1199L, c.3605delG), amplification or increased mRNA expression (Am J Surg Pathol 2017;41:781)
    • Subset of acral melanoma harbors the so called ALKATI isoform, arising from an alternative transcription initiation (Am J Surg Pathol 2016;40:786)
Molecular / cytogenetics description
  • Fluorescent in situ hybridization (FISH)
    • FISH using break apart (BA) probes is the most widely available technology for detection of ALK gene rearrangements in routine clinical practice
    • Currently, the Vysis ALK Break Apart FISH Probe Kit (Abbott Molecular, Des Plaines, Illinois, USA) is the only approved companion diagnostic tool for crizotinib based treatment eligibility
    • FISH BA positive result is established using a threshold of 15% tumor cells showing either signals separated by at least 2 probe diameters or 3’ isolated red signals (due to deletion of the 5' probe)
    • Isolated 3’ red signal pattern and other atypical signal patterns (5’ green signals or red doublet) can result in false positive and negative results when compared with detection by next generation sequencing
    • Polysomy including increased copy number or amplification detected by FISH does not predict response to therapy with targeted ALK inhibitors
    • Issues associated with discordant IHC and FISH or false negative FISH: complex deletions / rearrangements, cryptic insertions, alterations of ALK promoter and gene amplification
  • Next generation sequencing and mutation testing
Molecular / cytogenetics images

Contributed by A. Cristina Vargas, M.B.B.S., Ph.D.

ALK break apart FISH

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Break apart signal patterns for ALK rearrangement

Sample pathology report
  • ALK should be reported following the guidelines established for lung adenocarcinoma (e.g. template for biomarker testing established by the College of American Pathologists: Lungbiomarker v1.3.0.2). An example report following this template includes the following:
    • ALK rearrangement by molecular methods
      • Rearrangement identified
        • EML4-ALK
    • Polysomy:
      • Absent
    • ALK by immunohistochemistry
      • Positive: cytoplasmic and nuclear expression (Clone D5F3)
    • ALK rearrangement testing method(s)
      • In situ hybridization (fluorescence [FISH])
      • Immunohistochemistry
        • Ventana ALK (D5F3) immunohistochemistry (IHC) assay
  • Full template of report (College of American Pathologists: Lungbiomarker v1.3.0.2) available in CAP: Cancer Protocol Templates [Accessed 30 June 2021]
Board review style question #1

What is the expected diagnosis for a uterine spindle cell tumor with this histological appearance? The tumor displayed focal smooth muscle expression, strong ALK overexpression on IHC and an ALK translocation was confirmed by FISH.

  1. Endometrial stromal sarcoma
  2. Epitheliod myofibroblastic sarcoma
  3. Inflammatory myofibroblastic tumor
  4. Leiomyoma
  5. Spindle cell tumor (S100+ / SOX10- / CD34+) with ALK translocations
Board review style answer #1
C. Inflammatory myofibroblastic tumor

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Reference: ALK
Board review style question #2
What are the most common tumors harboring ALK translocations?

  1. Anaplastic large cell lymphoma, Spitz tumors, thyroid carcinomas and renal cell carcinomas
  2. Colorectal adenocarcinoma, inflammatory myofibroblastic tumors, anaplastic large cell lymphoma and Merkel cell carcinoma
  3. Lung adenocarcinoma, breast cancer, inflammatory myofibroblastic tumors, rhabdomyosarcoma and Spitz tumors
  4. Lung adenocarcinoma, inflammatory myofibroblastic tumors, anaplastic large cell lymphoma and Spitz tumors
  5. Melanoma, lung adenocarcinoma, inflammatory myofibroblastic tumors and diffuse large B cell lymphoma
Board review style answer #2
D. Lung adenocarcinoma, inflammatory myofibroblastic tumors, anaplastic large cell lymphoma and Spitz tumors

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Reference: ALK
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