Advertisement

Ki-67 Antigen in Lung Neuroendocrine Tumors: Unraveling a Role in Clinical Practice

      Classification of lung neuroendocrine (NE) tumors is a step-wise process with four tumor categories being identified by morphology, namely typical carcinoid (TC), atypical carcinoid, large-cell NE carcinoma, and small-cell lung carcinoma (SCLC). Ki-67 antigen or protein (henceforth simply Ki-67) has been largely studied in these tumors, but the clinical implications are so far not clear. A well-defined role has regarded the diagnostic use in the separation of TC and AC from SCLC in nonsurgical specimens, with monoclonal antibody MIB-1 resulting in the most used reagent after antigen retrieval procedures. Uncertainties, however, have arisen in its assessment, usually expressed as Ki-67 labeling index, because of some variability in obtaining either value of the fraction. A diagnostic role is currently lacking, even though there are significant differences in most cases between TC and AC, less so between large-cell NE carcinoma and SCLC. In addition, the prognostic role of Ki-67 is debated, likely due to methodological and biological reasons. The last challenge would be to identify an effective lung-specific grading system based on Ki-67 labeling index. In this review article, five relevant issues to Ki-67 have been addressed by using a question-answer methodology, with relevant key points discussing major interpretation issues. The conclusion is that Ki-67 is a feasible and potentially meaningful marker in lung NE tumors, but more data are needed to determine its ideal function in this setting of tumors.

      Key Words

      In the lung, neoplasms with neuroendocrine (NE) morphology and differentiation encompass four histologically defined variants, namely typical carcinoid (TC), atypical carcinoid, large-cell NE carcinoma (LCNEC), and small-cell carcinoma (SCLC).
      • Travis W
      • Brambilla E
      • Muller-Hermelink H
      • Harris C
      ,
      • Travis W
      • Colby T
      • Corrin B
      • Shimosato Y
      • Brambilla E
      According to epidemiologic, genetic, and clinical data, pulmonary NE tumors may be assembled for prognosis and therapy purposes into a three-tier clinicopathological scheme, according to which TC are low-grade malignant tumors with long life expectation and usual surgical treatment; AC, intermediate-grade malignant tumors with more aggressive clinical course and multimodality therapy; and LCNEC and SCLC, high-grade malignant tumors with overlapping dismal prognosis and multimodality or exclusive medical treatment.
      • Travis W
      • Brambilla E
      • Muller-Hermelink H
      • Harris C
      • Travis W
      • Colby T
      • Corrin B
      • Shimosato Y
      • Brambilla E
      • Travis WD
      • Gal AA
      • Colby TV
      • et al.
      Reproducibility of neuroendocrine lung tumor classification.
      • Travis WD
      • Rush W
      • Flieder DB
      • et al.
      Survival analysis of 200 pulmonary neuroendocrine tumors with clarification of criteria for atypical carcinoid and its separation from typical carcinoid.
      • Gridelli C
      • Rossi A
      • Airoma G
      • et al.
      Treatment of pulmonary neuroendocrine tumours: state of the art and future developments.
      • McMullan DM
      • Wood DE
      Pulmonary carcinoid tumors.
      • Asamura H
      • Kameya T
      • Matsuno Y
      • et al.
      Neuroendocrine neoplasms of the lung: a prognostic spectrum.
      • Righi L
      • Volante M
      • Rapa I
      • et al.
      Mammalian target of rapamycin signaling activation patterns in neuroendocrine tumors of the lung.
      Subtyping pulmonary NE tumors is a step-wise process in which the four histologic variants are primarily separated by the number of mitoses per 2 mm2 and the presence of necrosis. Immunohistochemistry (IHC) for NE markers along with NE morphology is required to separate LCNEC from conventional non–small-cell lung carcinoma (NSCLC), and the distinction from SCLC is primarily based on cytological characteristics, including cell size.
      • Travis W
      • Brambilla E
      • Muller-Hermelink H
      • Harris C
      • Travis W
      • Colby T
      • Corrin B
      • Shimosato Y
      • Brambilla E
      • Travis WD
      • Gal AA
      • Colby TV
      • et al.
      Reproducibility of neuroendocrine lung tumor classification.
      ,
      • Franks TJ
      • Galvin JR
      Lung tumors with neuroendocrine morphology: essential radiologic and pathologic features.
      • Rekhtman N
      Neuroendocrine tumors of the lung: an update.
      • Litzky L
      Pulmonary neuroendocrine tumors.
      NSCLC lacking NE morphology such as adenocarcinoma or squamous cell carcinoma but with NE differentiation by IHC or electron microscopy has not been shown consistently to have different prognosis or response to treatment, so this is not accepted as a distinct class of lung cancer.
      • Travis W
      • Brambilla E
      • Muller-Hermelink H
      • Harris C
      ,
      • Travis W
      • Colby T
      • Corrin B
      • Shimosato Y
      • Brambilla E
      Ki-67 antigen, also known as simply Ki-67 or MKI67 antigen identified by monoclonal antibody Ki-67, is a 359-kD non-histone nuclear protein with short half-life, which is encoded by the 15 exon-spanning MKI67 gene mapping to chromosome 10q26.2. This protein plays an essential role in the control and timing of cell proliferation,
      • Duchrow M
      • Schlüter C
      • Key G
      • et al.
      Cell proliferation-associated nuclear antigen defined by antibody Ki-67: a new kind of cell cycle-maintaining proteins.
      • Duchrow M
      • Schlüter C
      • Wohlenberg C
      • Flad HD
      • Gerdes J
      Molecular characterization of the gene locus of the human cell proliferation-associated nuclear protein defined by monoclonal antibody Ki-67.
      • Fonatsch C
      • Duchrow M
      • Rieder H
      • Schlüter C
      • Gerdes J
      Assignment of the human Ki-67 gene (MK167) to 10q25-qter.
      • Schlüter C
      • Duchrow M
      • Wohlenberg C
      • et al.
      The cell proliferation-associated antigen of antibody Ki-67: a very large, ubiquitous nuclear protein with numerous repeated elements, representing a new kind of cell cycle-maintaining proteins.
      • Bruno S
      • Darzynkiewicz Z
      Cell cycle dependent expression and stability of the nuclear protein detected by Ki-67 antibody in HL-60 cells.
      • Schonk DM
      • Kuijpers HJ
      • van Drunen E
      • et al.
      Assignment of the gene(s) involved in the expression of the proliferation-related Ki-67 antigen to human chromosome 10.
      which undergoes a complex mechanism of post-translational phosphorylation and dephosphorylation by cell cycle key regulators leading to its subcellular redistribution from the interior of the nucleus/nucleolus to the perichromosomal layer and heterochromatin during mitosis and meiosis and vice versa
      • Endl E
      • Gerdes J
      Posttranslational modifications of the KI-67 protein coincide with two major checkpoints during mitosis.
      • Kreitz S
      • Fackelmayer FO
      • Gerdes J
      • Knippers R
      The proliferation-specific human Ki-67 protein is a constituent of compact chromatin.
      • Scholzen T
      • Endl E
      • Wohlenberg C
      • et al.
      The Ki-67 protein interacts with members of the heterochromatin protein 1 (HP1) family: a potential role in the regulation of higher-order chromatin structure.
      • Traut W
      • Endl E
      • Scholzen T
      • Gerdes J
      • Winking H
      The temporal and spatial distribution of the proliferation associated Ki-67 protein during female and male meiosis.
      • Saiwaki T
      • Kotera I
      • Sasaki M
      • Takagi M
      • Yoneda Y
      In vivo dynamics and kinetics of pKi-67: transition from a mobile to an immobile form at the onset of anaphase.
      Functionally, Ki-67 expression is finely tuned by specific microRNAs
      • Hou YY
      • Cao WW
      • Li L
      • et al.
      MicroRNA-519d targets MKi67 and suppresses cell growth in the hepatocellular carcinoma cell line QGY-7703.
      and produced during the entire cell cycle with a maximum in the G2 and M phases.
      • Baisch H
      • Gerdes J
      Identification of proliferating cells by Ki-67 antibody.
      ,
      • Gerdes J
      • Lemke H
      • Baisch H
      • et al.
      Cell cycle analysis of a cell proliferation-associated human nuclear antigen defined by the monoclonal antibody Ki-67.
      However, it may be found at sites linked to the ribosomal RNA transcription machinery with a tight chromatin-associated function in both interphase and mitotic cells, although this finding does not argue against its valuable function as proliferation marker.
      • Bullwinkel J
      • Baron-Luhr B
      • Ludemann A
      • et al.
      Ki-67 protein is associated with ribosomal RNA transcription in quiescent and proliferating cells.
      ,
      • Starborg M
      • Gell K
      • Brundell E
      • Höög C
      The murine Ki-67 cell proliferation antigen accumulates in the nucleolar and heterochromatic regions of interphase cells and at the periphery of the mitotic chromosomes in a process essential for cell cycle progression.
      The name Ki-67 derives from the city of Kiel in Germany where the antibody was first raised and the number 67 from the clone position in the original 96-well plate generated immunizing mice with nuclei of the lymphoma cell line L428.
      • Gerdes J
      • Schwab U
      • Lemke H
      • Stein H
      Production of a mouse monoclonal antibody reactive with a human nuclear antigen associated with cell proliferation.
      ,
      • Gerdes J
      • Li L
      • Schlueter C
      • et al.
      Immunobiochemical and molecular biologic characterization of the cell proliferation-associated nuclear antigen that is defined by monoclonal antibody Ki-67.
      As the original monoclonal antibody to Ki-67 worked on frozen or fresh material only, subsequent antibodies have been developed to react with Ki-67 in formalin-fixed and paraffin-embedded material in a huge variety of malignancies, among which endocrine tumors in different anatomical sites.
      • Pelosi G
      • Zamboni G
      Proliferation markers and their uses in the study of endocrine tumors.
      These reagents included polyclonal Ki-67
      • Key G
      • Petersen JL
      • Becker MH
      • et al.
      New antiserum against Ki-67 antigen suitable for double immunostaining of paraffin wax sections.
      and monoclonal MIB-1-3-5,
      • Cattoretti G
      • Becker MH
      • Key G
      • et al.
      Monoclonal antibodies against recombinant parts of the Ki-67 antigen (MIB 1 and MIB 3) detect proliferating cells in microwave-processed formalin-fixed paraffin sections.
      • Key G
      • Becker MH
      • Baron B
      • et al.
      New Ki-67-equivalent murine monoclonal antibodies (MIB 1-3) generated against bacterially expressed parts of the Ki-67 cDNA containing three 62 base pair repetitive elements encoding for the Ki-67 epitope.
      • Kubbutat MH
      • Key G
      • Duchrow M
      • et al.
      Epitope analysis of antibodies recognising the cell proliferation associated nuclear antigen previously defined by the antibody Ki-67 (Ki-67 protein).
      IND.64,
      • Key G
      • Meggetto F
      • Becker MH
      • et al.
      Immunobiochemical characterization of the antigen detected by monoclonal antibody IND.64. Evidence that IND.64 reacts with the cell proliferation associated nuclear antigen previously defined by Ki-67.
      JG-67-2a,
      • Kubbutat MH
      • Key G
      • Duchrow M
      • et al.
      Epitope analysis of antibodies recognising the cell proliferation associated nuclear antigen previously defined by the antibody Ki-67 (Ki-67 protein).
      Ki-S1,
      • Rudolph P
      • Lappe T
      • Schubert C
      • et al.
      Diagnostic assessment of two novel proliferation-specific antigens in benign and malignant melanocytic lesions.
      Ki-S3,
      • Heidebrecht HJ
      • Buck F
      • Haas K
      • Wacker HH
      • Parwaresch R
      Monoclonal antibodies Ki-S3 and Ki-S5 yield new data on the ‘Ki-67’ proteins.
      Ki-S5,
      • Rudolph P
      • Lappe T
      • Schubert C
      • et al.
      Diagnostic assessment of two novel proliferation-specific antigens in benign and malignant melanocytic lesions.
      ,
      • Kreipe H
      • Wacker HH
      • Heidebrecht HJ
      • et al.
      Determination of the growth fraction in non-Hodgkin's lymphomas by monoclonal antibody Ki-S5 directed against a formalin-resistant epitope of the Ki-67 antigen.
      and Ki-S11
      • Rudolph P
      • Kellner U
      • Chassevent A
      • et al.
      Prognostic relevance of a novel proliferation marker, Ki-S11, for soft-tissue sarcoma. A multivariate study.
      antibodies, with most studies confirming the validity of results obtained with these reagents in the measurement of proliferative activity in routinely processed tissues and even cytological samples.
      • Schwarting R
      • Gerdes J
      • Niehus J
      • Jaeschke L
      • Stein H
      Determination of the growth fraction in cell suspensions by flow cytometry using the monoclonal antibody Ki-67.
      • Scott RJ
      • Hall PA
      • Haldane JS
      • et al.
      A comparison of immunohistochemical markers of cell proliferation with experimentally determined growth fraction.
      • Sasaki K
      • Matsumura K
      • Tsuji T
      • Shinozaki F
      • Takahashi M
      Relationship between labeling indices of Ki-67 and BrdUrd in human malignant tumors.
      • Pelosi G
      • Bresaola E
      • Manfrin E
      • et al.
      Immunocytochemical detection of cell proliferation-related antigens in cytologic smears of human malignant neoplasms using PC10, reactive with proliferating cell nuclear antigen, and Ki-67. A comparative study.
      In more recent years, clone Mib-1 has been emerging as the most reliable and consistent reagent to recognize Ki-67 in paraffin sections
      • Cattoretti G
      • Becker MH
      • Key G
      • et al.
      Monoclonal antibodies against recombinant parts of the Ki-67 antigen (MIB 1 and MIB 3) detect proliferating cells in microwave-processed formalin-fixed paraffin sections.
      ,
      • Key G
      • Becker MH
      • Baron B
      • et al.
      New Ki-67-equivalent murine monoclonal antibodies (MIB 1-3) generated against bacterially expressed parts of the Ki-67 cDNA containing three 62 base pair repetitive elements encoding for the Ki-67 epitope.
      and its wide commercial availability allowed many investigative studies and meta-analyses to be performed on the clinical implications of Ki-67 to assess proliferative activity in different malignancies,
      • Chalkidou A
      • Landau DB
      • Odell EW
      • et al.
      Correlation between Ki-67 immunohistochemistry and 18F-fluorothymidine uptake in patients with cancer: A systematic review and meta-analysis.
      ,
      • de Azambuja E
      • Cardoso F
      • de Castro Jr, G
      • et al.
      Ki-67 as prognostic marker in early breast cancer: a meta-analysis of published studies involving 12,155 patients.
      including lung.
      • Martin B
      • Paesmans M
      • Mascaux C
      • et al.
      Ki-67 expression and patients survival in lung cancer: systematic review of the literature with meta-analysis.
      In NE tumor pathology, Ki-67 was first clinically investigated as prognostic factor in the pancreas,
      • Pelosi G
      • Zamboni G
      • Doglioni C
      • et al.
      Immunodetection of proliferating cell nuclear antigen assesses the growth fraction and predicts malignancy in endocrine tumors of the pancreas.
      • Pelosi G
      • Bresaola E
      • Bogina G
      • et al.
      Endocrine tumors of the pancreas: Ki-67 immunoreactivity on paraffin sections is an independent predictor for malignancy: a comparative study with proliferating-cell nuclear antigen and progesterone receptor protein immunostaining, mitotic index, and other clinicopathologic variables.
      • Rindi G
      • Falconi M
      • Klersy C
      • et al.
      TNM staging of neoplasms of the endocrine pancreas: results from a large international cohort study.
      then exported to many other types of intestinal NE tumors
      • Chaudhry A
      • Oberg K
      • Wilander E
      A study of biological behavior based on the expression of a proliferating antigen in neuroendocrine tumors of the digestive system.
      ,
      • Rindi G
      • Luinetti O
      • Cornaggia M
      • Capella C
      • Solcia E
      Three subtypes of gastric argyrophil carcinoid and the gastric neuroendocrine carcinoma: a clinicopathologic study.
      until it was incorporated into the grading system of digestive tract NE neoplasms in the 2010 World Health Organization (WHO) classification.
      • Rindi G
      • Klöppel G
      • Alhman H
      • All Other Frascati Consensus Conference Participants
      • European Neuroendocrine Tumor Society (ENETS)
      • et al.
      TNM staging of foregut (neuro)endocrine tumors: a consensus proposal including a grading system.
      ,
      • Bosman F
      • Carneiro F
      • Hruban R
      • Theise N
      However, Ki-67 has entered the clinical practice of other tumors, such as breast cancer, this outlining its role in the molecular classification
      • Guiu S
      • Michiels S
      • André F
      • et al.
      Molecular subclasses of breast cancer: how do we define them? The IMPAKT 2012 Working Group Statement.
      and clinical management of these oncologic patients.
      • Dowsett M
      • Nielsen TO
      • A'Hern R
      • International Ki-67 in Breast Cancer Working Group
      • et al.
      Assessment of Ki67 in breast cancer: recommendations from the International Ki67 in Breast Cancer working group.
      In this evolving scenario of increasing clinical appraisal, it is not surprising that Ki-67 has been widely studied even in NE tumors of the lung.
      • Arbiser ZK
      • Arbiser JL
      • Cohen C
      • Gal AA
      Neuroendocrine lung tumors: grade correlates with proliferation but not angiogenesis.
      • Aslan DL
      • Gulbahce HE
      • Pambuccian SE
      • Manivel JC
      • Jessurun J
      Ki-67 immunoreactivity in the differential diagnosis of pulmonary neuroendocrine neoplasms in specimens with extensive crush artifact.
      • Costes V
      • Marty-Ané C
      • Picot MC
      • et al.
      Typical and atypical bronchopulmonary carcinoid tumors: a clinicopathologic and KI-67-labeling study.
      • Grimaldi F
      • Muser D
      • Beltrami CA
      • et al.
      Partitioning of bronchopulmonary carcinoids in two different prognostic categories by ki-67 score.
      • Igarashi T
      • Jiang SX
      • Kameya T
      • et al.
      Divergent cyclin B1 expression and Rb/p16/cyclin D1 pathway aberrations among pulmonary neuroendocrine tumors.
      • Iyoda A
      • Hiroshima K
      • Moriya Y
      • et al.
      Pulmonary large cell neuroendocrine carcinoma demonstrates high proliferative activity.
      • Lin O
      • Olgac S
      • Green I
      • Zakowski MF
      • Klimstra DS
      Immunohistochemical staining of cytologic smears with MIB-1 helps distinguish low-grade from high-grade neuroendocrine neoplasms.
      • Pelosi G
      • Pasini F
      • Sonzogni A
      • et al.
      Prognostic implications of neuroendocrine differentiation and hormone production in patients with Stage I nonsmall cell lung carcinoma.
      • Pelosi G
      • Rodriguez J
      • Viale G
      • Rosai J
      Typical and atypical pulmonary carcinoid tumor overdiagnosed as small-cell carcinoma on biopsy specimens: a major pitfall in the management of lung cancer patients.
      • Rugge M
      • Fassan M
      • Clemente R
      • et al.
      Bronchopulmonary carcinoid: phenotype and long-term outcome in a single-institution series of Italian patients.
      • Skov BG
      • Holm B
      • Erreboe A
      • Skov T
      • Mellemgaard A
      ERCC1 and Ki67 in small cell lung carcinoma and other neuroendocrine tumors of the lung: distribution and impact on survival.
      • Tsuta K
      • Kalhor N
      • Raso MG
      • Wistuba II
      • Moran CA
      Oncocytic neuroendocrine tumors of the lung: histopathologic spectrum and immunohistochemical analysis of 15 cases.
      • Walts AE
      • Ines D
      • Marchevsky AM
      Limited role of Ki-67 proliferative index in predicting overall short-term survival in patients with typical and atypical pulmonary carcinoid tumors.
      • Zahel T
      • Krysa S
      • Herpel E
      • et al.
      Phenotyping of pulmonary carcinoids and a Ki-67-based grading approach.
      • Al-Khafaji B
      • Noffsinger AE
      • Miller MA
      • et al.
      Immunohistologic analysis of gastrointestinal and pulmonary carcinoid tumors.
      • Helpap B
      • Köllermann J
      Immunohistochemical analysis of the proliferative activity of neuroendocrine tumors from various organs. Are there indications for a neuroendocrine tumor-carcinoma sequence?.
      • Laitinen KL
      • Soini Y
      • Mattila J
      • Pääkkö P
      Atypical bronchopulmonary carcinoids show a tendency toward increased apoptotic and proliferative activity.
      However, clarifying its limits and defining practical applications can help clinicians and pathologists to better understand the potential lesson of Ki-67 in the management of NE lung tumor patients.

      MATERIALS AND METHODS

      A general overview of articles thus far published on the issue of Ki-67 as an operational IHC marker of cell proliferation in lung NE tumors is shown in Table 1. The term “NE tumor” will be synonymously and interchangeably used with the more correct alternative “NE neoplasm” to encompass the whole spectrum of lung NE tumors.
      • Klimstra DS
      • Modlin IR
      • Coppola D
      • Lloyd RV
      • Suster S
      The pathologic classification of neuroendocrine tumors: a review of nomenclature, grading, and staging systems.
      Only articles dealing with the 1999 or 2004 WHO classifications
      • Travis WD
      • Gal AA
      • Colby TV
      • et al.
      Reproducibility of neuroendocrine lung tumor classification.
      ,
      • Travis WD
      • Rush W
      • Flieder DB
      • et al.
      Survival analysis of 200 pulmonary neuroendocrine tumors with clarification of criteria for atypical carcinoid and its separation from typical carcinoid.
      or equivalent systems
      • Costes V
      • Marty-Ané C
      • Picot MC
      • et al.
      Typical and atypical bronchopulmonary carcinoid tumors: a clinicopathologic and KI-67-labeling study.
      ,
      • Al-Khafaji B
      • Noffsinger AE
      • Miller MA
      • et al.
      Immunohistologic analysis of gastrointestinal and pulmonary carcinoid tumors.
      have been considered because they are more homogeneous for the definition of NE tumor categories.
      • Travis W
      • Brambilla E
      • Muller-Hermelink H
      • Harris C
      ,
      • Travis W
      • Colby T
      • Corrin B
      • Shimosato Y
      • Brambilla E
      ,
      • Travis WD
      • Rush W
      • Flieder DB
      • et al.
      Survival analysis of 200 pulmonary neuroendocrine tumors with clarification of criteria for atypical carcinoid and its separation from typical carcinoid.
      ,
      • Beasley MB
      • Thunnissen FB
      • Brambilla E
      • et al.
      Pulmonary atypical carcinoid: predictors of survival in 106 cases.
      A list of key questions was developed with regard to technical issues, diagnostic and prognostic implication, tumor grading, and relevance to therapy, and these formed the basis for the literature review. Our research was limited to the English literature available in PubMed by variably crossing different research terms, such as Ki-67 or Ki67 (either antigen or protein), MIB1, MIB-1, antibody, NE, tumor, neoplasm, pulmonary, lung, carcinoid, typical, atypical, LCNEC, SCLC, prognosis, survival, or therapy. As a whole, 2067 lung NE tumors were retrieved corresponding to 25 independent studies (Table 1). Our work did not intend to perform a quantitative meta-analysis but rather to provide a critical reappraisal of the literature addressing frequently asked questions on NE lung tumor pathology and Ki-67 in daily clinical practice. Accordingly, a question-answer methodology has been pursued in the article, with relevant key points summing up major interpretation issues at the end of each answer. In the literature, the term hot spot has been used to indicate tumor areas with the highest concentration of nuclear decoration for Ki-67, whereas the term cold spot has been exploited by some studies to indicate the opposite phenomenon of tumor areas showing the minimal concentration of Ki-67 immunoreactive tumor cells
      • Tsuta K
      • Liu DC
      • Kalhor N
      • Wistuba II
      • Moran CA
      Using the mitosis-specific marker anti-phosphohistone H3 to assess mitosis in pulmonary neuroendocrine carcinomas.
      (Table 2).
      Table 1Literature Data on pKi-67 Immunostaining in Pulmonary Neuroendocrine Tumors
       StudyAuthorsSpecimen TypeNo. CasesAntibodySourceDilutionRetrieval Analysis SystemOutput ValueReferring ValueCutoff Value %Diagnostic ImplicationsClinical Implications on Outcome
       1Wart, Virchows Arch 2013Resection20MIB-1Dako1:400YesManual count and automated evaluation on ×40-digitalized slides% as labeling indexMedian
      • Range,
      • 0.1%–20%
      Differences between TC and AC; close correlation between KI-67 hotspot and overall evaluation; better interobserver agreement than mitotic count; close correlation of mitosis vs. hotspot or overall Ki-67
       2Zheng, Acta Cytol 2013Cytology5530.9VentanaPredilutedYesManual count% as labeling indexMeanNoAvoiding misdiagnosis of TC/AC as poorly differentiated NE tumors
       3Walts, Mod Pathol 2012Resection10130.9Ventanai.n.a.YesAutomated system% as labeling indexMean5Differences between TC and ACPrognostic stratification, not independent of histology
       4Zahel, Virchows Arch 2012Resection193MIB-1Dako1:200YesManual count% as labeling indexMean2.5 and 5.8Differences between TC and ACPrognostic stratification, better G1/G2 than histology
       5Grimaldi, Front Endocrinol 2011Resection106MOB-1Dakoi.n.a.i.n.a.Manual count% as labeling indexMean4Differences between TC and ACPrognostic stratification, independent of histology
       6
      • Li, Appl
      • Immunohistochem Mol Morphol 2011
      Resection and biopsy32MOB-1Dako1:100YesManual countKi-67 cumulative score (intensity 1 to 3+ by percentage of tumor cells in each grade)MeanNoDifferences across the entire spectrum, but not between TC and AC or between LCNEC and SCLC
       7Tsuta et al., Hum Pathol 2011Resection15MOB-1Dako1:100i.n.a.Manual count% as labeling indexMeanNo
       8Tsuta et al., Am J Clin Pathol 2011Resection113MOB-1Dako1:100YesManual count% as labeling indexMeanNoNo differences between TC and AC or between LCNEC and SCLC; correlation of KI-67 labeling index with mitosis and anti-phosphohistone H3 immunohistochemistry
       9Skov, J Thorac Oncol 2010Resection and biopsy276MIB-1Dako1:1000YesManual count% as labeling indexMean>8Differences between TC and ACNo prognostic stratification for TC and AC or LCNEC and SCLC
       10Das-Neves-Pereira, Eur J Cardiothorac SurgResection330MIB-1Dako1:1800i.n.aManual countStaining index (density of stained cells in areas of tumor tissue)i.n.a.NoPredictor of metastasis at univariate but not multivariate analysis
       11Rugge, Clin Cancer Res 2008Resection67MIB-1Dako1:100YesManual count% as labeling indexMean5.4Differences between TC and ACPrognostic stratification, independent of histology
       12Aslan, Am J Clin Path 2005Resection and biopsy20MM1VentanaPredilutedYesManual count% as labeling indexMedianNoAvoiding misdiagnosis of TC/AC as SCLC in crush artifacts
       13Pelosi, Am J Surg Pathol 2005Resection220MIB-1Dako1:200YesManual count% as labeling indexMedianNoDifferences between TC and AC and LCNEC and SCLC
      Pelosi, Am J Surg Pathol 2005Resection and biopsy16MOB-1Dako1:200YesManual count% as labeling indexMean20Avoiding misdiagnosis of TC/AC as SCLC in crush artifacts
       14Igarashi, Mod Path 2004Resection111MOB-1Dako1:100YesManual count% as labeling indexMeanNoDifferences between TC and AC but not LCNEC and SCLCWorse prognosis within overall but not individual tumor categories
       15Iyoda, Arm Thor Surg 2004Resection20MOB-1Dakoi.n.a.i.n.a.Manual count% as labeling indexMeanNo
       16Lin, 2003Cytology40MOB-1Immunotechi.n.a.YesManual count% as labeling indexi.n.a.25 (LG) and >50 (HG)Avoiding misdiagnosis of TC/AC as SCLC in crush artifacts
       17Pelosi, Cancer 2003Resection11MOB-1Dako1:100YesManual count% as labeling indexMeanNoWorse prognosis as tumor category compared with conventional NSCLC
       18Pelosi, Lung Cancer 2003Resection128MIB-1Immunotech1:400YesManual count% as labeling indexMean5 (LG) and 55 (HG)Association with LN metastasis-promoting fascin expression in TC/AC group
       19Van Eeden, Hum Pathol 2002Resection and biopsy10Rabbit polyclonalDako1:1200YesManual count% as labeling indexCategorical <25% (negative) and >25% (positive)Correlation between KI-67 and mitotic count in both tumor groupsNo worse prognosis within the group of TC and AC
       20Arbiser, Mod Path 2001Resection20MIB-1Immunotech1:50YesManual count% as labeling indexMeanNoNo distinction between TC and AC or between SCLC and LCNEC
       21Helpap, Virchows Arch 2001Resection31MIB-1Dianova1:50YesManual count% as labeling indexMeanNoDistinction between TC and AC; avoiding misdiagnosing TC/AC as high-grade NEC
       22Laitinen, Cancer 2000Resection31MIB-1Immunotech1:25YesManual count% as labeling indexi.n.a.NoDistinction between TC and AC; association with BCL-2 expression and apoptotic index
       23
      • Granberg, J Clin
      • Endocrinol Metab 2000
      Resection and biopsy43MIB-1Immunotech1:100YesManual count% as labeling indexi.n.a.NoWorse prognosis
       24Al-Khafaji, Hum Pathol 1998Resection11MIB-1AMAC1:50YesManual count% as labeling indexMean<10% and ≤50% No differences between metastatizing and nonmetastatizing tumors, independent of histology, but tumors with up to 50% Ki-67–labeled cells were atypical
       25Costes, Hum Path 1995Resection47MIB-1Immunotechi.n.a.YesAutomated system % as labeling indexMean4Distinction between TC and ACPrognostic stratification, independent of histology at multivariate analysis
      Table 2Distribution of pKi-67 Labeling Index According to Histology and Selection Criteria of Lung Neuroendocrine Tumor Cells
      pKi-67 Labeling Index
       StudyAuthorsHow to Get ResultsTC (n)AC (n)LCNEC (n)SCLC (n)
       1Wart, Virchows Arch 2013Hot spot fields at ×40 and whole tissue slideIndividual data not availableIndividual data not available
       2Zheng, Acta Cytol 2013i.n.a.
      Not declared hot spot areas.
      3 (11)
      Biopsy samples.
      7 (8)
      Biopsy samples.
      60 (2)
      Biopsy samples.
      87 (34)
      Biopsy samples.
       3Walts, Mod Pathol 2012Eight hot spot fields at ×20
      Declared hot spot areas.
      3.7 (78)
      Surgical specimens.
      18.8 (31)
      Surgical specimens.
       4Zahel, Virchows Arch 2012Four hot spot fields at ×40
      Declared hot spot areas.
      1.8 mean to 2.5 hot (111)
      Surgical specimens.
      3.7 mean to 5.8 hot (82)
      Surgical specimens.
       5Grimaldi, Front Endocrinol 20112000 cells
      Not declared hot spot areas.
      2.9 (75)
      Surgical specimens.
      9.5 (31)
      Surgical specimens.
       6Li, Appl Immunohistochem Mol Morphol 2011i.n.a.[0.06 (11)][0.41 (6)][1.29 (8)][1.83 (7)]
       7Tsuta et al., Hum Pathol 20111000 cells
      Not declared hot spot areas.
      0.87 (6)
      Surgical specimens.
      9.9 (1)
      Surgical specimens.
       8Tsuta et al., Am J Clin Pathol 20111000 cells in cold and hot spot fields
      Declared hot spot areas.
      0.1 cold to 2 hot (66)
      Surgical specimens.
      0.7 cold to 7.2 hot (12)
      Surgical specimens.
      36.8 cold to 55.9 hot (20)
      Surgical specimens.
      29 cold to 53.6 hot (15)
      Surgical specimens.
       9Skov, J Thorac Oncol 2010400 cells
      Not declared hot spot areas.
      1.8 (48)
      Biopsy samples.
      4 (15)
      Biopsy samples.
      25.5 (27)
      Biopsy samples.
      41.8 (186)
      Biopsy samples.
       10Das-Neves-Pereira, Eur J Cardiothorac Surg1000 cells in hot spot fieldsIndividual data not available
       11Rugge, Clin Cancer Res 20082000 cells
      Not declared hot spot areas.
      1.6 (58)
      Surgical specimens.
      8.8 (9)
      Surgical specimens.
       12Aslan, Am J Clin Path 2005i.n.a.
      Not declared hot spot areas.
      1 (7)
      Biopsy samples.
      60 (13)
       13Pelosi, Am J Surg Pathol 20052000 cells in hot spot fields at ×400 magnification2.3 (100)
      Surgical specimens.
      9 (36)
      Surgical specimens.
      47.5 (52)
      Surgical specimens.
      64.5 (32)
      Surgical specimens.
      Pelosi, Am J Surg Pathol 2005Whole tissue section for biopsy; hot spot fields at ×40 for surgical specimen counting 2000 tumor cells1 (2) on biopsy; 5.8 (1) on surgical specimen
      Biopsy samples.
      6 (5) on biopsy; 11.8 (4) on surgical specimen
      Biopsy samples.
      81.8 (9) on biopsy
      Biopsy samples.
       14Igarashi, Mod Path 20041000 cells in fields with average labeling incidence1.3 (13)
      Surgical specimens.
      8.6 (5)
      Surgical specimens.
      52.2 (44)
      Surgical specimens.
      54.6 (49)
      Surgical specimens.
       15Iyoda, Ann Thor Surg 20041000 cells in randomly selected fields
      Not declared hot spot areas.
      41.9 (20)
      Surgical specimens.
       16Lin, 2003Quintiles (<5%; 6–25%; 26–50%; 51–75%; >75%)<25 (low-grade NE tumors)>50 (high-grade NE tumors)
       17Pelosi, Cancer 20032000 cells in hot spot fields
      Declared hot spot areas.
      <5% (29) and32.8 (11)
      Surgical specimens.
       18Pelosi, Lung Cancer 20032000 cells in hot spot fields>5% (32)<55% (31) and >55% (36)
       19Van Eeden, Hum Pathol 2002Positive cells per 1 mm
      • Travis W
      • Colby T
      • Corrin B
      • Shimosato Y
      • Brambilla E
      <25% (4)<25% (4)>25% (2)
       20Arbiser, Mod Path 20011000 cells
      Not declared hot spot areas.
      0.4 (5)
      Surgical specimens.
      0.8 (5)
      Surgical specimens.
      25 (5)
      Surgical specimens.
      42 (5)
      Surgical specimens.
       21Helpap, Virchows Arch 20012000 cells in hot spot fields0.9 (i.n.a.)5.6 (i.n.a.)66.3 (i.n.a.)
       22Laitinen, Cancer 2000i.n.a.
      Not declared hot spot areas.
      <1 (21)
      Surgical specimens.
      10–20 (10)
      Surgical specimens.
       23Granberg, J Clin Endocrinol Metab 2000i.n.a.
      Not declared hot spot areas.
      0.9 (43)
      Biopsy samples.
       24Al-Khafaji, Hum Pathol 1998i.n.a.<10 (6)≤50% (5)
       25Costes, Hum Path 19952 mm
      • Travis W
      • Colby T
      • Corrin B
      • Shimosato Y
      • Brambilla E
      at ×25 with randomly selected fields
      Not declared hot spot areas.
      0.5 (31)
      Surgical specimens.
      2.4 (16)
      Surgical specimens.
      Surgical specimens only
      • 1.72
        Not declared hot spot areas (in parenthesis the corresponding number of tumors).
        /2.58
        Declared hot spot areas (in parenthesis the corresponding number of tumors).
      • (n = 209
        Not declared hot spot areas (in parenthesis the corresponding number of tumors).
        /355
        Declared hot spot areas (in parenthesis the corresponding number of tumors).
        )
      8.04
      Not declared hot spot areas (in parenthesis the corresponding number of tumors).
      /9.13
      Declared hot spot areas (in parenthesis the corresponding number of tumors).
      (n = 77
      Not declared hot spot areas (in parenthesis the corresponding number of tumors).
      /161
      Declared hot spot areas (in parenthesis the corresponding number of tumors).
      )
      • 47.57
        Not declared hot spot areas (in parenthesis the corresponding number of tumors).
        /47.24
        Declared hot spot areas (in parenthesis the corresponding number of tumors).
      • (n = 69
        Not declared hot spot areas (in parenthesis the corresponding number of tumors).
        /83
        Declared hot spot areas (in parenthesis the corresponding number of tumors).
        )
      53.43761.02
      Declared hot spot areas (in parenthesis the corresponding number of tumors).
      (n = 54747
      Declared hot spot areas (in parenthesis the corresponding number of tumors).
      )
      Surgical specimens and biopsies1.65
      Not declared hot spot areas (in parenthesis the corresponding number of tumors).
      /2.60
      Declared hot spot areas (in parenthesis the corresponding number of tumors).
      (n = 3187358
      Declared hot spot areas (in parenthesis the corresponding number of tumors).
      )
      7.41
      Not declared hot spot areas (in parenthesis the corresponding number of tumors).
      /11.04
      Declared hot spot areas (in parenthesis the corresponding number of tumors).
      (n = 100
      Not declared hot spot areas (in parenthesis the corresponding number of tumors).
      /170
      Declared hot spot areas (in parenthesis the corresponding number of tumors).
      )
      41.51
      Not declared hot spot areas (in parenthesis the corresponding number of tumors).
      /47.57
      Declared hot spot areas (in parenthesis the corresponding number of tumors).
      (n = 91
      Not declared hot spot areas (in parenthesis the corresponding number of tumors).
      /83
      Declared hot spot areas (in parenthesis the corresponding number of tumors).
      )
      • 50.16
        Not declared hot spot areas (in parenthesis the corresponding number of tumors).
        /64.36
        Declared hot spot areas (in parenthesis the corresponding number of tumors).
      • (n =287
        Not declared hot spot areas (in parenthesis the corresponding number of tumors).
        /56
        Declared hot spot areas (in parenthesis the corresponding number of tumors).
        )
      TC, typical carcinoid; AC, atypical carcinoid; LCNEC, large-cell neuroendocrine carcinoma; SCLC, small-cell lung cancer; i.n.a., information not available; NE, neuroendocrine
      a Surgical specimens.
      b Biopsy samples.
      c Declared hot spot areas.
      d Not declared hot spot areas.
      e Not declared hot spot areas (in parenthesis the corresponding number of tumors).
      f Declared hot spot areas (in parenthesis the corresponding number of tumors).
      Question 1. Are there relevant technical issues to Ki-67 IHC and evaluation of results?
      Answer: Yes, there is no uniform methodology for Ki-67 IHC and evaluation of results, but most studies pinpointed monoclonal antibody MIB-1 on paraffin sections after antigen retrieval procedures and the assessment of a Ki-67 labeling index (LI) as the most widely agreed-upon methodologies, which have been optimized within each laboratory by longstanding experience on this marker.
      Although there are no systematic investigations comparing different antibodies against Ki-67 in clinically worked up NE lung tumors, clone MIB-1 on paraffin sections has been used in all but three articles,
      • Aslan DL
      • Gulbahce HE
      • Pambuccian SE
      • Manivel JC
      • Jessurun J
      Ki-67 immunoreactivity in the differential diagnosis of pulmonary neuroendocrine neoplasms in specimens with extensive crush artifact.
      ,
      • Walts AE
      • Ines D
      • Marchevsky AM
      Limited role of Ki-67 proliferative index in predicting overall short-term survival in patients with typical and atypical pulmonary carcinoid tumors.
      ,
      • Van E
      • eden S
      • Quaedvlieg PF
      • Taal BG
      • et al.
      Classification of low-grade neuroendocrine tumors of midgut and unknown origin.
      with different antibody dilutions (ranging from 1:25 to 1:1800) and antigen retrieval procedures being adopted within each laboratory usually by utilizing heat-induced unmasking systems in saline buffer and/or following specific manufacturer’s instructions (Table 1). Although two articles used prediluted reagents,
      • Aslan DL
      • Gulbahce HE
      • Pambuccian SE
      • Manivel JC
      • Jessurun J
      Ki-67 immunoreactivity in the differential diagnosis of pulmonary neuroendocrine neoplasms in specimens with extensive crush artifact.
      ,
      • Zheng G
      • Ettinger DS
      • Maleki Z
      Utility of the quantitative Ki-67 proliferation index and CD56 together in the cytologic diagnosis of small cell lung carcinoma and other lung neuroendocrine tumors.
      five others did not provide details on antibody dilutions being applied to
      • Costes V
      • Marty-Ané C
      • Picot MC
      • et al.
      Typical and atypical bronchopulmonary carcinoid tumors: a clinicopathologic and KI-67-labeling study.
      ,
      • Grimaldi F
      • Muser D
      • Beltrami CA
      • et al.
      Partitioning of bronchopulmonary carcinoids in two different prognostic categories by ki-67 score.
      ,
      • Iyoda A
      • Hiroshima K
      • Moriya Y
      • et al.
      Pulmonary large cell neuroendocrine carcinoma demonstrates high proliferative activity.
      ,
      • Lin O
      • Olgac S
      • Green I
      • Zakowski MF
      • Klimstra DS
      Immunohistochemical staining of cytologic smears with MIB-1 helps distinguish low-grade from high-grade neuroendocrine neoplasms.
      ,
      • Walts AE
      • Ines D
      • Marchevsky AM
      Limited role of Ki-67 proliferative index in predicting overall short-term survival in patients with typical and atypical pulmonary carcinoid tumors.
      and four lacked information about the antigen retrieval procedures in use.
      • Grimaldi F
      • Muser D
      • Beltrami CA
      • et al.
      Partitioning of bronchopulmonary carcinoids in two different prognostic categories by ki-67 score.
      ,
      • Iyoda A
      • Hiroshima K
      • Moriya Y
      • et al.
      Pulmonary large cell neuroendocrine carcinoma demonstrates high proliferative activity.
      ,
      • Tsuta K
      • Kalhor N
      • Raso MG
      • Wistuba II
      • Moran CA
      Oncocytic neuroendocrine tumors of the lung: histopathologic spectrum and immunohistochemical analysis of 15 cases.
      ,
      • Das-Neves-Pereira JC
      • Bagan P
      • Milanez-de-Campos JR
      • et al.
      Individual risk prediction of nodal and distant metastasis for patients with typical bronchial carcinoid tumors.
      Quantification of Ki-67 expression has been accomplished on surgical resection specimens by manual counting in all but three studies, in which automated systems of assessment were exploited.
      • Costes V
      • Marty-Ané C
      • Picot MC
      • et al.
      Typical and atypical bronchopulmonary carcinoid tumors: a clinicopathologic and KI-67-labeling study.
      ,
      • Walts AE
      • Ines D
      • Marchevsky AM
      Limited role of Ki-67 proliferative index in predicting overall short-term survival in patients with typical and atypical pulmonary carcinoid tumors.
      ,
      • Warth A
      • Fink L
      • Fisseler-Eckhoff A
      • Pulmonary Pathology Working Group of the German Society of Pathology
      • et al.
      Interobserver agreement of proliferation index (Ki-67) outperforms mitotic count in pulmonary carcinoids.
      A high overall agreement of manual Ki-67 LI evaluation and an automated evaluation method upon scanned slides have recently substantiated the value, reproducibility, and easiness of Ki-67 LI upon manual counting.
      • Warth A
      • Fink L
      • Fisseler-Eckhoff A
      • Pulmonary Pathology Working Group of the German Society of Pathology
      • et al.
      Interobserver agreement of proliferation index (Ki-67) outperforms mitotic count in pulmonary carcinoids.
      Small biopsy and cytology samples were used in six
      • Aslan DL
      • Gulbahce HE
      • Pambuccian SE
      • Manivel JC
      • Jessurun J
      Ki-67 immunoreactivity in the differential diagnosis of pulmonary neuroendocrine neoplasms in specimens with extensive crush artifact.
      ,
      • Pelosi G
      • Rodriguez J
      • Viale G
      • Rosai J
      Typical and atypical pulmonary carcinoid tumor overdiagnosed as small-cell carcinoma on biopsy specimens: a major pitfall in the management of lung cancer patients.
      ,
      • Skov BG
      • Holm B
      • Erreboe A
      • Skov T
      • Mellemgaard A
      ERCC1 and Ki67 in small cell lung carcinoma and other neuroendocrine tumors of the lung: distribution and impact on survival.
      ,
      • Van E
      • eden S
      • Quaedvlieg PF
      • Taal BG
      • et al.
      Classification of low-grade neuroendocrine tumors of midgut and unknown origin.
      ,
      • Granberg D
      • Wilander E
      • Oberg K
      • Skogseid B
      Prognostic markers in patients with typical bronchial carcinoid tumors.
      ,
      • Li F
      • Ye B
      • Hong L
      • Xu H
      • Fishbein MC
      Epigenetic modifications of histone h4 in lung neuroendocrine tumors.
      and two studies,
      • Lin O
      • Olgac S
      • Green I
      • Zakowski MF
      • Klimstra DS
      Immunohistochemical staining of cytologic smears with MIB-1 helps distinguish low-grade from high-grade neuroendocrine neoplasms.
      ,
      • Zheng G
      • Ettinger DS
      • Maleki Z
      Utility of the quantitative Ki-67 proliferation index and CD56 together in the cytologic diagnosis of small cell lung carcinoma and other lung neuroendocrine tumors.
      respectively, to witness the applicability of these materials to accomplish Ki-67 evaluation. Although two works have imaginatively expressed Ki-67 results as either cumulative score
      • Li F
      • Ye B
      • Hong L
      • Xu H
      • Fishbein MC
      Epigenetic modifications of histone h4 in lung neuroendocrine tumors.
      or staining index
      • Das-Neves-Pereira JC
      • Bagan P
      • Milanez-de-Campos JR
      • et al.
      Individual risk prediction of nodal and distant metastasis for patients with typical bronchial carcinoid tumors.
      by including the intensity of immunoreactivity or the density of stained cells in tumor tissue areas, respectively, all the remaining studies used the percentage of nuclear-stained tumor cells to substantiate a Ki-67 LI.
      • Arbiser ZK
      • Arbiser JL
      • Cohen C
      • Gal AA
      Neuroendocrine lung tumors: grade correlates with proliferation but not angiogenesis.
      • Aslan DL
      • Gulbahce HE
      • Pambuccian SE
      • Manivel JC
      • Jessurun J
      Ki-67 immunoreactivity in the differential diagnosis of pulmonary neuroendocrine neoplasms in specimens with extensive crush artifact.
      • Costes V
      • Marty-Ané C
      • Picot MC
      • et al.
      Typical and atypical bronchopulmonary carcinoid tumors: a clinicopathologic and KI-67-labeling study.
      • Grimaldi F
      • Muser D
      • Beltrami CA
      • et al.
      Partitioning of bronchopulmonary carcinoids in two different prognostic categories by ki-67 score.
      • Igarashi T
      • Jiang SX
      • Kameya T
      • et al.
      Divergent cyclin B1 expression and Rb/p16/cyclin D1 pathway aberrations among pulmonary neuroendocrine tumors.
      • Iyoda A
      • Hiroshima K
      • Moriya Y
      • et al.
      Pulmonary large cell neuroendocrine carcinoma demonstrates high proliferative activity.
      • Lin O
      • Olgac S
      • Green I
      • Zakowski MF
      • Klimstra DS
      Immunohistochemical staining of cytologic smears with MIB-1 helps distinguish low-grade from high-grade neuroendocrine neoplasms.
      • Pelosi G
      • Pasini F
      • Sonzogni A
      • et al.
      Prognostic implications of neuroendocrine differentiation and hormone production in patients with Stage I nonsmall cell lung carcinoma.
      • Pelosi G
      • Rodriguez J
      • Viale G
      • Rosai J
      Typical and atypical pulmonary carcinoid tumor overdiagnosed as small-cell carcinoma on biopsy specimens: a major pitfall in the management of lung cancer patients.
      • Rugge M
      • Fassan M
      • Clemente R
      • et al.
      Bronchopulmonary carcinoid: phenotype and long-term outcome in a single-institution series of Italian patients.
      • Skov BG
      • Holm B
      • Erreboe A
      • Skov T
      • Mellemgaard A
      ERCC1 and Ki67 in small cell lung carcinoma and other neuroendocrine tumors of the lung: distribution and impact on survival.
      • Tsuta K
      • Kalhor N
      • Raso MG
      • Wistuba II
      • Moran CA
      Oncocytic neuroendocrine tumors of the lung: histopathologic spectrum and immunohistochemical analysis of 15 cases.
      • Walts AE
      • Ines D
      • Marchevsky AM
      Limited role of Ki-67 proliferative index in predicting overall short-term survival in patients with typical and atypical pulmonary carcinoid tumors.
      • Zahel T
      • Krysa S
      • Herpel E
      • et al.
      Phenotyping of pulmonary carcinoids and a Ki-67-based grading approach.
      • Al-Khafaji B
      • Noffsinger AE
      • Miller MA
      • et al.
      Immunohistologic analysis of gastrointestinal and pulmonary carcinoid tumors.
      • Helpap B
      • Köllermann J
      Immunohistochemical analysis of the proliferative activity of neuroendocrine tumors from various organs. Are there indications for a neuroendocrine tumor-carcinoma sequence?.
      • Laitinen KL
      • Soini Y
      • Mattila J
      • Pääkkö P
      Atypical bronchopulmonary carcinoids show a tendency toward increased apoptotic and proliferative activity.
      ,
      • Tsuta K
      • Liu DC
      • Kalhor N
      • Wistuba II
      • Moran CA
      Using the mitosis-specific marker anti-phosphohistone H3 to assess mitosis in pulmonary neuroendocrine carcinomas.
      • Van E
      • eden S
      • Quaedvlieg PF
      • Taal BG
      • et al.
      Classification of low-grade neuroendocrine tumors of midgut and unknown origin.
      • Zheng G
      • Ettinger DS
      • Maleki Z
      Utility of the quantitative Ki-67 proliferation index and CD56 together in the cytologic diagnosis of small cell lung carcinoma and other lung neuroendocrine tumors.
      ,
      • Warth A
      • Fink L
      • Fisseler-Eckhoff A
      • Pulmonary Pathology Working Group of the German Society of Pathology
      • et al.
      Interobserver agreement of proliferation index (Ki-67) outperforms mitotic count in pulmonary carcinoids.
      ,
      • Granberg D
      • Wilander E
      • Oberg K
      • Skogseid B
      Prognostic markers in patients with typical bronchial carcinoid tumors.
      ,
      • Pelosi G
      • Pasini F
      • Fraggetta F
      • et al.
      Independent value of fascin immunoreactivity for predicting lymph node metastases in typical and atypical pulmonary carcinoids.
      However, the way to select immunoreactive tumor cells differed somewhat among the diverse studies, with six of them even not providing useful contributory information for further evaluation.
      • Aslan DL
      • Gulbahce HE
      • Pambuccian SE
      • Manivel JC
      • Jessurun J
      Ki-67 immunoreactivity in the differential diagnosis of pulmonary neuroendocrine neoplasms in specimens with extensive crush artifact.
      ,
      • Al-Khafaji B
      • Noffsinger AE
      • Miller MA
      • et al.
      Immunohistologic analysis of gastrointestinal and pulmonary carcinoid tumors.
      ,
      • Laitinen KL
      • Soini Y
      • Mattila J
      • Pääkkö P
      Atypical bronchopulmonary carcinoids show a tendency toward increased apoptotic and proliferative activity.
      ,
      • Zheng G
      • Ettinger DS
      • Maleki Z
      Utility of the quantitative Ki-67 proliferation index and CD56 together in the cytologic diagnosis of small cell lung carcinoma and other lung neuroendocrine tumors.
      ,
      • Granberg D
      • Wilander E
      • Oberg K
      • Skogseid B
      Prognostic markers in patients with typical bronchial carcinoid tumors.
      ,
      • Li F
      • Ye B
      • Hong L
      • Xu H
      • Fishbein MC
      Epigenetic modifications of histone h4 in lung neuroendocrine tumors.
      Briefly, Ki-67 LI was assessed in nine studies pinpointing hot spot
      • Pelosi G
      • Pasini F
      • Sonzogni A
      • et al.
      Prognostic implications of neuroendocrine differentiation and hormone production in patients with Stage I nonsmall cell lung carcinoma.
      ,
      • Pelosi G
      • Rodriguez J
      • Viale G
      • Rosai J
      Typical and atypical pulmonary carcinoid tumor overdiagnosed as small-cell carcinoma on biopsy specimens: a major pitfall in the management of lung cancer patients.
      ,
      • Walts AE
      • Ines D
      • Marchevsky AM
      Limited role of Ki-67 proliferative index in predicting overall short-term survival in patients with typical and atypical pulmonary carcinoid tumors.
      ,
      • Zahel T
      • Krysa S
      • Herpel E
      • et al.
      Phenotyping of pulmonary carcinoids and a Ki-67-based grading approach.
      ,
      • Helpap B
      • Köllermann J
      Immunohistochemical analysis of the proliferative activity of neuroendocrine tumors from various organs. Are there indications for a neuroendocrine tumor-carcinoma sequence?.
      ,
      • Tsuta K
      • Liu DC
      • Kalhor N
      • Wistuba II
      • Moran CA
      Using the mitosis-specific marker anti-phosphohistone H3 to assess mitosis in pulmonary neuroendocrine carcinomas.
      ,
      • Das-Neves-Pereira JC
      • Bagan P
      • Milanez-de-Campos JR
      • et al.
      Individual risk prediction of nodal and distant metastasis for patients with typical bronchial carcinoid tumors.
      ,
      • Warth A
      • Fink L
      • Fisseler-Eckhoff A
      • Pulmonary Pathology Working Group of the German Society of Pathology
      • et al.
      Interobserver agreement of proliferation index (Ki-67) outperforms mitotic count in pulmonary carcinoids.
      ,
      • Pelosi G
      • Pasini F
      • Fraggetta F
      • et al.
      Independent value of fascin immunoreactivity for predicting lymph node metastases in typical and atypical pulmonary carcinoids.
      or average labeling frequency fields
      • Igarashi T
      • Jiang SX
      • Kameya T
      • et al.
      Divergent cyclin B1 expression and Rb/p16/cyclin D1 pathway aberrations among pulmonary neuroendocrine tumors.
      after scanning the entire tumor area at low magnification, whereas the quantification of positive tumor cells per 1 mm2
      • Van E
      • eden S
      • Quaedvlieg PF
      • Taal BG
      • et al.
      Classification of low-grade neuroendocrine tumors of midgut and unknown origin.
      or 2 mm2
      • Costes V
      • Marty-Ané C
      • Picot MC
      • et al.
      Typical and atypical bronchopulmonary carcinoid tumors: a clinicopathologic and KI-67-labeling study.
      or randomly selected areas
      • Iyoda A
      • Hiroshima K
      • Moriya Y
      • et al.
      Pulmonary large cell neuroendocrine carcinoma demonstrates high proliferative activity.
      was declared by others to better accomplish Ki-67 LI. In another study, both hot and cold tumor areas were screened, in the same tumor samples, at low magnification to count 1000 tumor nuclei and a separate evaluation was provided for comparison.
      • Tsuta K
      • Liu DC
      • Kalhor N
      • Wistuba II
      • Moran CA
      Using the mitosis-specific marker anti-phosphohistone H3 to assess mitosis in pulmonary neuroendocrine carcinomas.
      Evaluation of stained tumor cells on whole tissue sections of biopsy samples has also been used to maximize information obtainable from small material and avoid selection biases.
      • Pelosi G
      • Rodriguez J
      • Viale G
      • Rosai J
      Typical and atypical pulmonary carcinoid tumor overdiagnosed as small-cell carcinoma on biopsy specimens: a major pitfall in the management of lung cancer patients.
      No particular details on the selection criteria of tumor cells but only the global number of tumor cells being assessed were included in other investigations, which did not thus contribute to unveiling this issue.
      • Arbiser ZK
      • Arbiser JL
      • Cohen C
      • Gal AA
      Neuroendocrine lung tumors: grade correlates with proliferation but not angiogenesis.
      ,
      • Grimaldi F
      • Muser D
      • Beltrami CA
      • et al.
      Partitioning of bronchopulmonary carcinoids in two different prognostic categories by ki-67 score.
      ,
      • Rugge M
      • Fassan M
      • Clemente R
      • et al.
      Bronchopulmonary carcinoid: phenotype and long-term outcome in a single-institution series of Italian patients.
      • Skov BG
      • Holm B
      • Erreboe A
      • Skov T
      • Mellemgaard A
      ERCC1 and Ki67 in small cell lung carcinoma and other neuroendocrine tumors of the lung: distribution and impact on survival.
      • Tsuta K
      • Kalhor N
      • Raso MG
      • Wistuba II
      • Moran CA
      Oncocytic neuroendocrine tumors of the lung: histopathologic spectrum and immunohistochemical analysis of 15 cases.
      Interestingly, all studies dealing with LI determination were performed on biopsy or surgical specimens, except for two cytology investigations that expressed results either by quintiles
      • Lin O
      • Olgac S
      • Green I
      • Zakowski MF
      • Klimstra DS
      Immunohistochemical staining of cytologic smears with MIB-1 helps distinguish low-grade from high-grade neuroendocrine neoplasms.
      or by Ki-67 LI.
      • Zheng G
      • Ettinger DS
      • Maleki Z
      Utility of the quantitative Ki-67 proliferation index and CD56 together in the cytologic diagnosis of small cell lung carcinoma and other lung neuroendocrine tumors.
      All tumor cells showing specific nuclear staining for Ki-67 were considered positive regardless of decoration patterns (diffuse, speckled, nucleolar, mitosis featuring), which are due to the differential expression of the protein during cell cycle progression.
      • Baisch H
      • Gerdes J
      Identification of proliferating cells by Ki-67 antibody.
      ,
      • Gerdes J
      • Lemke H
      • Baisch H
      • et al.
      Cell cycle analysis of a cell proliferation-associated human nuclear antigen defined by the monoclonal antibody Ki-67.
      Expectedly, a significant correlation of Ki-67 LI with mitotic count
      • Grimaldi F
      • Muser D
      • Beltrami CA
      • et al.
      Partitioning of bronchopulmonary carcinoids in two different prognostic categories by ki-67 score.
      ,
      • Rugge M
      • Fassan M
      • Clemente R
      • et al.
      Bronchopulmonary carcinoid: phenotype and long-term outcome in a single-institution series of Italian patients.
      ,
      • Zahel T
      • Krysa S
      • Herpel E
      • et al.
      Phenotyping of pulmonary carcinoids and a Ki-67-based grading approach.
      ,
      • Tsuta K
      • Liu DC
      • Kalhor N
      • Wistuba II
      • Moran CA
      Using the mitosis-specific marker anti-phosphohistone H3 to assess mitosis in pulmonary neuroendocrine carcinomas.
      ,
      • Van E
      • eden S
      • Quaedvlieg PF
      • Taal BG
      • et al.
      Classification of low-grade neuroendocrine tumors of midgut and unknown origin.
      ,
      • Warth A
      • Fink L
      • Fisseler-Eckhoff A
      • Pulmonary Pathology Working Group of the German Society of Pathology
      • et al.
      Interobserver agreement of proliferation index (Ki-67) outperforms mitotic count in pulmonary carcinoids.
      or expression of cyclin B1
      • Igarashi T
      • Jiang SX
      • Kameya T
      • et al.
      Divergent cyclin B1 expression and Rb/p16/cyclin D1 pathway aberrations among pulmonary neuroendocrine tumors.
      or histone H3 (a surrogate marker of mitoses)
      • Tsuta K
      • Liu DC
      • Kalhor N
      • Wistuba II
      • Moran CA
      Using the mitosis-specific marker anti-phosphohistone H3 to assess mitosis in pulmonary neuroendocrine carcinomas.
      has been described in NE tumors of both the lung and the pancreas
      • Pelosi G
      • Bresaola E
      • Bogina G
      • et al.
      Endocrine tumors of the pancreas: Ki-67 immunoreactivity on paraffin sections is an independent predictor for malignancy: a comparative study with proliferating-cell nuclear antigen and progesterone receptor protein immunostaining, mitotic index, and other clinicopathologic variables.
      in virtue of the strong colinearity of the two indicators of cycling cells with variable correlation coefficients likely due to biological and technical reasons.
      • Tsuta K
      • Liu DC
      • Kalhor N
      • Wistuba II
      • Moran CA
      Using the mitosis-specific marker anti-phosphohistone H3 to assess mitosis in pulmonary neuroendocrine carcinomas.
      ,
      • Warth A
      • Fink L
      • Fisseler-Eckhoff A
      • Pulmonary Pathology Working Group of the German Society of Pathology
      • et al.
      Interobserver agreement of proliferation index (Ki-67) outperforms mitotic count in pulmonary carcinoids.
      More critical is the question of how Ki-67 LI should be calculated, because different methods have been provided to establish the optimal denominator, that is, the number of cells to be counted. Four to eight histological fields at ×20
      • Walts AE
      • Ines D
      • Marchevsky AM
      Limited role of Ki-67 proliferative index in predicting overall short-term survival in patients with typical and atypical pulmonary carcinoid tumors.
      or ×40
      • Zahel T
      • Krysa S
      • Herpel E
      • et al.
      Phenotyping of pulmonary carcinoids and a Ki-67-based grading approach.
      ,
      • Warth A
      • Fink L
      • Fisseler-Eckhoff A
      • Pulmonary Pathology Working Group of the German Society of Pathology
      • et al.
      Interobserver agreement of proliferation index (Ki-67) outperforms mitotic count in pulmonary carcinoids.
      magnification, histological fields with average labeling incidence,
      • Igarashi T
      • Jiang SX
      • Kameya T
      • et al.
      Divergent cyclin B1 expression and Rb/p16/cyclin D1 pathway aberrations among pulmonary neuroendocrine tumors.
      2-mm2 tumor areas taken at ×25 magnification,
      • Costes V
      • Marty-Ané C
      • Picot MC
      • et al.
      Typical and atypical bronchopulmonary carcinoid tumors: a clinicopathologic and KI-67-labeling study.
      1-mm2 tumor areas not otherwise specified,
      • Van E
      • eden S
      • Quaedvlieg PF
      • Taal BG
      • et al.
      Classification of low-grade neuroendocrine tumors of midgut and unknown origin.
      or 400 to 2000 tumor cells being consecutively counted
      • Arbiser ZK
      • Arbiser JL
      • Cohen C
      • Gal AA
      Neuroendocrine lung tumors: grade correlates with proliferation but not angiogenesis.
      ,
      • Grimaldi F
      • Muser D
      • Beltrami CA
      • et al.
      Partitioning of bronchopulmonary carcinoids in two different prognostic categories by ki-67 score.
      • Igarashi T
      • Jiang SX
      • Kameya T
      • et al.
      Divergent cyclin B1 expression and Rb/p16/cyclin D1 pathway aberrations among pulmonary neuroendocrine tumors.
      • Iyoda A
      • Hiroshima K
      • Moriya Y
      • et al.
      Pulmonary large cell neuroendocrine carcinoma demonstrates high proliferative activity.
      ,
      • Pelosi G
      • Pasini F
      • Sonzogni A
      • et al.
      Prognostic implications of neuroendocrine differentiation and hormone production in patients with Stage I nonsmall cell lung carcinoma.
      • Pelosi G
      • Rodriguez J
      • Viale G
      • Rosai J
      Typical and atypical pulmonary carcinoid tumor overdiagnosed as small-cell carcinoma on biopsy specimens: a major pitfall in the management of lung cancer patients.
      • Rugge M
      • Fassan M
      • Clemente R
      • et al.
      Bronchopulmonary carcinoid: phenotype and long-term outcome in a single-institution series of Italian patients.
      • Skov BG
      • Holm B
      • Erreboe A
      • Skov T
      • Mellemgaard A
      ERCC1 and Ki67 in small cell lung carcinoma and other neuroendocrine tumors of the lung: distribution and impact on survival.
      • Tsuta K
      • Kalhor N
      • Raso MG
      • Wistuba II
      • Moran CA
      Oncocytic neuroendocrine tumors of the lung: histopathologic spectrum and immunohistochemical analysis of 15 cases.
      ,
      • Helpap B
      • Köllermann J
      Immunohistochemical analysis of the proliferative activity of neuroendocrine tumors from various organs. Are there indications for a neuroendocrine tumor-carcinoma sequence?.
      ,
      • Tsuta K
      • Liu DC
      • Kalhor N
      • Wistuba II
      • Moran CA
      Using the mitosis-specific marker anti-phosphohistone H3 to assess mitosis in pulmonary neuroendocrine carcinomas.
      ,
      • Das-Neves-Pereira JC
      • Bagan P
      • Milanez-de-Campos JR
      • et al.
      Individual risk prediction of nodal and distant metastasis for patients with typical bronchial carcinoid tumors.
      ,
      • Pelosi G
      • Pasini F
      • Fraggetta F
      • et al.
      Independent value of fascin immunoreactivity for predicting lymph node metastases in typical and atypical pulmonary carcinoids.
      have been used for assessing Ki-67 LI, which may account for some discrepant results and preclude a direct cross-study comparison.
      • Walts AE
      • Ines D
      • Marchevsky AM
      Limited role of Ki-67 proliferative index in predicting overall short-term survival in patients with typical and atypical pulmonary carcinoid tumors.
      Another source of variability may derive from evaluating results of Ki-67 LI as mean
      • Arbiser ZK
      • Arbiser JL
      • Cohen C
      • Gal AA
      Neuroendocrine lung tumors: grade correlates with proliferation but not angiogenesis.
      ,
      • Costes V
      • Marty-Ané C
      • Picot MC
      • et al.
      Typical and atypical bronchopulmonary carcinoid tumors: a clinicopathologic and KI-67-labeling study.
      • Grimaldi F
      • Muser D
      • Beltrami CA
      • et al.
      Partitioning of bronchopulmonary carcinoids in two different prognostic categories by ki-67 score.
      • Igarashi T
      • Jiang SX
      • Kameya T
      • et al.
      Divergent cyclin B1 expression and Rb/p16/cyclin D1 pathway aberrations among pulmonary neuroendocrine tumors.
      • Iyoda A
      • Hiroshima K
      • Moriya Y
      • et al.
      Pulmonary large cell neuroendocrine carcinoma demonstrates high proliferative activity.
      ,
      • Pelosi G
      • Pasini F
      • Sonzogni A
      • et al.
      Prognostic implications of neuroendocrine differentiation and hormone production in patients with Stage I nonsmall cell lung carcinoma.
      • Pelosi G
      • Rodriguez J
      • Viale G
      • Rosai J
      Typical and atypical pulmonary carcinoid tumor overdiagnosed as small-cell carcinoma on biopsy specimens: a major pitfall in the management of lung cancer patients.
      • Rugge M
      • Fassan M
      • Clemente R
      • et al.
      Bronchopulmonary carcinoid: phenotype and long-term outcome in a single-institution series of Italian patients.
      • Skov BG
      • Holm B
      • Erreboe A
      • Skov T
      • Mellemgaard A
      ERCC1 and Ki67 in small cell lung carcinoma and other neuroendocrine tumors of the lung: distribution and impact on survival.
      • Tsuta K
      • Kalhor N
      • Raso MG
      • Wistuba II
      • Moran CA
      Oncocytic neuroendocrine tumors of the lung: histopathologic spectrum and immunohistochemical analysis of 15 cases.
      • Walts AE
      • Ines D
      • Marchevsky AM
      Limited role of Ki-67 proliferative index in predicting overall short-term survival in patients with typical and atypical pulmonary carcinoid tumors.
      • Zahel T
      • Krysa S
      • Herpel E
      • et al.
      Phenotyping of pulmonary carcinoids and a Ki-67-based grading approach.
      • Al-Khafaji B
      • Noffsinger AE
      • Miller MA
      • et al.
      Immunohistologic analysis of gastrointestinal and pulmonary carcinoid tumors.
      • Helpap B
      • Köllermann J
      Immunohistochemical analysis of the proliferative activity of neuroendocrine tumors from various organs. Are there indications for a neuroendocrine tumor-carcinoma sequence?.
      ,
      • Tsuta K
      • Liu DC
      • Kalhor N
      • Wistuba II
      • Moran CA
      Using the mitosis-specific marker anti-phosphohistone H3 to assess mitosis in pulmonary neuroendocrine carcinomas.
      ,
      • Zheng G
      • Ettinger DS
      • Maleki Z
      Utility of the quantitative Ki-67 proliferation index and CD56 together in the cytologic diagnosis of small cell lung carcinoma and other lung neuroendocrine tumors.
      ,
      • Li F
      • Ye B
      • Hong L
      • Xu H
      • Fishbein MC
      Epigenetic modifications of histone h4 in lung neuroendocrine tumors.
      ,
      • Pelosi G
      • Pasini F
      • Fraggetta F
      • et al.
      Independent value of fascin immunoreactivity for predicting lymph node metastases in typical and atypical pulmonary carcinoids.
      or median
      • Aslan DL
      • Gulbahce HE
      • Pambuccian SE
      • Manivel JC
      • Jessurun J
      Ki-67 immunoreactivity in the differential diagnosis of pulmonary neuroendocrine neoplasms in specimens with extensive crush artifact.
      ,
      • Pelosi G
      • Rodriguez J
      • Viale G
      • Rosai J
      Typical and atypical pulmonary carcinoid tumor overdiagnosed as small-cell carcinoma on biopsy specimens: a major pitfall in the management of lung cancer patients.
      ,
      • Warth A
      • Fink L
      • Fisseler-Eckhoff A
      • Pulmonary Pathology Working Group of the German Society of Pathology
      • et al.
      Interobserver agreement of proliferation index (Ki-67) outperforms mitotic count in pulmonary carcinoids.
      thresholds, whereas other studies provided either poorly manageable categorical variables
      • Van E
      • eden S
      • Quaedvlieg PF
      • Taal BG
      • et al.
      Classification of low-grade neuroendocrine tumors of midgut and unknown origin.
      or no useful information.
      • Lin O
      • Olgac S
      • Green I
      • Zakowski MF
      • Klimstra DS
      Immunohistochemical staining of cytologic smears with MIB-1 helps distinguish low-grade from high-grade neuroendocrine neoplasms.
      ,
      • Laitinen KL
      • Soini Y
      • Mattila J
      • Pääkkö P
      Atypical bronchopulmonary carcinoids show a tendency toward increased apoptotic and proliferative activity.
      ,
      • Das-Neves-Pereira JC
      • Bagan P
      • Milanez-de-Campos JR
      • et al.
      Individual risk prediction of nodal and distant metastasis for patients with typical bronchial carcinoid tumors.
      ,
      • Greenberg RS
      • Baumgarten DA
      • Clark WS
      • Isacson P
      • McKeen K
      Prognostic factors for gastrointestinal and bronchopulmonary carcinoid tumors.
      Reproducibility studies on Ki-67 LI evaluation by repeating the measurements in randomly selected carcinoid subsets
      • Walts AE
      • Ines D
      • Marchevsky AM
      Limited role of Ki-67 proliferative index in predicting overall short-term survival in patients with typical and atypical pulmonary carcinoid tumors.
      or comparing manual mitotic count with Ki-67 LI by different pulmonary pathologists in the same tumor samples
      • Warth A
      • Fink L
      • Fisseler-Eckhoff A
      • Pulmonary Pathology Working Group of the German Society of Pathology
      • et al.
      Interobserver agreement of proliferation index (Ki-67) outperforms mitotic count in pulmonary carcinoids.
      revealed encouraging results, with less than 1.5% of variability
      • Walts AE
      • Ines D
      • Marchevsky AM
      Limited role of Ki-67 proliferative index in predicting overall short-term survival in patients with typical and atypical pulmonary carcinoid tumors.
      and an outperformance of Ki-67 LI over mitotic count with regard to interobserver agreement.
      • Warth A
      • Fink L
      • Fisseler-Eckhoff A
      • Pulmonary Pathology Working Group of the German Society of Pathology
      • et al.
      Interobserver agreement of proliferation index (Ki-67) outperforms mitotic count in pulmonary carcinoids.
      Relevant key points: At variance with the gastroenteropancreatic system,
      • Tang LH
      • Gonen M
      • Hedvat C
      • Modlin IM
      • Klimstra DS
      Objective quantification of the Ki67 proliferative index in neuroendocrine tumors of the gastroenteropancreatic system: a comparison of digital image analysis with manual methods.
      there are no comparative studies evaluating different methods to perform and express Ki-67 results in lung NE tumors. However, most published investigations agreed on the opportunity of measuring Ki-67 LI in hot spot areas, taking into account all nuclear signals after visual scrutiny of the entire tumor. This would apply especially to TC or AC, whereas Ki-67 decoration is usually much more uniform in high-grade NE tumors. For practical purposes, Ki-67 LI should be calculated in surgical specimens by counting at least 2000 consecutive tumors cells in hot spot fields at ×40 magnification or 2 mm2 for consistency with the histological classification, possibly in the same tumor area as that used for assessing mitotic count. In biopsy or cytology samples, in which the number of tumor cells may be lower than 2000 and the 2-mm2 criterion unsuitable, it could be reasonable to calculate Ki-67 LI on all tumor cells. For experienced pathologists, manual counting of Ki-67 LI upon visual inspection or eyeball estimation differs little from more sophisticated, time-consuming, or cumbersome methods.
      • Tang LH
      • Gonen M
      • Hedvat C
      • Modlin IM
      • Klimstra DS
      Objective quantification of the Ki67 proliferative index in neuroendocrine tumors of the gastroenteropancreatic system: a comparison of digital image analysis with manual methods.
      Additional work and reproducibility studies are needed to address the optimal procedure for evaluating Ki-67 in lung NE tumors.
      Question 2. Is there a diagnostic role for Ki-67 LI in lung NE tumors?
      Answer: No, the classification on NE lung tumors is currently guided by morphology alone, but a practical utility for this marker has been emerging for separating TC/AC from high-grade NE tumors in limited diagnostic material.
      As outlined in Table 2, the weighted average of Ki-67 LI values across different studies in which this evaluation could be done differed between TC and AC but not between LCNEC and SCLC when considering both surgical specimens only or biopsy/cytology and surgical specimens as a whole, with minor differences if hot spot values of Ki-67 LI were taken into account. The distribution of Ki-67 LI values across the different categories of lung NE tumors according to the type of specimens (only excised specimens or excised and biopsy samples) and the way to select tumor cells (declared hot spot areas versus not declared hot spot areas) is shown in Table 2. In three large studies
      • Righi L
      • Volante M
      • Rapa I
      • et al.
      Mammalian target of rapamycin signaling activation patterns in neuroendocrine tumors of the lung.
      ,
      • Pelosi G
      • Rodriguez J
      • Viale G
      • Rosai J
      Typical and atypical pulmonary carcinoid tumor overdiagnosed as small-cell carcinoma on biopsy specimens: a major pitfall in the management of lung cancer patients.
      ,
      • Wang H
      • Iyoda A
      • Roh MS
      • et al.
      WHO histologic classification is an independent predictor of prognosis in lung neuroendocrine (NE) tumors but Ki-67 proliferation rate is not (abstract #1952).
      accounting for 628 surgically excised NE tumors of the lung (one of which in abstract form only),
      • Wang H
      • Iyoda A
      • Roh MS
      • et al.
      WHO histologic classification is an independent predictor of prognosis in lung neuroendocrine (NE) tumors but Ki-67 proliferation rate is not (abstract #1952).
      the value of Ki-67 LI ranged from 2.3% to 4.15% in 211 TC, 9% to 17.8% in 131 AC, 47.5% to 70.0% in 153 LCNEC, and 64.5% to 77.5% in 133 SCLC, in substantial agreement with the expected proliferation rates of these tumors.
      Significant differences in the Ki-67 LI distribution have been described in several studies between TC and AC,
      • Costes V
      • Marty-Ané C
      • Picot MC
      • et al.
      Typical and atypical bronchopulmonary carcinoid tumors: a clinicopathologic and KI-67-labeling study.
      • Grimaldi F
      • Muser D
      • Beltrami CA
      • et al.
      Partitioning of bronchopulmonary carcinoids in two different prognostic categories by ki-67 score.
      • Igarashi T
      • Jiang SX
      • Kameya T
      • et al.
      Divergent cyclin B1 expression and Rb/p16/cyclin D1 pathway aberrations among pulmonary neuroendocrine tumors.
      ,
      • Pelosi G
      • Rodriguez J
      • Viale G
      • Rosai J
      Typical and atypical pulmonary carcinoid tumor overdiagnosed as small-cell carcinoma on biopsy specimens: a major pitfall in the management of lung cancer patients.
      • Rugge M
      • Fassan M
      • Clemente R
      • et al.
      Bronchopulmonary carcinoid: phenotype and long-term outcome in a single-institution series of Italian patients.
      • Skov BG
      • Holm B
      • Erreboe A
      • Skov T
      • Mellemgaard A
      ERCC1 and Ki67 in small cell lung carcinoma and other neuroendocrine tumors of the lung: distribution and impact on survival.
      ,
      • Walts AE
      • Ines D
      • Marchevsky AM
      Limited role of Ki-67 proliferative index in predicting overall short-term survival in patients with typical and atypical pulmonary carcinoid tumors.
      ,
      • Zahel T
      • Krysa S
      • Herpel E
      • et al.
      Phenotyping of pulmonary carcinoids and a Ki-67-based grading approach.
      ,
      • Helpap B
      • Köllermann J
      Immunohistochemical analysis of the proliferative activity of neuroendocrine tumors from various organs. Are there indications for a neuroendocrine tumor-carcinoma sequence?.
      ,
      • Laitinen KL
      • Soini Y
      • Mattila J
      • Pääkkö P
      Atypical bronchopulmonary carcinoids show a tendency toward increased apoptotic and proliferative activity.
      ,
      • Warth A
      • Fink L
      • Fisseler-Eckhoff A
      • Pulmonary Pathology Working Group of the German Society of Pathology
      • et al.
      Interobserver agreement of proliferation index (Ki-67) outperforms mitotic count in pulmonary carcinoids.
      between LCNEC and SCLC,
      • Pelosi G
      • Rodriguez J
      • Viale G
      • Rosai J
      Typical and atypical pulmonary carcinoid tumor overdiagnosed as small-cell carcinoma on biopsy specimens: a major pitfall in the management of lung cancer patients.
      or across the entire spectrum of lung NE tumors,
      • Li F
      • Ye B
      • Hong L
      • Xu H
      • Fishbein MC
      Epigenetic modifications of histone h4 in lung neuroendocrine tumors.
      whereas other authors did not support this correlation at all
      • Arbiser ZK
      • Arbiser JL
      • Cohen C
      • Gal AA
      Neuroendocrine lung tumors: grade correlates with proliferation but not angiogenesis.
      ,
      • Al-Khafaji B
      • Noffsinger AE
      • Miller MA
      • et al.
      Immunohistologic analysis of gastrointestinal and pulmonary carcinoid tumors.
      ,
      • Tsuta K
      • Liu DC
      • Kalhor N
      • Wistuba II
      • Moran CA
      Using the mitosis-specific marker anti-phosphohistone H3 to assess mitosis in pulmonary neuroendocrine carcinomas.
      or limited the failure to poorly differentiated NE tumors only.
      • Igarashi T
      • Jiang SX
      • Kameya T
      • et al.
      Divergent cyclin B1 expression and Rb/p16/cyclin D1 pathway aberrations among pulmonary neuroendocrine tumors.
      Proposed cutoff thresholds of Ki-67 LI ranged from 2.5 to approximately 30% for carcinoids,
      • Costes V
      • Marty-Ané C
      • Picot MC
      • et al.
      Typical and atypical bronchopulmonary carcinoid tumors: a clinicopathologic and KI-67-labeling study.
      ,
      • Grimaldi F
      • Muser D
      • Beltrami CA
      • et al.
      Partitioning of bronchopulmonary carcinoids in two different prognostic categories by ki-67 score.
      ,
      • Lin O
      • Olgac S
      • Green I
      • Zakowski MF
      • Klimstra DS
      Immunohistochemical staining of cytologic smears with MIB-1 helps distinguish low-grade from high-grade neuroendocrine neoplasms.
      ,
      • Pelosi G
      • Rodriguez J
      • Viale G
      • Rosai J
      Typical and atypical pulmonary carcinoid tumor overdiagnosed as small-cell carcinoma on biopsy specimens: a major pitfall in the management of lung cancer patients.
      • Rugge M
      • Fassan M
      • Clemente R
      • et al.
      Bronchopulmonary carcinoid: phenotype and long-term outcome in a single-institution series of Italian patients.
      • Skov BG
      • Holm B
      • Erreboe A
      • Skov T
      • Mellemgaard A
      ERCC1 and Ki67 in small cell lung carcinoma and other neuroendocrine tumors of the lung: distribution and impact on survival.
      ,
      • Walts AE
      • Ines D
      • Marchevsky AM
      Limited role of Ki-67 proliferative index in predicting overall short-term survival in patients with typical and atypical pulmonary carcinoid tumors.
      ,
      • Zahel T
      • Krysa S
      • Herpel E
      • et al.
      Phenotyping of pulmonary carcinoids and a Ki-67-based grading approach.
      ,
      • Tsuta K
      • Liu DC
      • Kalhor N
      • Wistuba II
      • Moran CA
      Using the mitosis-specific marker anti-phosphohistone H3 to assess mitosis in pulmonary neuroendocrine carcinomas.
      ,
      • Van E
      • eden S
      • Quaedvlieg PF
      • Taal BG
      • et al.
      Classification of low-grade neuroendocrine tumors of midgut and unknown origin.
      ,
      • Warth A
      • Fink L
      • Fisseler-Eckhoff A
      • Pulmonary Pathology Working Group of the German Society of Pathology
      • et al.
      Interobserver agreement of proliferation index (Ki-67) outperforms mitotic count in pulmonary carcinoids.
      ,
      • Pelosi G
      • Pasini F
      • Fraggetta F
      • et al.
      Independent value of fascin immunoreactivity for predicting lymph node metastases in typical and atypical pulmonary carcinoids.
      with two studies detecting 50% or more in few AC,
      • Walts AE
      • Ines D
      • Marchevsky AM
      Limited role of Ki-67 proliferative index in predicting overall short-term survival in patients with typical and atypical pulmonary carcinoid tumors.
      ,
      • Al-Khafaji B
      • Noffsinger AE
      • Miller MA
      • et al.
      Immunohistologic analysis of gastrointestinal and pulmonary carcinoid tumors.
      whereas the separation of SCLC and LCNEC, if any,
      • Pelosi G
      • Rodriguez J
      • Viale G
      • Rosai J
      Typical and atypical pulmonary carcinoid tumor overdiagnosed as small-cell carcinoma on biopsy specimens: a major pitfall in the management of lung cancer patients.
      is of more limited clinical impact.
      • Travis W
      • Brambilla E
      • Muller-Hermelink H
      • Harris C
      ,
      • Righi L
      • Volante M
      • Tavaglione V
      • et al.
      Somatostatin receptor tissue distribution in lung neuroendocrine tumours: a clinicopathologic and immunohistochemical study of 218 ‘clinically aggressive' cases.
      In another study on 190 lung NE tumors published in an abstract form only, although there were differences in Ki-67 LI among diverse tumor categories, the incorporation of this marker as a primary criterion in the classification scheme of lung NE tumors was not further supported.
      • Wang H
      • Iyoda A
      • Roh MS
      • et al.
      WHO histologic classification is an independent predictor of prognosis in lung neuroendocrine (NE) tumors but Ki-67 proliferation rate is not (abstract #1952).
      A possible explanation why Ki-67 LI could not effectively split biologically adjacent tumor variants could be the imperfect correlation with mitotic count. This causes the frequency distributions of Ki-67 LI to consistently overlap between these adjacent tumor variants,
      • Walts AE
      • Ines D
      • Marchevsky AM
      Limited role of Ki-67 proliferative index in predicting overall short-term survival in patients with typical and atypical pulmonary carcinoid tumors.
      ,
      • Zahel T
      • Krysa S
      • Herpel E
      • et al.
      Phenotyping of pulmonary carcinoids and a Ki-67-based grading approach.
      ,
      • Tsuta K
      • Liu DC
      • Kalhor N
      • Wistuba II
      • Moran CA
      Using the mitosis-specific marker anti-phosphohistone H3 to assess mitosis in pulmonary neuroendocrine carcinomas.
      also taking into account the high interobserver variability existing, for example, in high-grade NE tumor subclassification.
      • den Bakker MA
      • Willemsen S
      • Grünberg K
      • et al.
      Small cell carcinoma of the lung and large cell neuroendocrine carcinoma interobserver variability.
      One of the most agreed-upon uses of Ki-67 LI with important clinical implications deals with the distinction of low to intermediate grade from poorly differentiated NE tumors (especially SCLC) in small biopsy or cytology samples,
      • Aslan DL
      • Gulbahce HE
      • Pambuccian SE
      • Manivel JC
      • Jessurun J
      Ki-67 immunoreactivity in the differential diagnosis of pulmonary neuroendocrine neoplasms in specimens with extensive crush artifact.
      ,
      • Lin O
      • Olgac S
      • Green I
      • Zakowski MF
      • Klimstra DS
      Immunohistochemical staining of cytologic smears with MIB-1 helps distinguish low-grade from high-grade neuroendocrine neoplasms.
      ,
      • Pelosi G
      • Rodriguez J
      • Viale G
      • Rosai J
      Typical and atypical pulmonary carcinoid tumor overdiagnosed as small-cell carcinoma on biopsy specimens: a major pitfall in the management of lung cancer patients.
      ,
      • Helpap B
      • Köllermann J
      Immunohistochemical analysis of the proliferative activity of neuroendocrine tumors from various organs. Are there indications for a neuroendocrine tumor-carcinoma sequence?.
      ,
      • Zheng G
      • Ettinger DS
      • Maleki Z
      Utility of the quantitative Ki-67 proliferation index and CD56 together in the cytologic diagnosis of small cell lung carcinoma and other lung neuroendocrine tumors.
      especially in the presence of crush artifacts or poor tissue preservation,
      • Dowsett M
      • Nielsen TO
      • A'Hern R
      • International Ki-67 in Breast Cancer Working Group
      • et al.
      Assessment of Ki67 in breast cancer: recommendations from the International Ki67 in Breast Cancer working group.
      ,
      • Igarashi T
      • Jiang SX
      • Kameya T
      • et al.
      Divergent cyclin B1 expression and Rb/p16/cyclin D1 pathway aberrations among pulmonary neuroendocrine tumors.
      ,
      • Lin O
      • Olgac S
      • Green I
      • Zakowski MF
      • Klimstra DS
      Immunohistochemical staining of cytologic smears with MIB-1 helps distinguish low-grade from high-grade neuroendocrine neoplasms.
      in which nuclear markers are more suitable for the diagnostic interpretation than cytoplasmic markers. In fact, nuclear markers are easier to scrutiny because there is no passive diffusion of cytoplasmic proteins into adjacent cells, but chromatin-related molecules, such as Ki-67, are likely to remain tightly associated to nuclear remnants even when filamentous changes occur due to tumor cell fragmentation. Thresholds up to 25% to 30% of Ki-67 LI have been quoted as a useful diagnostic adjunct to exclude poorly differentiated NE tumors, which are associated with an exceedingly high proliferation index,
      • Aslan DL
      • Gulbahce HE
      • Pambuccian SE
      • Manivel JC
      • Jessurun J
      Ki-67 immunoreactivity in the differential diagnosis of pulmonary neuroendocrine neoplasms in specimens with extensive crush artifact.
      ,
      • Lin O
      • Olgac S
      • Green I
      • Zakowski MF
      • Klimstra DS
      Immunohistochemical staining of cytologic smears with MIB-1 helps distinguish low-grade from high-grade neuroendocrine neoplasms.
      ,
      • Pelosi G
      • Rodriguez J
      • Viale G
      • Rosai J
      Typical and atypical pulmonary carcinoid tumor overdiagnosed as small-cell carcinoma on biopsy specimens: a major pitfall in the management of lung cancer patients.
      ,
      • Helpap B
      • Köllermann J
      Immunohistochemical analysis of the proliferative activity of neuroendocrine tumors from various organs. Are there indications for a neuroendocrine tumor-carcinoma sequence?.
      whereas thresholds of less than 3% would support a diagnosis of low-grade NE tumor and thresholds between 3% and 30% would indicate indeterminate tumors that most often consisted of AC with very few poorly differentiated tumors.
      • Tsuta K
      • Liu DC
      • Kalhor N
      • Wistuba II
      • Moran CA
      Using the mitosis-specific marker anti-phosphohistone H3 to assess mitosis in pulmonary neuroendocrine carcinomas.
      A comparative assessment of Ki-67 LI in biopsy
      • Pelosi G
      • Rodriguez J
      • Viale G
      • Rosai J
      Typical and atypical pulmonary carcinoid tumor overdiagnosed as small-cell carcinoma on biopsy specimens: a major pitfall in the management of lung cancer patients.
      or cytology samples
      • Zheng G
      • Ettinger DS
      • Maleki Z
      Utility of the quantitative Ki-67 proliferation index and CD56 together in the cytologic diagnosis of small cell lung carcinoma and other lung neuroendocrine tumors.
      and paired surgical specimens was available from two studies, with similar but not perfectly overlapping results in the setting of low to intermediate malignant lung NE tumors in keeping with those obtained in pancreatic NE tumors,
      • Larghi A
      • Capurso G
      • Carnuccio A
      • et al.
      Ki-67 grading of nonfunctioning pancreatic neuroendocrine tumors on histologic samples obtained by EUS-guided fine-needle tissue acquisition: a prospective study.
      likely owing to either sampling or methodological issues.
      • Pelosi G
      • Rodriguez J
      • Viale G
      • Rosai J
      Typical and atypical pulmonary carcinoid tumor overdiagnosed as small-cell carcinoma on biopsy specimens: a major pitfall in the management of lung cancer patients.
      ,
      • Zheng G
      • Ettinger DS
      • Maleki Z
      Utility of the quantitative Ki-67 proliferation index and CD56 together in the cytologic diagnosis of small cell lung carcinoma and other lung neuroendocrine tumors.
      Although it is mandatory to avoid major pitfall in the management of lung cancer patients and Ki-67 LI assessment is effective to assist this task,
      • Pelosi G
      • Rodriguez J
      • Viale G
      • Rosai J
      Typical and atypical pulmonary carcinoid tumor overdiagnosed as small-cell carcinoma on biopsy specimens: a major pitfall in the management of lung cancer patients.
      worth noting, however, is that Ki-67 LI on small biopsy sample may represent the only available data of cell proliferation for clinical decisions in inoperable patients.
      • Volante M
      • Righi L
      • Berruti A
      • Rindi G
      • Papotti M
      The pathological diagnosis of neuroendocrine tumors: common questions and tentative answers.
      Relevant key points: Ki-67 LI is not part of the current WHO diagnostic criteria for classifying lung NE tumors and should not be used to differentiate TC and AC owing to considerable overlapping in the distribution of Ki-67 indices between biologically adjacent NE tumor categories. The assessment of Ki-67 LI, however, is useful as a diagnostic adjunct in small biopsy or cytology specimens with poor preservation or crush artifact, to avoid misdiagnosing low- to intermediate-grade NE tumors as poorly differentiated NE carcinoma.
      • Aslan DL
      • Gulbahce HE
      • Pambuccian SE
      • Manivel JC
      • Jessurun J
      Ki-67 immunoreactivity in the differential diagnosis of pulmonary neuroendocrine neoplasms in specimens with extensive crush artifact.
      ,
      • Lin O
      • Olgac S
      • Green I
      • Zakowski MF
      • Klimstra DS
      Immunohistochemical staining of cytologic smears with MIB-1 helps distinguish low-grade from high-grade neuroendocrine neoplasms.
      ,
      • Pelosi G
      • Rodriguez J
      • Viale G
      • Rosai J
      Typical and atypical pulmonary carcinoid tumor overdiagnosed as small-cell carcinoma on biopsy specimens: a major pitfall in the management of lung cancer patients.
      ,
      • Helpap B
      • Köllermann J
      Immunohistochemical analysis of the proliferative activity of neuroendocrine tumors from various organs. Are there indications for a neuroendocrine tumor-carcinoma sequence?.
      ,
      • Zheng G
      • Ettinger DS
      • Maleki Z
      Utility of the quantitative Ki-67 proliferation index and CD56 together in the cytologic diagnosis of small cell lung carcinoma and other lung neuroendocrine tumors.
      Ki-67 LI does not serve to make specific diagnoses of lung NE subtypes, rather it very sensitively parallels the inherent proliferative properties of the tumors under evaluation.
      Question 3: Is there a prognostic role for Ki-67 LI?
      Answer: Possibly, Ki-67 LI has been emerging as a promising prognostic factor in excised specimens especially of low- to intermediate-grade lung NE tumors, although more data are needed to establish its ideal role.
      As indicated in Table 1, at least 12 articles have investigated the prognostic inference of Ki-67 LI in diverse categories of lung NE tumors, especially TC and AC,
      • Costes V
      • Marty-Ané C
      • Picot MC
      • et al.
      Typical and atypical bronchopulmonary carcinoid tumors: a clinicopathologic and KI-67-labeling study.
      • Grimaldi F
      • Muser D
      • Beltrami CA
      • et al.
      Partitioning of bronchopulmonary carcinoids in two different prognostic categories by ki-67 score.
      • Igarashi T
      • Jiang SX
      • Kameya T
      • et al.
      Divergent cyclin B1 expression and Rb/p16/cyclin D1 pathway aberrations among pulmonary neuroendocrine tumors.
      ,
      • Pelosi G
      • Pasini F
      • Sonzogni A
      • et al.
      Prognostic implications of neuroendocrine differentiation and hormone production in patients with Stage I nonsmall cell lung carcinoma.
      ,
      • Rugge M
      • Fassan M
      • Clemente R
      • et al.
      Bronchopulmonary carcinoid: phenotype and long-term outcome in a single-institution series of Italian patients.
      ,
      • Skov BG
      • Holm B
      • Erreboe A
      • Skov T
      • Mellemgaard A
      ERCC1 and Ki67 in small cell lung carcinoma and other neuroendocrine tumors of the lung: distribution and impact on survival.
      ,
      • Walts AE
      • Ines D
      • Marchevsky AM
      Limited role of Ki-67 proliferative index in predicting overall short-term survival in patients with typical and atypical pulmonary carcinoid tumors.
      ,
      • Zahel T
      • Krysa S
      • Herpel E
      • et al.
      Phenotyping of pulmonary carcinoids and a Ki-67-based grading approach.
      ,
      • Van E
      • eden S
      • Quaedvlieg PF
      • Taal BG
      • et al.
      Classification of low-grade neuroendocrine tumors of midgut and unknown origin.
      ,
      • Das-Neves-Pereira JC
      • Bagan P
      • Milanez-de-Campos JR
      • et al.
      Individual risk prediction of nodal and distant metastasis for patients with typical bronchial carcinoid tumors.
      ,
      • Pelosi G
      • Pasini F
      • Fraggetta F
      • et al.
      Independent value of fascin immunoreactivity for predicting lymph node metastases in typical and atypical pulmonary carcinoids.
      ,
      • Greenberg RS
      • Baumgarten DA
      • Clark WS
      • Isacson P
      • McKeen K
      Prognostic factors for gastrointestinal and bronchopulmonary carcinoid tumors.
      but results are sometimes conflicting and not conclusive yet to authorize a well-recognized role as a prognostic factor for Ki-67 LI in lung NE tumors. Some authors denied any relevance for this marker to pinpoint differences in patients’ life expectancy inside individual tumors categories,
      • Igarashi T
      • Jiang SX
      • Kameya T
      • et al.
      Divergent cyclin B1 expression and Rb/p16/cyclin D1 pathway aberrations among pulmonary neuroendocrine tumors.
      ,
      • Skov BG
      • Holm B
      • Erreboe A
      • Skov T
      • Mellemgaard A
      ERCC1 and Ki67 in small cell lung carcinoma and other neuroendocrine tumors of the lung: distribution and impact on survival.
      ,
      • Van E
      • eden S
      • Quaedvlieg PF
      • Taal BG
      • et al.
      Classification of low-grade neuroendocrine tumors of midgut and unknown origin.
      whereas others indicated a worse prognosis in TC
      • Granberg D
      • Wilander E
      • Oberg K
      • Skogseid B
      Prognostic markers in patients with typical bronchial carcinoid tumors.
      or purported a role as metastasis predictor alone
      • Das-Neves-Pereira JC
      • Bagan P
      • Milanez-de-Campos JR
      • et al.
      Individual risk prediction of nodal and distant metastasis for patients with typical bronchial carcinoid tumors.
      or upon fascin overexpression (a protein involved in cell migration).
      • Pelosi G
      • Pasini F
      • Fraggetta F
      • et al.
      Independent value of fascin immunoreactivity for predicting lymph node metastases in typical and atypical pulmonary carcinoids.
      Only six studies have indeed supported a prognostic role of Ki-67 LI in surgically excised TC and/or AC,
      • Costes V
      • Marty-Ané C
      • Picot MC
      • et al.
      Typical and atypical bronchopulmonary carcinoid tumors: a clinicopathologic and KI-67-labeling study.
      ,
      • Grimaldi F
      • Muser D
      • Beltrami CA
      • et al.
      Partitioning of bronchopulmonary carcinoids in two different prognostic categories by ki-67 score.
      ,
      • Rugge M
      • Fassan M
      • Clemente R
      • et al.
      Bronchopulmonary carcinoid: phenotype and long-term outcome in a single-institution series of Italian patients.
      ,
      • Walts AE
      • Ines D
      • Marchevsky AM
      Limited role of Ki-67 proliferative index in predicting overall short-term survival in patients with typical and atypical pulmonary carcinoid tumors.
      ,
      • Zahel T
      • Krysa S
      • Herpel E
      • et al.
      Phenotyping of pulmonary carcinoids and a Ki-67-based grading approach.
      ,
      • Granberg D
      • Wilander E
      • Oberg K
      • Skogseid B
      Prognostic markers in patients with typical bronchial carcinoid tumors.
      but results are far from being conclusive. In fact, Ki-67 LI seemed to accurately segregate TC and AC into two distinct prognostic categories by cutoff values between 2.5% and 5.8%,
      • Costes V
      • Marty-Ané C
      • Picot MC
      • et al.
      Typical and atypical bronchopulmonary carcinoid tumors: a clinicopathologic and KI-67-labeling study.
      ,
      • Grimaldi F
      • Muser D
      • Beltrami CA
      • et al.
      Partitioning of bronchopulmonary carcinoids in two different prognostic categories by ki-67 score.
      ,
      • Rugge M
      • Fassan M
      • Clemente R
      • et al.
      Bronchopulmonary carcinoid: phenotype and long-term outcome in a single-institution series of Italian patients.
      ,
      • Walts AE
      • Ines D
      • Marchevsky AM
      Limited role of Ki-67 proliferative index in predicting overall short-term survival in patients with typical and atypical pulmonary carcinoid tumors.
      ,
      • Zahel T
      • Krysa S
      • Herpel E
      • et al.
      Phenotyping of pulmonary carcinoids and a Ki-67-based grading approach.
      which turned out independent of morphology in three studies totaling 220 carcinoids.
      • Costes V
      • Marty-Ané C
      • Picot MC
      • et al.
      Typical and atypical bronchopulmonary carcinoid tumors: a clinicopathologic and KI-67-labeling study.
      ,
      • Grimaldi F
      • Muser D
      • Beltrami CA
      • et al.
      Partitioning of bronchopulmonary carcinoids in two different prognostic categories by ki-67 score.
      ,
      • Rugge M
      • Fassan M
      • Clemente R
      • et al.
      Bronchopulmonary carcinoid: phenotype and long-term outcome in a single-institution series of Italian patients.
      In another study dealing with 43 TC, patients with increased Ki-67 expression had significantly shorter survival time.
      • Granberg D
      • Wilander E
      • Oberg K
      • Skogseid B
      Prognostic markers in patients with typical bronchial carcinoid tumors.
      A note of caution, however, has been advanced on the limited role of Ki-67 LI in predicting survival of low to intermediate malignant lung NE tumors when lumping TC and AC, inasmuch as a threshold of 5% did not offer substantial better survival information over morphology, also within individual tumor categories.
      • Walts AE
      • Ines D
      • Marchevsky AM
      Limited role of Ki-67 proliferative index in predicting overall short-term survival in patients with typical and atypical pulmonary carcinoid tumors.
      Similar conclusions on the lack of an independent prognostic efficacy of Ki-67 LI in lung NE tumors have recently been published in an abstract form only.
      • Wang H
      • Iyoda A
      • Roh MS
      • et al.
      WHO histologic classification is an independent predictor of prognosis in lung neuroendocrine (NE) tumors but Ki-67 proliferation rate is not (abstract #1952).
      Interestingly, however, several studies have revealed that a Ki-67 LI cutoff of 4% to 5% could differentiate between lower and higher malignant NE tumors in the setting of TC and AC,
      • Costes V
      • Marty-Ané C
      • Picot MC
      • et al.
      Typical and atypical bronchopulmonary carcinoid tumors: a clinicopathologic and KI-67-labeling study.
      ,
      • Grimaldi F
      • Muser D
      • Beltrami CA
      • et al.
      Partitioning of bronchopulmonary carcinoids in two different prognostic categories by ki-67 score.
      ,
      • Rugge M
      • Fassan M
      • Clemente R
      • et al.
      Bronchopulmonary carcinoid: phenotype and long-term outcome in a single-institution series of Italian patients.
      ,
      • Walts AE
      • Ines D
      • Marchevsky AM
      Limited role of Ki-67 proliferative index in predicting overall short-term survival in patients with typical and atypical pulmonary carcinoid tumors.
      ,
      • Zahel T
      • Krysa S
      • Herpel E
      • et al.
      Phenotyping of pulmonary carcinoids and a Ki-67-based grading approach.
      ,
      • Pelosi G
      • Pasini F
      • Fraggetta F
      • et al.
      Independent value of fascin immunoreactivity for predicting lymph node metastases in typical and atypical pulmonary carcinoids.
      similar to what already demonstrated in analogous NE tumors of the pancreas.
      • Pelosi G
      • Bresaola E
      • Bogina G
      • et al.
      Endocrine tumors of the pancreas: Ki-67 immunoreactivity on paraffin sections is an independent predictor for malignancy: a comparative study with proliferating-cell nuclear antigen and progesterone receptor protein immunostaining, mitotic index, and other clinicopathologic variables.
      ,
      • Rindi G
      • Falconi M
      • Klersy C
      • et al.
      TNM staging of neoplasms of the endocrine pancreas: results from a large international cohort study.
      Although conceptually reasonable, no studies have so far addressed a role of Ki-67 LI in the prognostic stratification of poorly differentiated NE tumors, at variance with what has been proposed in other endocrine organs, such as the pancreas.
      • Bastrurk O
      • Yang Z
      • Tang L
      • et al.
      Increased (>20%) Ki-67 proliferation index in morphologically well differentiated pancreatic neuroendocrine tumors (PanNETs) correlates with decreased overall survival (abstract #1761).
      Relevant key points: Ki-67 LI has been proposed as a prognostic factor in excised specimens of TC and AC, with cutoff values ranging from 2.5% to 5.8%, sometimes but not always independent of morphology. The existence of conflicting results and the lack of widely agreed-upon cutoff thresholds to stratify these tumors preclude making a recommendation at this time. Additional information is needed to establish the ideal role of Ki-67 LI in the prognostic assessment of lung NE tumors, ideally helping to predict prognosis within individual tumor categories. Because there is not much variability in survival for TC, LCNEC, and SCLC, the tumor category where there would be the greatest potential to predict prognosis is within AC. When lumping TC and AC together, it is not surprising that Ki-67 may help with prognosis, but this is not really adding anything to existing diagnostic capabilities.
      Question 4: Is there an established role for Ki-67 LI in tumor grading?
      Answer: No, in lung NE tumors, the concept of tumor grading as a biological continuum paralleling increasing malignancy is tautologically included into the current WHO classification, according to which TC are considered low malignant, AC intermediate malignant, and LCNEC and SCLC high malignant tumors.
      In this setting, tumor grade of lung NE tumors refers to the degree of biologic aggressiveness and is related to, but different from, differentiation that is in turn defined by morphology.
      • Klimstra DS
      • Modlin IR
      • Coppola D
      • Lloyd RV
      • Suster S
      The pathologic classification of neuroendocrine tumors: a review of nomenclature, grading, and staging systems.
      The main reason why Ki-67 LI assessment cannot currently claim any primacy in the grading system of lung NE tumors over morphology to realize the clinical three-tier spectrum regards its suboptimal correlation with histological features used for classification, especially mitotic count
      • Tsuta K
      • Liu DC
      • Kalhor N
      • Wistuba II
      • Moran CA
      Using the mitosis-specific marker anti-phosphohistone H3 to assess mitosis in pulmonary neuroendocrine carcinomas.
      ,
      • Warth A
      • Fink L
      • Fisseler-Eckhoff A
      • Pulmonary Pathology Working Group of the German Society of Pathology
      • et al.
      Interobserver agreement of proliferation index (Ki-67) outperforms mitotic count in pulmonary carcinoids.
      and necrosis, which causes adjacent categories of traditionally assessed tumors to partially imbricate with each other. This also reflects the fact that morphology itself is insufficient in separating borderline/overlapping lesions for either low to intermediate malignant or high malignant tumors.
      • Travis WD
      • Gal AA
      • Colby TV
      • et al.
      Reproducibility of neuroendocrine lung tumor classification.
      ,
      • Asamura H
      • Kameya T
      • Matsuno Y
      • et al.
      Neuroendocrine neoplasms of the lung: a prognostic spectrum.
      ,
      • Marchevsky AM
      • Gal AA
      • Shah S
      • Koss MN
      Morphometry confirms the presence of considerable nuclear size overlap between “small cells” and “large cells” in high-grade pulmonary neuroendocrine neoplasms.