All specimens were pathologically reviewed and assessed retrospectively by two investigators (K.I and M.K). Maximum tumor size was determined based on H.E. slides and neuroendocrine differentiation was assessed by positive immunohistochemical staining for chromogranin A (diluted 1:200, clone DAK-A3, Dako, Glostrup, Denmark), synaptophysin (ready to use, DAK-A3, Dako, Glostrup, Denmark), and CD56 in all cases (diluted 1:50, clone 123C3, Dako, Glostrup, Denmark). Tumor specimens were considered positive for neuroendocrine markers if more than 5% of tumor cells were stained. All tumors are positive for at least two of three neuroendocrine markers. Elastica and D2–40 staining was used in all cases to assess lymph-vascular invasion (diluted 1:200, clone D2–40, Acris, Herford, Germany). The Ki-67 and mitotic index was evaluated, and grading was performed according to the WHO classification 201015 . Lymphatic invasion and venous invasion were assessed as reported previously27 (link). Lymph node metastasis was confirmed histologically using surgical specimens, and distant metastases were evaluated either radiologically or histologically.
Clone dak a3
Manufactured by Agilent Technologies
Sourced in Denmark
The Clone DAK-A3 is a laboratory instrument designed for DNA amplification and analysis. It is a key component in various molecular biology applications, such as PCR (Polymerase Chain Reaction) and genetic research. The device provides precise temperature control and cycling capabilities to facilitate the amplification of DNA samples.
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Lab products found in correlation
4 protocols using clone dak a3
1
Neuroendocrine Tumor Histopathological Assessment
2
Immunohistochemical Profiling of Neuroendocrine Tumors
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Immunohistochemical (IHC) staining was performed with the automated system Autostainer Link 48 (Dako Co, Carpinteria, CA) using primary antibodies to chromogranin (clone DAK-A3 DAKO), synaptophysin (clone 5438 DAKO) and CD56 (clone 123c DAKO), following the manufacturer's recommendations. Immunohistochemical neuroendocrine differentiation was considered when at least one of the three markers depicted cytoplasmic or membrane positive staining in tumor cells, even if it was focally.
As a surrogate of a transforming high risk HPV infection p16INK4a was detected using the CINtec histology kit (clone E6H4, mtm Laboratories, Heidelberg, Germany), following the manufacturers protocol. A pattern of diffuse nuclear and cytoplasmic staining (>50%) was considered a positive result.
In each run a negative and a positive control were included. These markers were performed when there was available material. Details on available material by marker are shown inFig. S1 of the supplementary material .
As a surrogate of a transforming high risk HPV infection p16INK4a was detected using the CINtec histology kit (clone E6H4, mtm Laboratories, Heidelberg, Germany), following the manufacturers protocol. A pattern of diffuse nuclear and cytoplasmic staining (>50%) was considered a positive result.
In each run a negative and a positive control were included. These markers were performed when there was available material. Details on available material by marker are shown in
3
Immunohistochemical Characterization of Tumors
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Antibodies (all from Dako North America, Carpintería, CA, USA) against cytokeratins (CKs) AE1/AE3 (clones AE1/AE3; dilution 1:50) and CK 5/6 (clone D5/16 B4; dilution 1:50) and against chromogranin (clone DAK-A3; dilution 1:100), synaptophysin (clone DAK-SYNAP; dilution 1:200), CD56 (clone 123C3; dilution 1:50), CD99 (clone 12E7; dilution 1:1000), neuronal specific enolase (clone BBS/NC/VI-H14; dilution 1:200), Ki67 (clone MIB-1; dilution 1:100) and PD-L1 (clone 22C3; dilution 1:50) were used for the characterization of each of the tumors through immunohistochemistry studies. Expression of PD-L1 was considered positive when there was ≥1% of tumor cells with membranous staining [10 (link)].
4
Immunohistochemical Evaluation of Neuroendocrine Markers
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Immunohistochemistry was performed using Bond™ Polymer Refine Detection kit (Leica Biosystems, Germany), in Leica Bond III platform. Antigen retrieval was performed for 10 min, with Epitope Retrieval Solution 1 for chromogranin and Epitope Retrieval Solution 2 for synaptophysin. Primary monoclonal antibodies for chromogranin (Clone DAK-A3, 1:1000 dilution, Dako, Denmark) and synaptophysin (Clone DAK-SYNAP, 1:100 dilution, Dako, Denmark) were used. Hematoxylin was used for nuclear counterstaining. Appropriate positive controls were used for each antibody and negative controls consisted on the omission of primary antibodies. Because all cases analyzed contained non-neoplastic prostatic epithelium, this served as (internal) control as appropriate.
Chromogranin and synaptophysin immunoexpression were assessed using a routine optical microscope by an experienced pathologist blinded to molecular data. Each marker was categorized as <1% positive neoplastic cells, 1–10% positive neoplastic cells and thereafter at 10% increments. Any cytoplasmic staining regardless of intensity was considered positive.
Chromogranin and synaptophysin immunoexpression were assessed using a routine optical microscope by an experienced pathologist blinded to molecular data. Each marker was categorized as <1% positive neoplastic cells, 1–10% positive neoplastic cells and thereafter at 10% increments. Any cytoplasmic staining regardless of intensity was considered positive.
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